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PO-CON1641E
Method development of high

throughput lipid analysis in foods
by direct analysis in real time
mass spectrometer (DART-MS)
ASMS 2016 ThP 002
Motoshi Sakakura1, Teruhisa Shiota1, Jun Watanabe2

1
AMR, Inc., Tokyo, JAPAN;
2
Shimadzu Corporation, Kyoto, JAPAN
Method development of high throughput lipid analysis in foods
by direct analysis in real time mass spectrometer (DART-MS)
Introduction
Acylglycerols included in a food play the role important to In this study, we analyzed food samples including
the food function such as flavor and nutrition. There were acylglyserols directly with DART (Direct Analysis in Real
some problems such as complex preparation steps and Time) and mass spectrometer with less sample
carry over to analytical instruments with an analysis of pretreatment and tried to detect the characteristic
acylglycerols, and it was difficult to analyze a great deal ingredients in samples and distinguish each sample.
of samples quickly.
Methods and Materials

Mass spectrometer LCMS-2020 (Shimadzu Corporation, it up to the DART gas stream about 10 seconds. Further,
Kyoto, Japan) equipped with real time direct analysis ion without pretreating a sample, it was set to the analysis.
source DART-OS (IonSense, Inc., Saugus, MA, USA) was LCMS-2020 can achieve the polarity switching time of
used for this study. Marketed soybean milk, butter and 15msec and the scanning speed of up to 15000u/sec,
margarine, etc. were used for food samples including therefore the loop time can be set at less than 1 second
acylglycerols. Samples were analyzed by making a small despite the relatively large scanning range of 10-1000u.
amount samples adherent onto a glass stick and holding
Figure 1 DART-MS System; LCMS-2020 equipped with DART-OS
2
Result
Various samples were analyzed by DART-MS. Analyzing time of each sample was about 10 seconds and analyzing
interval of each sample was less than 1 minute.
Positive Mass Spectum

From the results of food samples of plant origin like butter, many signals of triacylglycerides and
soybean milk and margarine, several signals of diacylglycerides which comprised of middle chain fatty
triacylglycerides (m/z 900 near here) and diacylglycerides acid as well as long chain fatty acid were detected, and
origin mainly comprised of oleic acid and the palmitic acid the spectrum pattern was indicated different from
were detected in the positive ion spectrum. In the case of vegetable fats and oils.
measuring food samples of animal origin like milk and
Inten. (x10,000)
6.0 DAG TAG

5.0
Soybean 4.0
milk 3.0
2.0
1.0
0.0
100 200 300 400 500 600 700 800 900 m/z
Inten. (x100,000)
1.00
TAG • DAG
0.75
Milk 0.50
0.25
0.00
100 200 300 400 500 600 700 800 900 m/z
Inten. (x100,000)
2.5
2.0
DAG TAG
1.5
Margarine
1.0
0.5
0.0
100 200 300 400 500 600 700 800 900 m/z
Inten. (x100,000)
2.0 TAG • DAG

1.5
Butter
1.0
0.5
0.0
100 200 300 400 500 600 700 800 900 m/z
Figure 2 Positive mass spectra of samples analyzed by DART-MS
3
The marketed food sample which was margarine added butter was analyzed. It had just mixed mass spectrum in which
some glyceride signals mainly comprised of oleic acid and the palmitic acid were detected and many glyceride signals
which comprised of middle and long chain fatty acids were detected.
Inten. (x1,000,000)
2.0 TAG • DAG DAG from TAG from

from animal plant plant
Margarine 1.5
added 1.0
butter
0.5
0.0
100 200 300 400 500 600 700 800 900 m/z
Figure 3 Positive mass spectum of margarine added butter analyzed by DART-MS
Negative Mass Spectrum

The negative mass spectra were indicated (figure 4). A signal was detected by m/z 215 in all spectra except for soybean
milk both. It was inferred that these signals were chloric adduct ion [M+Cl]- of monosaccharide (C6H12O6).
Inten. (x10,000)
2.0
1.5
Soybean
milk 1.0
0.5
0.0
50 100 150 200 250 300 350 400 450 500 550 600 650 m/z
Inten. (x100,000)
2.0
1.5
Milk 1.0
0.5
0.0
50 100 150 200 250 300 350 400 450 500 550 600 650 m/z
Inten. (x10,000)
4.0
3.0
Margarine 2.0
1.0
0.0
50 100 150 200 250 300 350 400 450 500 550 600 650 m/z
Inten. (x10,000)
8.0
7.0
6.0
5.0
Butter 4.0
3.0
2.0
1.0
0.0
50 100 150 200 250 300 350 400 450 500 550 600 650 m/z
Figure 4 Positive mass spectra of samples analyzed by DART-MS
4
Total ion current chromatograms (TIC) and extracted ion current chromatograms (XIC) of m/z 215 in negative mode
when each sample being analyzed were shown in figure 5. XIC signal of monosaccharide origin wasn't detected in
soybean milk. It was detected weakly in butter and hard in milk and margarine.
Soybean milk Milk Margarine Butter

(x1,000,000)
2:TIC(-)
3.0
2.5 TIC
Negative
2.0
1.5
(x100,000)
2:215.00(-)
2.0
1.5 XIC
1.0
m/z 215
Negative
0.5
0.0
2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 min
Figure 5 TIC chromatogram and XIC chromatogram (m/z 215)
High throughput lipid analysis in foods containing oils and fats was achieved by a mass spectrometer with high speed
polarity switching and high speed scanning integrated with DART ion source which could real-time-analyze and it was
possible to profile plant acylglycerol and animal acylglycerol.
Conclusions
Various food samples were analyzed by DART-MS;
High throughput lipid analysis in foods containing oils and fats was achieved by a mass spectrometer with high speed
polarity switching and high speed scanning.
From the results of food samples;

[Food samples of plant origin] several signals of triacylglycerides and diacylglycerides origin mainly comprised of oleic
acid and the palmitic acid were detected.
[Food samples of animal origin] many signals of triacylglycerides and diacylglycerides which comprised of middle and
long chain fatty acids were detected.
5
References
1. A. McANOY et.al, J. Am. Mass Spectrom., 2005, 16, 1498-1509.
2. B. Winther et.al, Lipids, 2011, 46, 25-36.
3. V. A.Tyurin et.al, Journal of Neurochemistry, 2008, 107, 1614-1633.
4. H. Mikuma, A. Kaneko, BUNSEKI KAGAKU, 2010, 59(5), 399-404.
Disclaimer: The products and applications in this presentations are intended for Research Use Only (RUO). Not for
use in diagnostic procedures.
First Edition: June, 2016
For Research Use Only. Not for use in diagnostic procedure.

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© Shimadzu Corporation, 2016
PO-CON1640E
Development of a flavor release

analysis method on volatile compounds
of citrus fruits by DART MS
ASMS 2016 ThP 005
Takehiko Sagawa1, Keiko Matsumoto2, Jun Watanabe2,

Chikako Takei3, Motoshi Sakakura4, Teruhisa Shiota4,
Hiroshi Matsufuji5
1
S & B Foods Inc., Tokyo, JAPAN;
2
Shimadzu Corporation, Kyoto, JAPAN;
3
BioChromato, Inc., Fujisawa, JAPAN;
4
AMR, Inc., Tokyo, JAPAN;
5
Nihon University, Fujisawa, JAPAN
Development of a flavor release analysis method on
volatile compounds of citrus fruits by DART MS
Introduction
Gas chromatography is typically used for analysis of the in volatile compounds and it is difficult to specify a target
volatile compounds represented by aroma compounds. compound by SIM analysis, we developed a simultaneous
Nevertheless it's important to find relationship with the analysis method using MRMs of flavor compounds which
results of chemical analysis and perceptive aroma, it is have the similar structure by LC-MS/MS at last this
hard to measurement flavor release from foods in real conference.
time. DART MS with closed-chamber interface system is Here, we tried to measure the flavor release phenomena
effective to monitor volatile compounds within seconds on volatile compounds of several kinds of citrus fruits.
successively. Although there are a lot of structural isomers

Limonene, gamma terpinene, octanal, linalool and alpha IonSense Inc., MA, USA), so MS conditions like
terpineol were used for volatile compounds. Limonene compound-dependent parameters and MRM transitions
and gamma terpinen (molecular weight 136), linalool and of each compound were optimized using volatile
alpha terpineol (molecular weight 154) are a structure compounds analysis system with DART.
isomer, respectively. Triple quadrupole mass spectrometer Next, the DART-MS/MS system was coupled with closed
LCMS-8030 (Shimadzu Corporation, Kyoto, Japan) was interface, Volatimeship (Bio Chromato, Inc., Japan) in
used for the analysis of these components. All compounds order to achieve high sensitivity and real time flavor
were ionized by APCI except for octanal, but all compounds response and then several kinds of citrus fruits were
were ionized with the DART (DART-OS ion source; analyzed.
limonene
C10H16 γ-terpinene
C10H16 octanal
Mw 136
Mw 136 C8H16O
Mw 128
linalool a-terpineol
C10H18O C10H18O
Mw 154 Mw 154
Figure 1 Structure of volatile compounds in this study
2
Ultra Fast Polarity Switching

• 15msec
Ultra Fast Scanning
• 15,000 u/sec
Ultra Fast MRM
• Max. 555 transition /sec
Figure 2 LCMS-8030 triple quadrupole mass spectrometer
Result
Method development for volatile compounds
Five volatile compounds were used for this study because and terpinene, 170, 186. All compounds were ionized
they were known as characteristic voltile compounds in high-sensitively at positive ion mode. For improvement of
citrus fruits. Limonene has an aroma of an orange peel, the selectivity of these compounds, MRM transitions of
octanal has a sweet aroma of citrus with low each compound were set using volatile compound
concentration, and linalool has an aroma of flower. analysis with DART. This was very easily implemented by
Regarding ionization ability of these components, all only bringing sample vials close to the DART ionization
compounds (their volatile) were ionized by DART. These area without injection. Then we found each compound
compounds were interestingly ionized, that is, they had specific MRM transitions; terpinene was detected
detected not molecular related ions (M+H) but several intensively at Q1/Q3=170/153 (positive) which transition
ions which might be fragment ions; precursor ion signals could specify terpinene.
of limonene were detected by positive m/z 135, 151, 153
Figure 3 Standard sample (vapor)

analysis by DART-MS
Sample vial opened its cap was placed

under DART gas flow.
3
Inten. (x10,000,000)
1.50
135.1
151.2
1.25
1.00
124.1
limonene 0.75
0.50
154.2
167.1
81.2
188.2
107.1
0.25
93.1
0.00
50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 m/z
Inten. (x10,000,000)
1.75
124.1
137.2
1.50
1.25
a-terpineol
1.00
153.2
0.75
81.1
0.50
154.2
188.2
138.2
169.2
0.25
0.00
50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 m/z
Figure 4 Positive mass spectra of limonene and a-terpineol
Analysis by DART-MS
Next, the DART-MS/MS system was coupled with closed sample cage and crushed, with analyzing the volatile
interface in order to achieve high sensitivity and real time compounds released from citrus fruit by closed DART
flavor response and then several kinds of citrus fruits system. Shonan Gold (local citrus fruit in Japan) and
were analyzed. Whole citrus fruit were put in the closed orange were analyzed successively.
Figure 5 Shonan Gold
4
Total analyzing time for each citrus fruit is within 5 fruits. The changes were different from each fruit. This
minutes. Terpinene, Limonene, Linalool and Terpineol system detected flavor compounds within sub-second
were detected at citrus fruits set into sample flask and resolution and may be useful for breeding and singularity
their intensity were changed just after squeezing the analysis of flavor fruits.
Shonan Gold Orange

(x10,000)
1:Terpinene 170 170.20>153.10(+) CE: -11.0
1.0 MRM 170/153

(positive)
Terpinene
0.5
0.0
(x100,000)
3:Linalool 154 154.20>81.10(+) CE: -12.0
3.0
MRM 154/81
2.0 (positive)
Linalool
1.0
0.0
(x100,000)
4:Limonene 135 135.20>107.10(+) CE: -16.0
1.5
1.0 MRM 135/107

(positive)
0.5 Limonene
0.0
(x1,000,000)
8:a-Terpineol 137.20>81.10(+) CE: -11.0
2.0
1.5
MRM 137/81
(positive)
1.0
Terpineol
0.5
0.0
17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 min
Set fruit in Squeezing Set fruit in Squeezing

sample sample
flask flask
Figure 5 MRM chromatograms of Shonan Gold and Orange
5
DART Volatimeship
He Gas
MS
squeezing N2 Gas
Figure 6 DART & Volatimeship System
Conclusions
Volatile compounds in citrus fruits could be detected with real time analysis by closed DART MS/MS system.
Disclaimer: The products and applications in this presentations are intended for Research Use Only (RUO). Not for

PO-CON1653E
A Combined MRM and SIM Method

for Direct Quantitative Determination
of Amino Acids in Various Samples
on LC/MS/MS
ASMS 2016 TP740
Zhe Sun1, Jie Xing1, Pei Yee Khoo2* and Zhaoqi Zhan1
1
Application Development & Support Centre,
Shimadzu (Asia Pacific) Pte Ltd, 79 Science Park Drive,
Singapore;
2
School of Physical and Mathematical Sciences,
Nanyang Technological University, Singapore;
*Student
A Combined MRM and SIM Method for
Direct Quantitative Determination of Amino Acids
in Various Samples on LC/MS/MS
Introduction
Quantitative analysis of amino acids in biological samples, applicability for various kinds of samples. We describe the
food and nutrition products are often required in various applications of the Intrada Amino Acid column with using
fields from research to manufacturing [1]. Recently, a combined MRM-SIM mode for direct analysis of 20
Imtakt introduced a new Intrada Amino Acid column, amino acids in a variety of samples on LC/MS/MS. The
which is composed of a mixed stationary phase of ion samples include from human plasma, serum, urines to
exchange and normal phase, for direct separation and wines, beers, vinegar, sports water & amino acid drink.
detection of amino acids on LC-MS without the need for The aim of using a combined SIM and MRM method is to
pre- or post-column derivatization [2]. This new method increase the detection sensitivity of certain amino acids
not only simplifies the analysis of amino acids drastically, which have low sensitivities in MRM mode, mainly Glycine
but also reduces the running cost and enhances the and a few other amino acids.
Experimental
Twenty amino acid standards in powders were obtained analyzed. The sample was de-proteinized by adding
from Sigma Aldrich. They were dissolved in 0.1N HCl MeOH/ACN (1:1) solvent in a ratio of 1:3 or 1:4, followed
solution to obtain individual stock solutions, except for by vortex and centrifugation at 13,000 rpm for 10 mins.
cystine and glutamine, which were dissolved in 1.0N HCl The supernatant was transferred and filtered before
solution. A mixed standard was prepared from the stocks LC/MS/MS analysis. An LCMS-8040 triple quadrupole
and was diluted using pure water serially to various coupled with an UFLC system (Shimadzu Corporation)
concentrations as calibrants. Two categories of samples, was employed in this work. The detailed conditions are
i.e., biological and beverage samples were collected and compiled in Table 1.
Table 1: Analytical conditions of twenty amino acids on LCMS-8040: HPLC (left) and MS/MS (right)
Column : Intrada Amino Acid (100 x3 mm, 3µm) Interface : ESI

Flow rate : 0.6 mL/min MS mode : Posi, MRM-SIM
Mobile phase : A: ACN/THF / 25mM ammonium formate / Block temp. : 400ºC
FA = 9 / 75 /16 / 0.3 (v) DL temp. : 300ºC
B: ACN / 100mM ammonium formate = 20 / 80 CID gas : Ar (230kPa)
Elution mode : Gradient elution, 0-3min (0% B) → 9min (17% B) Nebulizing gas flow : N2, 3 L/min
→ 16-18min (100% B) → 18.5min (0% B) Drying gas flow : N2, 15 L/min
Oven temp. : 35ºC
Injection vol. : 2.0 µL
2
Results and Discussion

Establishment of a combined MRM-SIM method for 20 amino acids
With the Intrada column, both MRM method and SIM below the concentration. While the SIM peak of glycine
method were applied for analysis of amino acids on exhibits high intensity at the same concentration and
LC/MS/MS and LC/MS independently [1,2]. Figure 1 could be detected at 5 nmon/mL in clear solution. Thus,
shows typical MRM chromatograms of a mixed standard quantitation of glycine could be relied on SIM mode for
of 20 amino acids on LCMS-8040 following the Imtakt lower concentration levels. In addition, a few more amino
method [1]. However, it was observed that glycine acids, namely, Thr, Asp, Ala, Ser, and Cys, exhibit higher
(m/z76) exhibited very low peak intensity in MRM mode SIM mode intensity and detection sensitivity than MRM
(76 > 30). As a result, the detection sensitivity remained mode in clear solutions (Figure 3). Based on these results,
poor. In this work, a combined MRM-SIM mode method a combined MRM-SIM method was established for
was adopted instead of only MRM. The SIM data was quantitation of amino acids rather than only MRM
acquired by the Q3 (Q1-q2-Q3) simultaneously with MRM method. The details of the MRM-SIM method established
data in the same analysis on LCMS-8040. are summarized in Table 2. The accuracy and repeatability
Several pairs of MRM and SIM peaks of amino acids are of the methods (not shown in the table) were evaluated
displayed in Figure 3. It can be seen that, the MRM peak and satisfied results were obtained. A few selected
of glycine was very small at 50 nmol/mL and disappeared calibration curves are displayed in Figure 2.
(x1,000,000) Area (x1,000,000) Area (x1,000,000) Area (x1,000,000)

8 8 8
Pro
4.0
Phe
MRM mode Leucine Methionine 2.0 Glutamic

3.0 (MRM) (MRM) Acid (MRM)
Ala
5.0 2.0
1.5
Ile
His
2.0
Trp
Leu
1.0
7 7 7
Arg
2.5 1.0
Val
Met
Asn
Ser
Tyr
1.0
Cys
Lys
Glu
0.5
Thr
Asp
6 6
0.0 5 5 56
0.0 34 0.0 34 0.0
2.5 5.0 7.5 10.0 12.5 15.0 17.5 min 0 50 Conc. 0 50 Conc. 0 50 Conc.
(x10,000,000) Area (x10,000,000) Area (x100,000) Area (x100,000)
2.0 8 7.5 8 5.0 8
Ile
Leu
SIM mode Proline Threonine Glycine

2.0
Phe
1.5 (MRM) (MRM) (SIM)

Met
1.5 5.0
Pro
Val
1.0
Cys
Asp
2.5
Tyr
Trp
1.0
7 7
His
2.5 7
Ser
0.5
Arg
Glu
AspAla
GlyGlu
0.5
Lys
Thr
6 6 6
0.0 2345
0.0 1
5 2345
0.0 1
0.0
2.5 5.0 7.5 10.0 12.5 15.0 17.5 min 0 50 Conc. 0 50 Conc. 0 50 Conc.
Figure 1: MRM and SIM chromatograms of 20 amino acids Figure 2: Representative MRM & SIM calibration curves
mixed standard (50 nmol/mL, 1 µL inj). of eight levels at 0.05-100 nmol/mL.
3
Q 120.10>74.00 1.52e5 Q 134.10>73.90 1.92e4 Q 106.10>60.20 3.68e4 Q 133.10>74.10 8.13e4 Q 241.00>151.95 1.17e5 Q 76.00>30.10 4.33e2
100.00 100.00 100.00 100.00 100.00 106.47

Thr Asp Ser Asn (Cys)2 Gly
% % % % % %
0.00 0.00 0.00 0.00 0.00 30.59

7.0 7.5 8.0 7.0 7.5 8.0 8.0 8.5 9.0 9.0 9.5 10.0 12.00 12.25 12.50 8.5 9.0 9.5
Q 120.10 1.49e6 Q 134.10 6.65e5 Q 106.10 5.44e5 Q 133.10 9.52e5 Q 241.00 1.78e6 Q 76.00 1.93e5
100.00 102.79 101.89 102.86 102.21 101.12

Thr Asp Ser Asn (Cys)2 Gly
% % % % % %
0.00 8.94 26.42 12.38 36.76 40.78

7.0 7.5 8.0 7 8 8.0 8.5 9.0 9.0 9.5 12.0 12.2 12.4 8.5 9.0 9.5
Figure 3: Individual chromatograms of selected amino acids (50 nmol/mL, 1 µL inj), comparing MRM (top) and SIM (bottom) modes.
Table 2: Summary of calibration linearity, LOQ and repeatability (%RSD) of the SIM-MRM quantification method.
RT MRM Method (nmo//mL) SIM Method (nmol/mL)

No. Name
(min) m/z Range R2 LOQ m/z Range R2 LOQ
1 Tryptophan 3.42 205.1>188.2 0.1-100 0.996 0.1 205.1 0.1-100 0.997 0.1
2 Phenylalanine 3.74 166.1>120.1 0.1-100 0.998 0.1 166.1 0.1-100 0.997 0.1
3 Tyrosine 4.06 182.1>136.2 0.5-100 0.998 0.5 182.1 0.5-100 0.999 0.5
4 Leucine 4.69 132.1>86.3 0.1-100 0.999 0.1 150.1 0.5-100 0.997 0.5
5 Methionine 4.91 150.1>56.1 0.5-100 0.999 0.5 132.1 0.1-100 0.998 0.1
6 Isoleucine 5.1 132.1>86.3 0.1-100 0.999 0.1 132.1 0.5-100 0.998 0.5
7 Valine 6.09 118.2>72.1 0.5-100 0.998 0.5 118.2 0.5-100 0.998 0.5
8 Glutamic Acid 7.06 148.1>84.1 0.5-100 1.000 0.5 148.1 0.5-100 0.998 0.5
9 Proline 7.29 116.1>70.1 0.1-100 0.999 0.1 116.1 0.1-100 0.999 0.1
10 Threonine 7.68 120.1>74.0 1.0-100 1.000 1 120.1 0.5-100 0.996 0.5
11 Aspartic acid 8.05 134.1>73.9 5.0-100 0.996 5 134.1 1.0-100 0.997 1.0
12 Alanine 8.25 90.1>44.1 5.0-100 0.994 5 90.1 0.5-100 0.993 0.5
13 Serine 8.94 106.1>60.2 5.0-100 0.999 5 106.1 1.0 -100 0.997 1.0
14 Glutamine 9.13 147.1>84.1 0.5-100 0.992 0.5 147.1 10-100 0.907 10
15 Glycine 9.38 76.0>30.0 25-100 0.993 25 76 5-100 0.993 5
16 Asparagine 9.56 133.1>74.1 5.0-100 0.999 5 133.1 5-100 0.998 5
17 Cystine 12.31 241.0>151.9 1-100 0.999 1 241 1-100 0.996 1
18 Histidine 16.57 156.1>110.1 0.5-100 0.992 0.5 156.1 0.5-100 0.991 0.5
19 Lysine 17.15 147.0>84.1 0.1-100 0.999 0.1 147 5-100 0.999 5
20 Arginine 18.1 175.1>70.1 0.5-100 0.999 0.5 175.1 0.1-100 0.998 0.5
4
Analysis of amino acid in biological and beverage samples

One of the purposes of this study is to evaluate the beverage samples), we could summarize the results and
robustness of the method for different samples (biological the method robustness in a few key points. First, the
and beverage samples). Without additional clean-up except quantitative results by MRM and SIM calibrations are well
deproteini-zation and filtering, the liquid sample was in agreement with each other. Second, glycine could be
injected to LCMS-8040. The results of six representative detected and quantified only by SIM method in all the
samples are compiled into Table 3 and the chromatograms samples. Third, two amino acids could not be detected by
of four samples are shown in Figure 4. From the analyses MRM, but were detected by SIM mode (Asp in urine, Cys
of 20 different samples (3 plasma, 1 serum, 7 urine, 9 in vinegar).
(x1,000,000) (x100,000)
3.0
1.5
(a) Human plasma (b) Urine
1.0 (MRM) 2.0 (MRM)
0.5 1.0
0.0 0.0
2.5 5.0 7.5 10.0 12.5 15.0 17.5 min 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min
(x10,000,000) (x1,000,000)
2.0
1.5 (a) Human plasma (b) Urine
1.5
(SIM) (SIM)
1.0
1.0
0.5
0.5
0.0 0.0
2.5 5.0 7.5 10.0 12.5 15.0 17.5 min 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min
(x1,000,000) (x1,000,000)
2.0 2.5
(c) Red wine 2.0

(d) Beer
1.5
(MRM) 1.5
(MRM)
1.0
1.0
0.5 0.5
0.0 0.0
5.0 7.5 10.0 12.5 15.0 17.5 min 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min
(x10,000,000) (x10,000,000)
1.5
1.5
(c) Red wine (d) Beer
1.0
(SIM) 1.0 (SIM)
0.5 0.5
0.0 0.0
2.5 5.0 7.5 10.0 12.5 15.0 17.5 min 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min
Figure 4: MRM (top) and SIM (bottom) profiles of (a) human plasma, (b) urine, (c) red wine and (d) beer sample.
Furthermore, the amino acid profiles in different samples rice wine). (d) In beer samples, Pro is in highest content
are outlined below. (a) In human plasma and serum, the and Cys is the lowest. (e) The Sports Water and Amino
contents of Glu and Ala are the highest, and Met is the Acid Drink bought from supermarket are with labeled
lowest. (b) Amino acid contents in urine are varied greatly contents of amino acids in the product bottles. The
across the 7 individuals. But Gln and Pro are detected quantitative results of amino acids are closed to the
consistently as the highest and lowest, respectively. (c) contents on labels. The Sports Water contains Leu, Ile and
Most amino acids are found in high contents except Typ Val. The Amino Acid Drink contains Try, Val, Leu, Ile, Thr
and Cys in all three types of wines (red, white and Chinese and Lys.
5
Table 3: Amino Acid profiles in biological and beverage samples determined by MRM-SIM method on LCMS-8040
Biological Sample Conc. (nmol/mL) Beverage Sample Conc. (nmol/mL)

No. Name m/z
Human Plasma Human Serum Urine Red Wine Beer Vinegar
205.1 141.5 34.8 109.9 3.0 111.1 ND
1 Tryptophan
205.1>188.2 134.8 31.5 100.5 2.5 87.7 ND
166.1 122.1 90.7 110.6 87.7 173.8 1010.0
2 Phenylalanine
166.1>120.1 122.2 89.6 93.5 79.0 159.6 766.1
182.1 117.6 93.9 91.8 92.4 135.5 47.9
3 Tyrosine
182.1>136.2 113.8 85.9 116.4 57.9 110.7 31.1
132.1 285.9 466.4 57.8 180.8 59.4 2988.2
4 Leucine
132.1>86.3 274.6 445.4 53.4 176.2 57.2 3248.1
150.1 3.6 0.9 111.1 33.7 7.2 90.6
5 Methionine
150.1>56.1 2.5 0.7 112.6 35.1 6.7 83.1
132.1 163.6 101.5 19.2 60.1 48.1 2357.2
6 Isoleucine
132.1>86.3 151.2 85.7 17.3 56.0 43.1 2010.5
118.2 360.0 375.9 67.3 110.6 255.7 3627.7
7 Valine
118.2>72.1 383.3 323.2 66.4 99.2 240.5 2783.3
148.1 1353.3 907.4 27.4 162.1 60.0 1942.5
8 Glutamic Acid
148.1>84.1 1358.1 904.5 18.6 167.5 56.5 1735.6
116.1 496.1 421.7 6.1 14724 3546.9 2851.6
9 Proline
116.1>70.1 495.7 418.8 6.9 19555 2699.9 3555.1
120.1 403.1 437.1 769.5 224.0 (235.0)* 2960.4
10 Threonine
120.1>74.0 303.0 375.7 549.2 183.8 5.7 1655.5
134.1 85.9 285.2 109.2 247.2 53.0 478.7
11 Aspartic acid
134.1>73.9 65.6 288.6 ND 296.8 42.0 116.3
90.1 937.5 1003.2 1184.1 997.9 1129.2 13420.1
12 Alanine
90.1>44.1 1053.1 1540.6 946.8 689.5 874.0 9828.1
106.1 350.0 574.1 1086.0 156.4 17.5 3205.1
13 Serine
106.1>60.2 344.8 631.0 1023.8 160.3 15.1 2721.7
147.1 195.6 155.7 3007.5 (56.5)* 210.1 10.3
14 Glutamine
147.1>84.1 254.0 282.8 3598.2 1.8 116.7 12.1
76 642.6 493.5 1515.2 488.6 366.3 5215.5
15 Glycine
76.0>30.1 ND ND ND ND ND ND
133.1 34.7 24.1 250.7 76.5 37.6 561.3
16 Asparagine
133.1>74.1 27.5 18.6 188.5 79.1 16.7 456.7
241 18.9 7.4 73.8 ND ND 7.9
17 Cystine
241.0>152.0 20.8 6.5 72.4 ND ND ND
156.1 238.4 45.1 2547.4 67.1 117.0 67.2
18 Histidine
156.1>110.1 246.3 49.6 2645.5 49.9 126.3 58.7
147 551.6 312.6 709.3 101.2 4.9 2135.3
19 Lysine
147.0>84.1 584.2 309.3 797.6 147.3 7.7 3374.8
175.1 923.3 171.2 86.3 152.9 197.4 1260.9
20 Arginine
175.1>70.1 924.0 170.4 85.2 161.3 194.0 1339.8
Note: ND = Not Detected; * The SIM peak has co-eluted component and the result is not accurate.
6
Conclusions
By using the Intrada Amino Acid column, a combined on LC-MS. The advantages of a combined MRM-SIM
MRM-SIM method has been established for detection and method on LC/MS/MS are the higher sensitivity for glycine
quantification of 20 amino acids in biological and beverage in SIM mode and overall enhanced reliability, robustness
samples. The Intrada Amino Acid column can separate and accuracy in comparison with the only MRM method or
effectively the amino acids without need of pre-column SIM method.
derivatization, which allow direct analysis of amino acids
References
1. Yazwa Itaru, Tachikawa Hiroshi, “Development of a novel amino acids analysis column for LC-MS without
derivatization”, Imtakt Corporation (2014)
2. Matsumoto K., Watanabe J., Yazawa I., “Simultaneous quantitative analysis of 20 amino acids in food samples
without derivatization using LC-MS/MS”, ASMS 2014, TP 510
Disclaimer: Shimadzu LCMS-8040, UFLC XR system and Labsolutions Insight are intended for Research Use Only
(RUO). Not for use in diagnostic procedures.

PO-CON1648E
Development of LC/MS/MS Method

for Screening and Quantitation
of 47 Synthetic Dyes under
Restricted Substance List in Textiles
ASMS 2016 TP375
Yin Ling Chew1; Jie Xing1; Leonard Guan Seng Lim2*;

Zhaoqi Zhan1
1
Shimadzu (Asia Pacific) Pte Ltd,
79 Science Park Drive #02-01/08, Singapore;
2
School of Physical & Mathematical Science,
Nanyang Technological University, Singapore;
*Student
Development of LC/MS/MS Method for Screening and
Quantitation of 47 Synthetic Dyes under Restricted
Substance List in Textiles
Introduction
Synthetic dyes such as azodyes and disperse dyes are introduced to ban the use of carcinogenic dyes and
widely used in the manufacture of various consumer restrict the amount of allergenic dyes allowed in textile
products such as textiles, leather, toys and plastics. Many making and consumer products. Various analysis methods
of these dyes are allergenic that may cause contact using HPLC, TLC and LC/MS/MS [2] are reported. We
dermatitis and others are carcinogenic. Many synthetic describe a fast and high sensitivity LC/MS/MS method for
dyes using in textiles and apparels are listed in the RSL [1] screening and quantitation of 47 synthetic dyes including
for protection of the consumers. Legislations such as EU 23 azodyes, 21 disperse dyes and 3 basic dyes in textile
2002/371/EC and OEKO-TEX Standard 100 were samples.
Experimental
Forty-seven dye compounds were obtained from Sigma 10,000 rpm. The supernatant was filtered with 0.22 µm
Aldrich, Dr. Ehrenstorfer, Merck Schuchardt, Fischer PTFE filter. It was then evaporated and reconstituted with
Scientfic, Accustandard, Institute of Leather Industry, equal volume of 95:5 water/methanol diluent before
Supelco, Fluka and Tokyo Chemical Industry. These analysis. An LCMS-8040 triple quadrupole UFMS system
compounds were dissolved in MeOH at 100 µg/mL as coupled with a Nexera UHPLC was used in this study. A
stock solutions which were used to prepare mixed Phenomenex, Kinetex UHPLC column (100 x 2.1 mm,
standards calibrant series and spiked samples. For sample 1.7 µm) was adopted and a gradient elution program was
preparation, 1 gram of clothing sample was extracted used for separation of the forty-seven compounds. The
using 20 mL of MeOH under sonicate at 50 ºC for 30 detailed conditions are compiled into Table 1.
minutes, followed by 10 minutes of centrifugation at
Table 1: Analytical conditions of forty-seven dye compounds on LCMS-8040
Column : Kinetex C18 100A (100 x 2.1mm, 1.7µm)

Flow Rate : 0.3 mL/min
Mobile Phase : A: H2O with 0.1% formic acid
B: ACN with 0.1% formic acid
Oven Temp. : 40ºC
Injection vol. : 5 µL
Elution Mode : Gradient elution, B%: 5% (0 .01 to 2 min) → 95% (12min)
→ 100% (12.01 to 17.50min) → 5% (17.51 to 20min)
Interface : ESI
MS mode : Positive & negative
Block Temp. : 400ºC
DL Temp. : 250ºC
CID Gas : Ar (230kPa)
Nebulizing Gas Flow : N2, 3 L/min
Drying Gas Flow : N2, 15 L/min
2

Establishment of MRM method for detection and quantitation of
47 dye compounds
The MRM auto-optimisation of the 47 dye compounds compounds. The LOQ ranges from 0.06 – 4.09 ng/mL
was carried out using Shimadzu workstation, while the LOD ranges from 0.02 – 1.35 ng/mL. The
LabSolutions. Two MRM transitions were produced for repeatability of the method was evaluated using
each compound used as quantifier and confirmation ions post-spiked calibrants at two concentrations, 10 ng/mL
(Table 2). A clothing sample free from the 47 dyes was and 50 ng/mL. The %RSD (n=6) at concentration 10
used as a blank matrix used in preparing post-spiked ng/mL ranges from 1.2 % to 16.3 % while the %RSD
calibrants for calibration curves construction. Every (n=6) at concentration 50 ng/mL ranges from 1.1 % to
post-spiked calibrant was injected thrice to obtain the 12.9 %. The linearity, LOD, LOQ and %RSD results are
average area for reliable results. Calibration curves with tabulated in Table 3.
good linearity (r2>0.993) were obtained for all 47 dye
Matrix effect and recovery of the method

The performance of the method in terms of matrix (ME: 42.1 %~49.5 %) but performed better at 50
effect and recovery includes was evaluated for the 47 ng/mL (ME: 71.3 %~ 87.7 %). On the other hand,
dyes studied too. The matrix effect and recoveries were another four dyes i.e., disperse blue 7, basic violet 3,
performed at two concentrations, 10 ng/mL and 50 disperse yellow 23 and disperse orange 149 exhibit
ng/mL. Most of the matrix effect ranges from 63.0 – significantly ion enhance-ment at 50 ng/mL (ME: 141.3
120.9 %. Four dyes, i.e., disperse red 17, disperse blue %~257.3 %) but performed better at 10 ng/mL (ME:
124, disperse blue 35, and disperse yellow 49 exhibit 74.2 %~120.9 %). The recovery was determined from
strong matrix effect at both concentrations (ME: 31.0 pre- and post-spiked samples of the 47 dyes, ranging
%~50.9 %). While, disperse orange and disperse from 81.8 % to 114.1 % at 10 ng/mL and from 84.9
orange 37 exhibit strong matrix effect at 10 ng/mL % to 104.1 % at 50 ng/mL.
Screening and quantitation of the targeted dyes in textile samples

Three light colour clothing samples labelled as 0B, 0G 0Y are free from the 47 dyes. Further studies on
and 0Y bought from the local stores were analysed sample extraction conditions and validation of the
using the method established. In sample 0G, disperse method to various textile samples need to be
red 1 was detected and quantified to be 31 ng/g, performed.
which is within the regulatory limits. Samples 0G and
3
Table 2: Names and information of targeted 47 dyes and MRM transitions on LCMS-8040
Quantifier ion Confirmation ion

No. Name CAS Formula
MRM Q1 (V) CE (V) Q3 (V) MRM Q1 (V) CE (V) Q3 (V)
1 2,4-toluenediamine 95-80-7 C7H10N2 123.10>106.05 -25.0 -20.0 -21.0 123.10>77.05 -25.0 -29.0 -29.0
2 Benzidine 92-87-5 C12H12N2 185.00>167.10 -30.0 -27.0 -30.0 185.00>168.10 -30.0 -20.0 -19.0
3 4,4'-oxydianiline 101-80-4 C12H12N2O 201.10>80.10 -30.0 -37.0 -16.0 201.10>108.10 -30.0 -20.0 -21.0
4 4,4'-diaminodiphenylmethane 101-77-9 C13H14N2 199.10>106.10 -30.0 -21.0 -21.0 199.10>77.10 -30.0 -50.0 -30.0
5 o-Anisidine 90-04-0 C7H9O1N1 124.00>109.05 -30.0 -25.0 -30.0 124.00>65.05 -30.0 -30.0 -30.0
6 o-toluidine 95-53-4 C7H9N1 108.10>91.10 -20.0 -21.0 -18.0 108.10>65.10 -20.0 -28.0 -26.0
7 p-Cresidine 120-71-8 C8H11O1N1 138.10>123.10 -30.0 -19.0 -24.0 138.10>77.05 -30.0 -34.0 -30.0
8 2,4'-Diaminoanisole 615-05-4 C7H10N2O 139.10>124.05 -14.0 -18.0 -25.0 139.10>107.05 -14.0 -17.0 -21.0
9 2,4-Xylidine 95-68-1 C8H11N 122.10>77.05 -30.0 -29.0 -30.0 122.10>107.05 -30.0 -20.0 -21.0
10 3,3'-dimethoxybenzidine 119-90-4 C14H16N2O2 (2HCl) 245.10>230.05 -17.0 -18.0 -25.0 245.10>187.10 -17.0 -33.0 -21.0
11 4- Chloroaniline 106-47-8 C6H6ClN 128.00>93.10 -30.0 -18.0 -18.0 128.00>75.05 -30.0 -34.0 -28.0
12 o-Tolidine 119-93-7 C14H16N2 213.10>180.05 -23.0 -36.0 -20.0 213.10>198.10 -23.0 -21.0 -23.0
13 4,4'-methylene-bis(2-methylaniline) 838-88-0 C15H18N2 227.10>120.10 -30.0 -26.0 -24.0 227.10>77.10 -30.0 -55.0 -29.0
14 2,6-Xylidine 87-62-7 C8H11N 122.10>77.05 -28.0 -27.0 -30.0 122.10>105.05 -28.0 -20.0 -21.0
15 2,4,5-Trimethylaniline 137-17-7 C9H13N1 136.10>121.10 -13.0 -20.0 -25.0 136.10>91.10 -13.0 -23.0 -19.0
16 2-Naphthylamine 91-59-8 C10H9N 144.10>127.05 -30.0 -24.0 -25.0 144.10>77.05 -30.0 -38.0 -29.0
17 4,4'-thiodianiline 139-65-1 C12H12N2S 217.10>124.05 -15.0 -23.0 -23.0 217.10>80.10 -15.0 -47.0 -30.0
18 4-Chloro-o-toluidine 95-69-2 C7H8ClN 142.10>107.10 -28.0 -18.0 -22.0 142.10>106.05 -28.0 -27.0 -21.0
19 Basic Red 9 569-61-9 C19H17N3.HCl 288.20>195.10 -30.0 -32.0 -20.0 288.20>167.10 -30.0 -54.0 -30.0
20 4-Aminobiphenyl 92-67-1 C12H11N 170.10>152.05 -30.0 -30.0 -30.0 170.10>153.10 -30.0 -23.0 -30.0
21 Basic Violet 14 632-99-5 C20H20ClN3.HCl 302.20>209.10 -30.0 -32.0 -23.0 302.20>195.10 -30.0 -34.0 -22.0
22 5-Nitro-o-toluidine 99-55-8 C7H8N2O2 153.00>107.10 -19.0 -17.0 -25.0 153.00>89.10 -19.0 -40.0 -30.0
23 Disperse Blue 7 3179-90-6 C18H18N2O6 359.10>283.05 -25.0 -34.0 -30.0 359.10>314.05 -25.0 -20.0 -23.0
24 Disperse Yellow 9 6373-73-5 C12H10N4O4 275.10>228.00 -20.0 -22.0 -25.0 275.10>258.10 -20.0 -15.0 -30.0
26 Disperse Red 11 2872-48-2 C15H12N2O3 269.10>226.10 -19.0 -30.0 -24.0 269.10>254.10 -19.0 -23.0 -28.0
27 Disperse Blue 102 12222-97-8 C15H19N5O4S 366.10>208.20 -27.0 -18.0 -24.0 366.10>147.15 -27.0 -33.0 -28.0
29 4-aminoazobenzene 60-09-3 C12H11N3 198.10>93.20 -30.0 -25.0 -19.0 198.10>77.10 -30.0 -21.0 -30.0
30 3,3'-dichlorobenzidine 91-94-1 C12H10Cl2N2 253.00>217.10 -18.0 -19.0 -23.0 253.00>182.05 -18.0 -30.0 -22.0
31 4,4'-methylene-bis-2-chloroaniline 101-14-4 C13H12N2Cl2 267.10>231.05 -19.0 -21.0 -25.0 267.10>140.05 -19.0 -25.0 -27.0
33 Disperse Orange 3 730-40-5 C12H10N4O2 243.10>122.05 -30.0 -17.0 -24.0 243.10>75.05 -30.0 -34.0 -28.0
34 Basic Violet 3 548-62-9 C25H30N3Cl 372.30>356.20 -30.0 -41.0 -24.0 372.30>340.15 -30.0 -55.0 -23.0
35 Disperse Yellow 3 2832-40-8 C15H15N2O2 270.10>107.10 -30.0 -24.0 -21.0 270.10>108.15 -30.0 -31.0 -21.0
36 Disperse Orange 11 82-28-0 C15H11NO2 238.10>165.05 -17.0 -34.0 -30.0 238.10>167.05 -17.0 -37.0 -30.0
37 Disperse Brown 1 23355-64-8 C16H15Cl3N4O4 433.00>153.00 -12.0 -41.0 -29.0 433.00>196.95 -12.0 -32.0 -23.0
40 Disperse Yellow 1 119-15-3 C12H9N3O5 274.20>227.20 19.0 27.0 30.0 274.20>243.05 19.0 20.0 24.0
41 Disperse Yellow 49 (Leather) 54824 - 37-2 C22H22N4O2 375.20>238.20 -27.0 -15.0 -28.0 375.20>208.15 -27.0 -39.0 -24.0
44 Disperse Orange 37 13301-61-6 C17H15Cl2N2O5 392.10>133.05 -29.0 -38.0 -25.0 392.10>351.00 -29.0 -22.0 -26.0
45 Disperse Yellow 23 6250-23-3 C18H14N4O 303.10>77.00 -22.0 -35.0 -30.0 303.10>105.05 -22.0 -21.0 -22.0
46 Disperse Orange 1 2581-69-3 C18H14N4O2 319.10>122.05 -30.0 -23.0 -23.0 319.10>169.10 -30.0 -24.0 -19.0
47 Disperse Orange 149 85136-74-9 C25H26N6O3 457.30>121.15 16.0 49.0 19.0 457.30>149.05 16.0 43.0 24.0
4
Table 3: Calibration and performance of MRM method for 47 dye compounds on LCMS-8040
%RSD (n=6)
No. Compound RT (min) Range (ppb) Linearity LOQ LOD
10 ppb 50 ppb
1 2,4-toluenediamine 1.148 1 - 1000 0.9992 0.68 0.22 1.92 3.73
2 Benzidine 1.506 1 - 1000 0.9960 0.52 0.17 4.89 3.88
3 4,4'-oxydianiline 1.268 0.5 - 200 0.9944 0.26 0.09 4.47 4.12
4 4,4'-diaminodiphenylmethane 1.583 0.5 - 1000 0.9992 0.35 0.12 1.28 2.31
5 o-Anisidine 1.733 0.2 - 200 0.9993 0.15 0.05 4.90 2.81
6 o-toluidine 2.049 0.2 - 200 0.9998 0.16 0.05 2.67 1.58
7 p-Cresidine 3.975 0.1 - 200 0.9992 0.06 0.02 3.08 1.36
8 2,4'-Diaminoanisole 3.975 1 - 200 0.9995 0.58 0.19 6.72 2.61
9 2,4-Xylidine 3.933 1 - 200 0.9994 0.73 0.24 1.65 2.33
10 3,3'-dimethoxybenzidine 4.514 0.2 - 200 0.9951 0.20 0.06 2.01 1.12
11 4- Chloroaniline 4.094 1 - 200 0.9997 0.78 0.26 3.75 1.93
12 o-Tolidine 4.395 0.5 - 200 0.9992 0.28 0.10 3.64 1.83
13 4,4'-methylene-bis(2-methylaniline) 4.484 0.5 - 200 0.9993 0.21 0.07 2.62 2.77
14 2,6-Xylidine 4.991 1 - 200 0.9995 0.85 0.28 3.21 3.23
15 2,4,5-Trimethylaniline 5.001 0.5 - 200 0.9995 0.29 0.10 3.22 1.30
16 2-Naphthylamine 5.472 0.2 - 500 0.9994 0.10 0.03 3.54 1.72
17 4,4'-thiodianiline 5.457 0.5 - 1000 0.9993 0.19 0.06 2.91 2.00
18 4-Chloro-o-toluidine 6.282 0.2 - 500 0.9997 0.15 0.05 1.99 2.08
19 Basic Red 9 6.139 0.05 - 100 0.9931 0.02 0.01 1.22 1.93
20 4-Aminobiphenyl 6.612 0.5 - 1000 0.9996 0.45 0.15 1.81 2.45
21 Basic Violet 14 6.479 0.05 - 100 0.9977 0.02 0.01 2.78 1.42
22 5-Nitro-o-toluidine 6.888 5 - 1000 0.9994 1.98 0.65 10.86 8.18
23 Disperse Blue 7 7.459 1 - 200 0.9991 0.71 0.24 3.10 1.33
24 Disperse Yellow 9 7.805 5 - 1000 0.9997 5.00 1.50 15.16 4.16
25 Disperse Blue 3 7.921 0.5 - 200 0.9994 0.28 0.09 5.14 1.91
26 Disperse Red 11 7.936 0.5 - 100 0.9967 0.20 0.06 3.04 2.32
27 Disperse Blue 102 8.294 2 - 200 0.9991 0.97 0.32 16.32 7.61
28 Disperse Red 17 8.489 0.5 - 200 0.9992 0.50 0.15 6.16 3.78
29 4-aminoazobenzene 8.815 2 - 200 0.9995 1.23 0.41 10.43 3.95
30 3,3'-dichlorobenzidine 8.789 2 - 200 0.9994 1.47 0.48 11.04 6.06
31 4,4'-methylene-bis-2-chloroaniline 8.978 2 - 200 0.9993 2.00 0.60 4.77 4.59
32 Disperse Blue 106 8.938 0.1 - 200 0.9994 0.04 0.01 10.34 3.56
33 Disperse Orange 3 9.128 0.1 - 200 0.9994 0.08 0.03 5.81 2.01
34 Basic Violet 3 9.2 0.05 - 200 0.9990 0.02 0.01 4.42 2.45
35 Disperse Yellow 3 9.203 0.5 - 200 0.9991 0.21 0.07 3.97 2.74
36 Disperse Orange 11 9.273 0.5 - 200 0.9992 0.34 0.11 11.59 4.69
37 Disperse Brown 1 9.289 1 - 200 0.9993 1.00 0.33 6.24 8.33
38 Disperse Red 1 9.571 0.2 - 100 0.9942 0.20 0.07 3.16 5.54
39 Disperse Blue 35 9.875 5 - 1000 0.9980 1.98 0.65 10.54 5.99
40 Disperse Yellow 1 8.438 2 - 200 0.9960 2.00 0.63 10.43 8.29
41 Disperse Yellow 49 (Leather) 10.099 0.5 - 200 0.9996 0.26 0.09 3.63 2.40
42 Disperse Blue 124 10.163 1 - 500 0.9981 0.78 0.26 4.70 4.74
43 Disperse Blue 26 10.779 5 - 200 0.9986 4.09 1.35 7.08 12.85
44 Disperse Orange 37 10.864 5 - 200 0.9970 4.05 1.34 4.31 3.35
45 Disperse Yellow 23 11.049 0.2 - 200 0.9969 0.17 0.06 5.19 1.75
46 Disperse Orange 1 11.195 0.2 - 200 0.9979 0.12 0.03 2.57 1.23
47 Disperse Orange 149 12.069 1 - 200 0.9975 0.73 0.24 4.80 7.42
5
Table 4: Matrix effects and recoveries of forty-seven dye compounds spiked in MeOH extract of blank textile matrix (white cloth).
Matrix Effect (%) Recovery (%)

No. Compound
10 ppb 50 ppb 10 ppb 50 ppb
1 2,4-toluenediamine 100.0 99.0 101.5 101.4
2 Benzidine 109.0 101.7 92.4 96.1
3 4,4'-oxydianiline 96.7 100.7 102 94.6
4 4,4'-diaminodiphenylmethane 109.1 105.7 99.3 95.6
5 o-Anisidine 119.0 109.3 107.2 96.8
6 o-toluidine 108.0 102.3 107.9 97.7
7 p-Cresidine 118.6 109.4 107.0 98.9
8 2,4'-Diaminoasole 117.7 106.3 102.6 98.8
9 2,4-Xylidine 116.2 106.4 107.0 97.3
10 3,3'-dimethoxybenzidine 111.1 102.6 102.5 97.8
11 4- Chloroaniline 109.0 108 111.4 98.1
12 o-Tolidine 113.6 108.7 101.6 95.6
13 4,4'-methylene-o- toluidine 97.8 105.5 103.9 98.4
14 2,6-Xylidine 109.5 104 106.6 98.9
15 2,4,5-Trimethylaniline 115.8 109.9 105.5 98.5
16 2-Naphthylamine 108.5 113.3 105.2 100
17 4,4'-thiodianiline 86.7 92.6 95.0 98.9
18 4-Chloro-o-toluidine 106.6 110 108.0 97.7
19 Basic Red 9* 109.6 92.5 81.8 86.3
20 4-Aminobiphenyl 97.2 100.0 102.3 98.0
21 Basic Violet 14* 114.3 93.6 86.5 87.1
22 5-Nitro-o-toluidine 104.0 116.4 110.1 100.3
23 Disperse Blue 7 114.5 141.3 100.5 89.0
24 Disperse Yellow 9 88.9 110.5 106.7 94.2
25 Disperse Blue 3 87.8 101.4 103.5 95.3
26 Disperse Red 11 89.8 86.9 90.4 91.4
27 Disperse Blue 102 76.0 80.9 100.2 88.8
28 Disperse Red 17 31.0 50.9 97.5 95.5
29 4-aminoazobenzene 78.1 90.4 96.5 98.4
30 3,3'-dichlorobenzidine 72.0 90.6 114.1 99.1
31 4,4'-methylene-bis-2-chloroaniline 82.1 89.6 110.4 103.0
32 Disperse Blue 106 82.9 88.7 94.2 95.9
33 Disperse Orange 3 68.2 73.9 100.4 95.3
34 Basic Violet 3 120.9 167.8 104.7 90.2
35 Disperse Yellow 3 72.5 82.5 97.7 94.4
36 Disperse Orange 11 82.9 89 101.3 94.9
37 Disperse Brown 1 63.0 94.1 109.6 98.0
38 Disperse Red 1 71.7 66.1 100.7 104.1
39 Disperse Blue 35 34.7 41.7 104.0 93.1
40 Disperse Yellow 1 113.2 105.5 101.3 100.1
41 Disperse Yellow 49 (Leather) 38.3 45.6 103.5 97.8
42 Disperse Blue 124 31.4 48.5 96.3 93.1
43 Disperse Blue 26 77.1 105.7 99.3 89.2
44 Disperse Orange 37 49.5 71.3 104.6 99.2
45 Disperse Yellow 23 74.2 187 105.7 84.9
46 Disperse Orange 1 42.1 87.7 104.6 85.7
47 Disperse Orange 149 114.1 257.3 107.0 87.6
6
(x100,000)
8.0
Peak 1
7.0
6.0
5.0
4.0
3.0
2.0 Peak 47
1.0
0.0
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 min
Figure 1: Scheduled MRM chromatograms of 47 dyes spiked in textile blank matrix at 20 ng/mL (ppb)
each compound (5 µL injection volume). Peak sequence refers to Table 3.
(x1,000)
8.0
Disperse Red 1
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
2.5 5.0 7.5 10.0 min
Figure 2: MRM chromatogram of sample 0G.
7
Conclusions
A fast and highly sensitive LC/MS/MS method has been time of the method is less than 20 mins. The LOQs of the
developed on LCMS-8040 for screening and quantitation method for these dyes are at 0.1~4.1 ng/mL, which are
of 47 restricted or banned synthetic dyes, including 23 fully complied with the current regulatory requirements.
azodyes, 21 disperse dyes and 3 basic dyes. The total run
References
1. C&A Europe, “C&A - Restricted Substance List (RSL), 2014; American Apparel and footwear Association, “Restricted
substances list (RSL), 2013”
2. J. Garcia-Lavandeira, E Blanco, C. Salgado and R. Cela, Talanta, 82 (2010) 261 – 269.
Disclaimer: Shimadzu LCMS-8040, UFLC XR system and Labsolutions Insight are intended for Research Use Only
(RUO). Not for use in diagnostic procedures.

PO-CON1685E
Rapid development of quantitative

method for determination of plant
growth regulators and streptomycin
in fruits using LC/MS/MS
ASMS 2016 WP 234
Anant Lohar, Shailendra Rane, Ashutosh Shelar,

Purushottam Sutar, Shailesh Damale, Deepti Bhandarkar,
Rashi Kochhar, Ajit Datar, Jitendra Kelkar
and Pratap Rasam
Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh
Chambers, Makwana Road, Marol, Andheri (E),
Mumbai-400059, Maharashtra, India.
Rapid development of quantitative method for
determination of plant growth regulators and streptomycin
Introduction
Plant growth regulators (PGRs) and antibiotics are method development exercises automatically. This results
commonly used by farmers for enhancing growth of in swift development of an optimum method which will
crops and to protect against infections respectively. suit all analytes.
However, extensive usages of these compounds is known Highly sensitive quantitative method was developed
to have adverse effect on human health[1],[2]. Each quickly using Nexera Method Scouting system for
standard has different linearity range as shown in Table 2. determination of eight PGRs and streptomycin (shown in
It is challenging to quickly develop multi-analyte Figure 1) on LCMS-8040 (shown in Figure 2), a triple
quantitation method for determination of varied quadrupole mass spectrometer from Shimadzu
compounds simultaneously[3]. One interesting approach Corporation, Japan.
can be Method Scouting, which allows user to conduct all
Chlormequat chloride Paraquat dichloride Diquat dibromide
Streptomycin 6 Benzyl adenine Mepiquat chloride
Ethephon 4 Chlorphenoxyacetic acid Gibberellic acid

(4 CPA)
Figure 1. Structures of PGRs and antibiotics
Method of analysis
Sample preparation
Preparation of aqueous calibration levels
The standards of PGRs and streptomycin procured from Sigma Aldrich were used for analysis. Mixed standard stock of
PGRs and streptomycin was prepared in the water : methanol (1:1 v/v). Further this stock was serially diluted to prepare
calibration levels ranging from 0.1 ng/mL to 1000 ng/mL in water : methanol (1:1 v/v).
2
Preparation of matrix matched calibration levels

Matrix matched standards were prepared in fresh collected and filtered through 0.2 micron filter. This
watermelon juice purchased from local market. In 50 mL filtered extract was used as a diluent for matrix matched
centrifuge tube, 10 mL of watermelon juice sample was calibration levels.
taken. To this sample, 10 mL of acidified methanol was The matrix matched calibration levels were prepared in a
added and vortexed for few minutes. Solution was then same way as aqueous standards.
centrifuged at 5000 rpm for 5 min. Supernatant was
Figure 2. LCMS-8040 triple quadrupole mass spectrometer
LC/MS/MS analysis
In a typical method scouting procedure, data was tested (shown in Figure 3). Various gradient programs
collected using a number of mobile phase and column were also tried. Automatic batch file was created using
combinations. Analyzers switching manually between Method Scouting software and data were acquired by
these combinations are limited by the number of work LabSolutions software (Shimadzu Corporation, Japan).
hours in a single day, making it impossible to perform Solvent blending is a powerful tool in order to save the
method scouting efficiently. The Nexera Method Scouting labor and time required for preparing mobile phases. For
System is capable of automatically investigating up to 96 example, a wide analysis range can be performed with
combinations of mobile phases and columns, without varied concentrations of the buffer and organic solvent by
such time restrictions, thereby significantly improving just assigning aqueous solutions to pump A and organic
method development productivity. solutions to pump B. When chromatographic elution
For primary method scouting, different mobile phases patterns are investigated with various compositions of the
were checked e.g. 0.1 % formic acid in water, 5 mM solvents, using an automated solvent blending eliminates
ammonium acetate, 10 mM ammonium acetate etc. as wastage of solvent and reduce labors.
aqueous phases and methanol, acetonitrile, 0.1 % formic By using solvent blending function, formic acid
acid in acetonitrile etc. as organic phases. Columns like concentration in 20 mM ammonium acetate was
C18 and HILIC of different makes and dimensions were optimized.
3
Figure 4. Solvent blending
Figure 3. Method scouting software
Table 1. Optimized LC/MS/MS conditions for PGRs and antibiotic analysis
Column : Grace Alltima HP HILIC (50 mm L x 2.1 mm I.D.; 3 µm)

Mobile phase : A: 20 mM ammonium acetate containing 0.05 % formic acid in water
B: 0.1 % formic acid in acetonitrile
Gradient program (B%) : 0.0-1.0 min → 80 (%); 1.0-2.5 min → 80-57 (%);
2.5-4.0 min → 5 (%); 4.0-4.1 min → 5 (%);
4.1-5.0 min → 80 (%); 5.0-12.0 min → 80 (%)
Flow rate : 0.8 mL/min
Oven temperature : 35 °C
Injection volume : 20 µL
MS interface : Electro Spray Ionization (ESI)
Nitrogen gas flow : Nebulizing gas 3 L/min; Drying gas 15 L/min
MS temperature : Desolvation line 300 °C; Heating block 400 °C
4
Results
LC/MS/MS analysis results of PGRs and streptomycin
The preliminary data (Figures 5A to 5D) showed that the software and 20 mM ammonium acetate containing 0.05
combination of 20 mM ammonium acetate containing % formic acid showed maximum response compared to
0.15 % formic acid and 0.1% formic acid in acetonitrile all other combinations especially for streptomycin (shown
with HILIC column gives good peak shapes and in Figure 6).
separation. This mobile phase composition was further The LC/MS/MS method was developed using method
optimized with solvent blending software (as shown in scouting for simultaneous quantitation of PGRs and
Figure 4). streptomycin. MRM transitions used for these compounds
It was observed that the formic acid concentration in are given in Table 2. Linearity studies were carried out
aqueous phase regulates the peak shapes. Therefore, using external standard calibration method and results of
automated batch file was created for optimization of linearity studies for both aqueous and matrix matched
formic acid concentration by using solvent blending standards are tabulated in Table 2.
(x10,000,000) (x10,000)
4.5 1:Streptomycin 2 TIC(+)
1.25 1:Streptomycin 2 TIC(+)(100.00)
2:Azhadirectin TIC(+)(100.00) 2:Azhadirectin TIC(+)
3:Paraquat TIC(+) 3:Paraquat TIC(+)
4:Mepiquate TIC(+) 4.0 4:Mepiquate TIC(+)
5:Diquat TIC(+)(20.00) 5:Diquat TIC(+)(20.00)
6:CCC TIC(+)(0.50) 6:CCC TIC(+)
1.00 7:6-BA TIC(+)
8:2,4 D TIC(-)(100.00)
3.5 7:6-BA TIC(+)
8:2,4 D TIC(-)(20.00)
9:4-CPA TIC(-)(100.00) 9:4-CPA TIC(-)
11:GA TIC(-)(50.00) 10:1-NA TIC(-)
12:Ethephon TIC(-)(50.00) 3.0 11:GA TIC(-)
12:Ethephon TIC(-)(20.00)
0.75
2.5
2.0
0.50
1.5
1.0
0.25
0.5
0.00 0.0
0.0 2.5 5.0 7.5 10.0 min 0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 5A. A=20 mM ammonium acetate 0.1% formic Figure 5B. A= 0.1 % formic acid in water
acid B= 0.15% formic acid in acetonitrile and B= methanol
(x1,000,000) (x100,000)
7.0 1:Streptomycin 2 TIC(+)
8.0 1:Streptomycin 2 TIC(+)(100.00)
2:Azhadirectin TIC(+)(50.00)
2:Azhadirectin TIC(+)
3:Paraquat TIC(+)(100.00) 3:Paraquat TIC(+)(50.00)
4:Mepiquate TIC(+) 4:Mepiquate TIC(+)
6.0 5:Diquat TIC(+)(50.00) 7.0 5:Diquat TIC(+)
6:CCC TIC(+) 6:CCC TIC(+)
7:6-BA TIC(+) 7:6-BA TIC(+)(0.50)
8:2,4 D TIC(-) 8:2,4 D TIC(-)
5.0 9:4-CPA TIC(-) 6.0 9:4-CPA TIC(-)
10:1-NA TIC(-) 10:1-NA TIC(-)(50.00)
11:GA TIC(-) 11:GA TIC(-)
12:Ethephon TIC(-) 5.0 12:Ethephon TIC(-)(20.00)
4.0
4.0
3.0
3.0
2.0
2.0
1.0 1.0
0.0 0.0
0.0 2.5 5.0 7.5 10.0 12.5 min 0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 5C. A= 10 mM ammonium formate and Figure 5D. A=10 mM ammonium acetate and
B= 0.1 % formic acid in methanol B= 0.1 % formic acid in methanol
5
(x10,000)
4.5 1:Streptomycin 2 TIC(+) 1_PGR_HILIC-G_0.05%FA in 20mMAA_0.1%FA in ACN_5_95_005.lcd
1:Streptomycin 2 TIC(+) 3_PGR_HILIC-G_0.15%FA in 20mMAA_0.1%FA in ACN_5_95_009.lcd
1:Streptomycin 2 TIC(+) 2_PGR_HILIC-G_0.2%FA in 20mMAA_0.1%FA in ACN_5_95_007.lcd
4.0 1:Streptomycin 2 TIC(+) 1_PGR_HILIC-G_0.1%FA in 20mMAA_0.1%FA in ACN_5_95_005.lcd
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min
Figure 6. Optimization of formic acid concentration by solvent blending
The matrix matched calibration levels were prepared and injected in triplicate. The calibration curve of PGRs and
streptomycin are shown in Figure 7 to Figure 15 and the correlation coefficient of > 0.99 was observed for all the
compounds.
Area (x100,000) Area (x10,000,000) Area (x1,000,000)

14 2.0
1.5 11
3.0
R2= 0.9947 R2= 0.9941 R2= 0.9982
13 1.5
1.0
2.0
12 4
1.0
10
0.5 9
1.0 11 3
0.5 8
10
9 2 7
8 1
56
0.0 0.0 0.0
0 250 500 750 Conc. 0.0 1.0 2.0 3.0 Conc. 0 100 200 Conc.
Figure 7. Streptomycin Figure 8. Paraquat Figure 9. Mepiquat
6
Area (x1,000,000) Area (x10,000,000) Area (x1,000,000)

5 12 13
1.0
R = 0.9927
2 1.0 R = 0.9954
2
R = 0.9942
2
4
1.0 12
11
0.5 3 0.5
0.5
11
2 910
1 8 910
7 8
0.0 0.0
56
0.0
567
0.0 25.0 50.0 Conc. 0 250 Conc. 0 250 500 Conc.
Figure 10. Diquat Figure 11. Chlormequat Figure 12. 6 Benzyl adenine
Area (x10,000) Area (x10,000) Area (x1,000)

14 14 14
7.5
R = 0.9952
2
R = 0.9911
2
R = 0.9967
2
13
1.0 2.0
13 13
5.0
12
12 12
0.5 1.0
2.5
11 11
11
10 10
9 10 9
0.0 78 0.0 0.0
0 250 500 750 Conc. 0 250 500 750 Conc. 0 250 500 750 Conc.
Figure 13. Ethephon Figure 14. Gibberellic acid Figure 15. 4 CPA
Table 2. Result table of aqueous and matrix matched calibration standards
Correlation coefficient (r2)

Linearity range
Sr.No. Name of molecule MRM transition Polarity Aqueous Matrix matched
in ppb
standard standards
1 Streptomycin 582.00>176.05 Positive 50-1000 0.9947 0.9983

2 Paraquat 186.00>171.05 Positive 5-75 0.9941 0.9941
3 Diquat 183.90>128.05 Positive 5-100 0.9927 0.9927
4 6 Benzyl adenine 225.80>91.00 Positive 5-750 0.9972 0.9942
5 Mepiquat 114.00>98.15 Positive 5-250 0.9982 0.9954
6 Chlormequat 122.10>58.15 Positive 5-500 0.9958 0.9954
7 Gibberellic acid 345.05>239.10 Negative 100-1000 0.9992 0.9911
8 Ethephon 143.00>106.90 Negative 50-1000 0.9948 0.9952
9 4 CPA 184.70>127.05 Negative 100-1000 0.9977 0.9967
7
Conclusion
• The Nexera Method Scouting System enhances method development efficiency.
• An automated solvent blending system can provide the same level of accuracy as manual premixing.
• With the help of method scouting and solvent blending techniques, it was very easy to develop a quantitative
multi-residue analytical method for different class of compounds.
References
[1] Sorensen MT, Danielsen V. International journal of Androl, (2006), 129-33.
[2] Ana Coste, Laurian Vlase. Plant Cell Tiss Organ Cult 106, (2011), 279-288.
[3] X. Esperza, E. Moyano,. Journal of Chromatography A, Volume 1216, (2009), 4402-4406.
Disclaimer : The product and applications in this poster are intended for Research Use Only (RUO). Not for use in
diagnostic procedures.

PO-CON1687E
Trace level quantitative

determination of phthalates from
high risk dosage pharmaceutical
formulations using LC/MS/MS
ASMS 2016 WP 715
Purushottam Sutar, Anant Lohar, Ashutosh Shelar,

Deepti Bhandarkar, Rashi Kochhar, Shailendra Rane,
Shailesh Damale, Ajit Datar, Pratap Rasam
and Jitendra Kelkar
Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh
Trace level quantitative determination of phthalates
from high risk dosage pharmaceutical formulations
using LC/MS/MS
Introduction
Phthalates are plasticizers which find their use in variety (mentioned in Table 1) which act by directly affecting the
of applications like packaging material in pharmaceutical site of action in the body.
preparations, plastic toys, personal-care products etc. Hence a highly sensitive LC/MS/MS method has been
Since they are used as additives, they can leach out from developed for trace level quantitative determination of
the polymeric matrix. Studies have demonstrated that phthalates (shown in Figure 1) from high risk dosage
phthalates exposure can cause endocrine disruption, pharmaceutical formulations using LCMS-8040, a triple
cancer, autism etc. quadrupole mass spectrometer from Shimadzu
These hazards make it mandatory to quantify phthalates Corporation, Japan.
especially in high risk dosage pharmaceutical formulations
Table 1. Examples of packaging concerns for common classes of drug products[1]
Degree of concern Likelihood of packaging component-dosage formulation interaction

associated with the route
of administration High Medium Low
Inhalations Aerosols and Solutions; Sterile powders and powders for

Highest
Injections and Injectables Suspensions Injection; Inhalation Powders
Ophthalmic Solutions and Suspensions;

High Transdermal Ointments and Patches;
Nasal Aerosols and Sprays
Topical Solutions and Suspensions;

Oral Tablets and Oral/
Low Topical and Lingual Aerosols; Oral Topical Powders; Oral powders
(Hard and soft Gelatin) Capsules
solutions and Suspensions
Bis methyl glycol phthalate Dimethyl phthalate Diethyl phthalate
Dipropyl phthalate Diallyl phthalate Dicyclohexyl phthalate
Benzyl butyl phthalate Dipentyl phthalate Diisodecyl phthalate
Figure 1. Structures of phthalates
2
using LC/MS/MS
Method of analysis
Sample preparation
Preparation of calibration levels
Phthalate mix standards procured from Sigma Aldrich were prepared in acetonitrile with concentration levels mentioned
in Table 2.
Table 2. Calibration levels for individual phthalate standards
Bis methyl
Dimethyl Diethyl Dipropyl Diallyl Dicyclohexyl Benzylbutyl Dipentyl Diisodecyl
glycol
phthalate phthalate phthalate phthalate phthalate phthalate phthalate phthalate
phthalate
(ppb) (ppb) (ppb) (ppb) (ppb) (ppb) (ppb) (ppb)
(ppb)
5 to 1000 2.5 to 500 2.5 to 500 1 to 100 2.5 to 100 1 to 250 1 to 100 10 to 250 5 to 500
Preparation of samples
Market samples of ear drops, eyes drops and nasal solution (two each) were diluted in 50 parts of acetonitrile and
injected on LCMS-8040.
Preparation of recovery levels

Three separate sample vials of one of the representative and 100 µL of each of the three solutions were diluted to
market samples were spiked with an aliquot of 10 µL ,50 1000 µL with acetonitrile in three separate vials resulting
µL ,100 µL from 10 ppm mixed phthalates standard stock in recovery levels of 10 ppb, 50 ppb and 100 ppb
solution. The resultant solutions were vortexed for 5 min respectively.
LC/MS/MS analysis
Phthalates mixture were analyzed using Ultra High Performance Liquid Chromatography (UHPLC) Nexera coupled with
LCMS-8040 (as shown in Figure 2) triple quadrupole system from Shimadzu Corporation, Japan.
Figure 2. LCMS-8040 triple quadrupole mass spectrometer by Shimadzu
3
using LC/MS/MS
LCMS-8040 triple quadrupole mass spectrometer, sets a far below the theoretical Analytical Evaluation Threshold
new benchmark in triple quadrupole technology with an (AET) for the market samples used for this analysis[2]. All
unsurpassed sensitivity (UFsensitivity), ultra fast scanning the calibration levels and market samples were analyzed
speed of 15,000 u/sec (UFscanning) and polarity using LC/MS/MS conditions mentioned in Table 3. Also,
switching speed of 15 msec (UFswitching) which has recovery study was carried out.
helped in achieving the Limit of Quantitation (LOQ) levels
Table 3. LC/MS/MS conditions for phthalates mixture
Column : Shim-pack XR-ODS (50 mm L x 3 mm I.D.; 2.2 µm)

Guard column : Phenomenex Security Guard ULTRA cartridge
Mobile phase : A: water
Gradient program (B %) : 0.0-2.0 min → 45 (%); 2.0-4.0 min → 45-95 (%); 4.0-8.0 min → 95 (%);
8.0-8.1 min → 95-45 (%); 8.1-12.0 min → 45 (%)
Results
LC/MS/MS analysis results of phthalate mixture
LC/MS/MS method was developed for trace level calibration curves for individual phthalates are shown in
quantitative determination of phthalates from high risk Figures 3 and 4 respectively.
dosage pharmaceutical formulations. Linearity study was The LOQ levels achieved using this method was far below
carried out using external standard calibration method for the estimated AET and the data for market samples
nine phthalates with % accuracy and correlation shows the presence of only diethyl phthalate in most of
coefficient value within acceptance criteria. The MRM the samples whereas rest of the phthalates are not
transitions and results of linearity study of these detected (ND) or below the quantification limit (BLOQ) as
compounds are given in Table 4. The overlay mentioned in Table 5. Results of recovery studies are
chromatograms for blank and LOQ level along with the tabulated in Table 6.
4
using LC/MS/MS
Table 4. Details of MRM transitions and linearity results
% RSD for area

Retention MRM LOQ Average % Linearity
Compound counts at LOQ
time (min) transitions (ppb) accuracy (n=5) (r2)
(n=5)
Bis methyl glycol phthalate 1.21 283.15>207.05 5.0 4.81 100.56 0.9958
Dimethyl phthalate 1.20 195.05>163.00 2.5 7.52 100.31 0.9977
Diethyl phthalate 2.36 223.15>149.00 2.5 5.18 100.65 0.9954
Dipropyl phthalate 4.27 251.20>149.00 1.0 8.00 96.45 0.9962
Diallyl phthalate 3.62 247.15>189.00 2.5 4.86 100.68 0.9945
Dicyclohexyl phthalate 5.38 331.20>149.00 1.0 8.35 100.29 0.9932
Benzylbutyl phthalate 4.81 313.20>91.10 1.0 7.98 100.85 0.9939
Dipentyl phthalate 5.30 307.20>149.00 10.0 2.27 100.37 0.9953
Diisodecyl phthalate 8.61 447.15>85.10 5.0 2.56 100.92 0.9920
Table 5. Results of market samples
Eye drops Ear drops Nasal solution

Compound
Sample 1 (ppb) Sample 2 (ppb) Sample 1 (ppb) Sample 2 (ppb) Sample 1 (ppb) Sample 2 (ppb)
Bis methyl glycol phthalate ND ND ND ND ND ND
Dimethyl phthalate ND ND ND ND ND ND
Diethyl phthalate BLOQ BLOQ 58.23 101.99 40.46 54.98
Dipropyl phthalate ND ND 0.85 0.89 ND ND
Diallyl phthalate ND ND ND ND ND ND
Dicyclohexyl phthalate ND ND ND ND ND ND
Benzylbutyl phthalate ND ND ND ND ND ND
Dipentyl phthalate BLOQ 10.61 BLOQ BLOQ BLOQ BLOQ
Diisodecyl phthalate ND ND ND ND ND ND
Table 6. Results of recovery study
Concentration of phthalate standard (ppb)

Low level Middle level High level
Blank
Compound
sample Spiked Observed Spiked Observed Spiked Observed
% % %
amount amount amount amount amount amount
Recovery Recovery Recovery
(ppb) (ppb) (ppb) (ppb) (ppb) (ppb)
Bis methyl glycol phthalate ND 10 8.20 81.90 50 50.59 101.20 100 90.62 90.60
Dimethyl phthalate ND 10 10.94 109.35 50 48.84 97.65 100 99.91 99.90
Diethyl phthalate BLOQ 10 9.85 98.50 50 53.46 106.90 100 111.05 111.05
Dipropyl phthalate ND 10 10.31 103.10 50 45.51 91.05 100 88.17 88.20
Diallyl phthalate ND 10 9.91 99.05 50 47.06 94.15 100 92.73 92.75
Dicyclohexyl phthalate ND 10 10.54 105.40 50 54.67 109.30 100 98.99 99.00
Benzylbutyl phthalate ND 10 9.43 94.30 50 45.51 91.05 100 85.27 85.30
Dipentyl phthalate BLOQ 10 10.84 108.45 50 49.09 98.15 100 92.53 92.55
Diisodecyl phthalate ND 10 10.90 109.05 50 48.76 97.50 100 94.02 94.00
5
using LC/MS/MS
(x100) (x1,000) (x10,000)

6.0
6.0 5.5 1.50
Benzyl butyl phthalate 5.0 Dicyclohexyl phthalate Dipentyl phthalate
5.0 4.5 1.25
4.0
4.0 3.5 1.00
3.0
3.0 2.5 0.75
2.0
2.0 1.5 0.50
1.0
1.0 0.5 0.25
0.0
0.0 0.00
-0.5
0.0 2.5 5.0 7.5 10.0 min 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min 2.5 5.0 7.5 10.0 min
(x1,000) (x100) (x1,000)
2.00 2.50
1.75
Diallyl phthalate 2.25 Diisodecyl 3.0
Diethyl phthalate
1.50
2.00 phthalate 2.5
1.75
1.25 2.0
1.50
1.00 1.25
1.5
0.75 1.00
0.75 1.0
0.50
0.50
0.25 0.5
0.25
0.00 0.00 0.0
2.5 5.0 7.5 10.0 min 2.5 5.0 7.5 10.0 min 2.5 5.0 7.5 10.0 min
(x1,000) (x1,000) (x100)
6.0
1.25
5.5
Dimethyl phthalate 1.50 Dipropyl phthalate 5.0
Bis methyl glycol phthalate
1.00
1.25 4.5
4.0
0.75 1.00 3.5
3.0
0.75 2.5
0.50
2.0
0.50
1.5
0.25
0.25 1.0
0.5
0.00 0.00 0.0
2.5 5.0 7.5 10.0 min 2.5 5.0 7.5 10.0 min 2.5 5.0 7.5 10.0 min
Figure 3. Overlay chromatograms of blank and LOQ
Area (x100,000) Area (x1,000,000) Area (x1,000,000)

9 10 7
4.0 2.0
4.0
3.0 3.0 1.5
8
6
2.0 7 2.0 9 1.0
6 Benzyl butyl phthalate Dicyclohexyl phthalate Dipentyl phthalate

1.0 5 0.5 5
r2 = 0.9939 r2 = 0.9932 r2 = 0.9953
1.0 8 4
4 7 3
6 2
3 5 1
2 4
0.0 1 0.0 1
23 0.0
0.0 25.0 50.0 75.0 Conc. 0 50 100 150 200 Conc. 0 50 100 150 200 Conc.
Area (x100,000) Area (x100,000) Area (x1,000,000)
8 8 10
6.0 4.0
7.5
5.0
3.0
4.0
5.0
7 7 9
3.0 2.0
6
2.5 5 Diallyl phthalate 2.0 Diisodecyl phthalate Diethyl phthalate

r2 = 0.9945 r2 = 0.9920 r2 = 0.9954
4 1.0 8
6
3 1.0
7
2 45
4 56
0.0
1 12 3 123
0.0 0.0
0.0 25.0 50.0 75.0 Conc. 0 100 200 300 400 Conc. 0 100 200 300 400 Conc.
Area (x1,000,000) Area (x1,000,000) Area (x1,000,000)
10 9 10
1.25
1.25
2.0
1.00
1.00
1.5
0.75
9 0.75 9
8
1.0 7
Dimethyl phthalate 0.50
Dipropyl phthalate 0.50 Bis methyl glycol
6
phthalate
0.5 8 r2 = 0.9977 5 r2 = 0.9962 8
r2 = 0.9958
0.25 0.25
7 4 7
56 3
34 12 56
0.0 12 0.00 1234
0.00
0 100 200 300 400 Conc. 0.0 25.0 50.0 75.0 Conc. 0 250 500 750 Conc.
Figure 4. Calibration curves of standards
6
using LC/MS/MS
Conclusion
• The theoretical AET for all the sample considering Safety Concern Threshold (SCT) as 0.15 µg/day is found to be
around 2 µg/mL i.e. 2000 ppb, whereas the estimated AET using this sample preparation method is about 1 µg/mL
i.e.1000 ppb.
• LOQ in the range of 1 ppb to 10 ppb was achieved for different phthalates which is well below the Estimated AET level.
• Analysis of market samples showed that these samples are free from most of the mentioned phthalates except
diethyl phthalate, which is also present at levels far below the estimated AET.
References
[1] FDA Guidance for Industry - Container Closure Systems for Packaging Human Drugs and Biologics (1999).
[2] Safety Thresholds and Best Practices for Extractables and Leachables in Orally Inhaled and Nasal Drug Products,
(2006).
Disclaimer : The product and applications in this poster are intended for Research Use Only (RUO). Not for use in

PO-CON1659E
LCMS-MS Method for Evaluation

of PPCPs in Environmental Water
ASMS 2016 MP169
Katie Pryor1, Jerry Byrne1, Rachel Lieberman1,

Christopher Gilles1
1
Shimadzu Scientific Instruments, Columbia, MD;
LCMS-MS Method for Evaluation of PPCPs
in Environmental Water
Novel Aspects
A rapid five minute method to analyze and quantify multiple pharmaceutical and personal care products in
environmental water by LC-MS/MS.
Introduction
Pharmaceutical and personal care products (PPCPs) environment is still unknown causing the need for more
encompass a family of compounds used by individuals for research on the topic. These contaminates are introduced
health and cosmetic purposes, as well as compounds into local waterways via sewage plants and natural
used by the agricultural industry to maintain health of disposal (ie; animal excrement and landfill waste). This
livestock. The number of individuals who are using these poster demonstrates a method for evaluating PPCPs at
products is increasing which has caused an increase in parts per billion levels in local waterways using liquid
environmental detection. The effect on humans and the chromatography tandem mass spectrometry (LC-MS/MS).
Methods
Sixteen compounds and one internal standard were in positive mode to ionize all of the compounds. Each
analyzed from various drug classes including analyte was optimized and separated using a binary
broad-spectrum antibiotics, antifungals, stimulants, gradient with reversed phase chromatography on a
opiates, alkaloids, antihistamines, biguanides, and Restek Raptor C18 column, in a single 6.5 minute
progestins. Heated Electrospray Ionization (hESI) was used method.
Chromatography Parameters LCMS-8050 Parameters
Column : Raptor C18 Nebulizing Gas : 10 L/min

Column Temp : 40 °C Interface Temp : 350 °C
Autosampler Temp : 15 °C DL Temp : 100 °C
Injection Volume : 1 µl Heat Block Temp : 150 °C
Flow Rate : 0.5 mL/min Drying Gas Flow : 4 L/min
2
Results- Neat Sample

Name Ret. Time Target Transition Reference Ions
Metformin 0.415 130.00>60.00 71.00
Cotinine 1.681 177.30>80.15 98.20
Codiene 1.865 300.20>165.05 165.05
Minocycline 2.521 458.30>441.05 352.05
1,7- Dimethylxathine 2.528 181.20>124.25 55.10
Tetracycline 2.768 445.10>410.10 427.00
Oxytetracycline 2.829 461.20>426.05 443.00
Ampicillin 2.943 350.15>106.10 192.25
Chlortetracycline 3.549 479.20>444.00 462.00
Doxycycline 3.967 445.20>428.00 410.05
Diphenhydramine 4.012 256.30>167.20 165.25
Diphenhydramine D3 4.02 260.60>168.25 166.10
Anhydrotetracycline 4.205 427.20>410.00 269.05
Clarithormycin 4.342 748.50>158.25 590.25
Miconazole 4.539 417.00>159.15 159.15
Virginiamycin 4.541 548.30>287.00 284.10
Norgestimate 5.007 370.30>310.15 310.15
Diphenhydramine
20000000
17500000
Clarithormycin
Virginiamycin
15000000
Diphenhydramine D3
12500000
10000000
Metformin
7500000
1,7- Dimethylxathine
Anhydrotetracycline
Miconazole
Doxycycline
Chlortetracycline
Oxytetracycline
Tetracycline
5000000
Norgestimate
Cotinine
Minocycline
Codiene
Ampicillin
2500000
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 min
3
Results- River Water

Samples were obtained from three local water ways and any of the target analytes except for Sample 1.
prepared for analysis. Two samples were collected at each Miconazole was detected in Sample 1 at a concentration
location. All of the samples were filtered with a 0.2 µm of 0.32 ppb which is below the lower limit of quantitation
syringe filter and injected. None of the samples contained for this compound.
275000
250000
225000
200000
175000 17:417.00>159.15(+)
25000 17:417.00>159.15(+)
150000 17:417.00>123.20(+)
125000 22500
100000
75000 20000
50000
17500
25000
0
15000
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 min
12500
River Water Source 1 10000
7500
5000
2500
4.2 4.3 4.4 4.5 4.6
Source 1: Miconazole
0.32 ppb
325000
300000
275000
250000
225000
200000
175000
150000 140000 14:427.20>410.00(+)
125000 14:427.20>269.05(+)
100000 130000 14:427.20>154.25(+)
75000
120000
50000
25000 110000
0
100000
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 min
90000
River Water Source 2 80000
70000
300000
275000 60000
250000 50000
225000
200000 40000
175000
30000
150000
125000 20000
100000
75000 10000
50000
0
25000
0 -10000
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 min 3.75 4.00 4.25 4.50
River Water Source 3 Source 2: Anhydrotetracycline

with Ion Ratio failure
4
Results- Calibration Curve of Neat Samples

The calibration curve was made up in mobile phase starting Virginiamycin, Metformin, and Minocyline exhibited source
conditions and spiked using a stock solution of the saturation and required a quadratic line of fit.
analytes. Virginiamycin’s linear range is 0.1 - 50 ppb, while
All of the analytes had a %RSD of less than 20% at the Metformin’s linear range is 0.1 – 100 ppb, and Minocyline
LOQ and all of the limits of quantitation were below 5 ppb. has a linear range of 5 - 100 ppb.
Area Ratio
Area Ratio 6000 7:445.10>410.10(+) 11000 5:181.20>124.25(+)
8.0 5500 7:445.10>427.00(+) 5:181.20>42.05(+)
6.0
7.0 Tetracycline 5000 1,7-Dimethylxathine 10000

9000
4500 5.0
6.0 8000
4000
7000
3500 4.0
5.0
3000 6000
4.0 2500 3.0 5000
3.0 2000 4000

1500 2.0 3000
2.0
1000 2000
500 1.0
1.0 1000
0
Conc. Ratio Conc. Ratio 0
0.0 0.0
0.0 25.0 75.0 125.0 175.0 225.0 2.25 2.50 2.75 3.00 0.0 25.0 75.0 125.0 175.0 225.0 2.00 2.25 2.50 2.75
Tetracyline 1,7-Dimethylxathine
LOQ: 0.5 ppb LOQ: 1 ppb
Area Ratio (x10) Area Ratio (x10)
11000 3:177.30>80.15(+)
6500 15:748.50>158.25(+) 2.25 3:177.30>98.20(+)
15:748.50>590.25(+) 10000 3:177.30>53.20(+)
Clarithomycin Cotinine
3.5 6000 15:748.50>83.10(+)
2.00
5500 9000
3.0 5000 1.75 8000
4500
1.50 7000
2.5 4000
6000
3500 1.25
2.0 3000 5000
2500 1.00
4000
1.5
2000 0.75 3000
1500
1.0 2000
1000 0.50
500 1000
0.5 0.25
0 0
Conc. Ratio Conc. Ratio
0.0 0.00
0.0 25.0 75.0 125.0 175.0 225.0 4.00 4.25 4.50 4.75 0.0 25.0 75.0 125.0 175.0 225.0 1.25 1.50 1.75 2.00
Clarithomycin Cotinine
LOQ: 0.1 ppb LOQ: 0.75 ppb
Name LOQ (ppb) Linear Range (ppb) LOQ %RSD

Metformin 0.1 0.1-100 2.62
Cotinine 0.75 0.75-250 10.79
Codiene 0.25 0.25-250 13.14
Minocycline 5 5-100 11.64
1,7- Dimethylxathine 1 1-250 6.62
Tetracycline 0.5 0.5-250 9.91
Oxytetracycline 2.5 2.5-250 11.86
Ampicillin 1 1-250 13.19
Chlortetracycline 5 5-250 1
Doxycycline 1 1-250 9.54
Diphenhydramine 0.25 0.25-250 3.14
Anhydrotetracycline 5 5-250 4.64
Clarithormycin 0.1 0.1-250 14.3
Miconazole 0.5 0.5-250 17.75
Virginiamycin 0.1 0.1-50 16.38
Norgestimate 5 5-250 5.98
5
Conclusion
A robust and rapid 6.5 minute method for evaluating samples. The samples may have been too diluted from
pharmaceuticals and personal care products was the excessive amount of rain in the area before sampling
developed using ultra-fast liquid chromatography mass occurred.
spectrometry. Most analytes had an lower limit of The method can be used to further test other river water
quantitation at a level between 0.1 ppb and 5 ppb. samples when there is a more stable water table to test
Miconazole was the only drug detected in the river water from.
For research purposes only. Not for use in diagnostic procedures.

PO-CON1652E
Development of Automated Screening

and Quantitation Approach on
Novel On-Line SFE-SFC-MS/MS Platform
– (I) For 23 Restricted Perflurocompounds
in Textiles
ASMS 2016 MP 283
Jie Xing, Jun Xiang Lee, Peiting Zeng and Zhaoqi Zhan
Singapore
Development of Automated Screening and Quantitation
Approach on Novel On-Line SFE-SFC-MS/MS Platform –
(I) For 23 Restricted Perflurocompounds in Textiles
Introduction
Supercritical Fluid Chromatography (SFC) with carbon analysis of 510 residual pesticides in agricultural products
dioxide as eluent has attracted attention recently because [1]. One of the main advantages of the Nexera UC
of its advantages in low running cost, non-toxicity and platform allows to set up on-line method to analyse
wider coverage of analytes in terms of polarity. The directly different types and forms of un-pretreated
combination of SFC with SFE (E=Extraction) has extended samples. We describe the development of an approach
the applications to fully-automated sample pre-treatment on the Nexera UC platform, aiming at screening and
and analysis as demonstrated by the Nexera UC system quantitation of 23 perflurocompounds (PFCs) listed under
introduced by Shimadzu recently. The novel the Restricted Substance List (RSL) in textile, leather and
SFE-SFC-MS/MS system has been used successfully for consumer goods industries [2].
Experimental
A total of 23 PFCs and 2 internal standards (IS), M-PFOS the Nexera UC system employed in this study is shown in
and M-PFOA (refer to Table 2) were obtained from Sigma Figure 1. The system can be operated for on-line
Aldrich, Wellington Laboratories and Apollo Scientific [3]. SFE-SFC-MS/MS experiments or only for SFC-MS/MS
Textile samples were cut into smaller pieces and weighed. analysis. The mobile phase is supercritical fluid CO2, with
The sample (60 mg) was loaded into a 0.2 mL stainless addition of organic modifier like MeOH. Thus, both
steel SFE vessel tightly before proceeding to isocratic and gradient elution modes can be chosen. The
online-SFE-SFC-MSMS analysis. A schematic diagram of details of the analytical conditions are compiled into Table 1.
Make up solvent pump
SFE Unit C: Make up

solvent
sfCO2 pump Splitter

A: sfCO2 Column
Cylinder MS/MS
Auto-
sampler Column oven BPR-A
BPR-B
SFC Unit connected to MS/MS
Extraction vessel
B: Modifier Modifier
solvent pump drain
Figure 1: Schematic diagram of Nexera UC system for SFE-SFC-MS/MS analysis of un-pretreated samples
2
Table 1: Analytical conditions of 23 PFCs and 2 internal standard on Nexera UC with LCMS-8050
Column : Shim-pack UC-X Sil (250 mmL. x 2.1mm I.D., 3µm)

0.2 mL/min (make up pump of MS)
Mobile Phase : A : Supercritical Fluid Carbon dioxide (sfCO2)
B : Methanol with 5 mM ammonium formate
C : Methanol with 5 mM ammonium formate
Temp. : Column Oven: 40ºC; SFE unit: 40ºC or RT
Injection vol. : SFC: 5 µL; SFE-SFC: 200 µL or 3% split ratio
Elution Mode : Gradient elution, LC program 7 minute
0%B (0.00mins to 0.30mins)
→ 50%B (4.00mins to 4.50mins)
→ 0%B (4.70mins to 6.00mins)
Interface : ESI
MS mode : Negative
DL Temp. : 250ºC
Interface Temp. : 300ºC
Nebulizing Gas Flow : N2, 3 L/min
Heating Gas Flow : 0 Air, 10 L/min

Establishment of SFC-MS/MS method
A SFC-MS/MS method was established for the targeted standards with linearity (r2>0.97) for all 23 PFCs (Table 2).
23 PFCs with 2 IS first. A Shim-pack UC-X Sil column was Instead of using the concentration, absolute amount (pg)
used and a gradient elution program was adopted. Two was used for the calibration curves. The LOQ ranges from
MRM transitions (if available) were used for each PFC, 0.03 ~1 ng/mL while the LOD ranges from 0.01 ~ 0.32
one as quantifier ion and the other for confirmation. The ng/mL. The repeatability of the method was evaluated at
SFC-MS/MS chromatograms and calibration curves (only two concentrations, 5 and 25 pg. The %RSD results of
PFOA and PFOS are displayed) are shown in Figure 2. the post-spiked samples are tabulated in Table 2.
Calibration curves were built based on the two internal
3
(x10,000) (x10,000) Area (x100,000)

8.0 2.0
7.0
(c) PFOA
MPFOS
(a) 23 PFCs and 2 IS (b)
MPFOA
7.0
6.0
1.0
6.0
5.0
5.0
4.0 0.0
4.0 0 50 100 pg.
3.0
Area (x100,000)
PFOS
3.0
2.0
2.0 (c) PFOS
PFOA
2.0
1.0
1.0 1.0
0.0
0.0
-1.0 0.0
0.0 1.0 2.0 3.0 4.0 5.0 min 3.25 min 0 50 100 pg.
Figure 2: (a) MRM chromatograms of 23 PFC mixed standards with 2 IS, 5 pg for each compound;
(b) Zoomed MRM peaks of PFOA and PFOS with their IS;
(c) Calibration curves of PFOA (5~125 pg) and PFOS (2.5~125 pg) based on quantifier ions as shown in table 3.
Table 2: Calibration curves and performance values of the MRM method for quantitative determination of 23 PFCs on SFC-MS/MS.
Absolute amounts (in pg) of analytes are used for convenience (injection volume: 5 µL)
RT Range LOD LOQ RSD (%), n=6

No. Name MRM transition R2
(min) (pg) (pg) (pg) (5 pg) (25 pg)
1 N-EtFOSA-M 1.613 526.10>169.00 5 ~ 125 0.9732 < 2.5 5 34.4 32.3
2 N-MeFOSA-M 1.692 512.00>169.15 2.5 ~ 125 0.9966 < 2.5 5 47.1 18.1
3 H4PFUnA 1.695 491.10>367.00 2.5 ~ 125 0.9877 1.2 3.6 59.9 20.7
4 FOSA 1.831 498.00>77.90 2.5 ~ 125 0.9926 < 2.5 5 23.5 7.9
5 PFODA 3.171 913.00>868.90 2.5 ~ 125 0.9916 0.45 1.3 43.9 12.1
6 PFDHxA 3.2 813.00>768.80 2.5 ~ 125 0.9985 0.25 0.8 16.3 8.3
7 PFDS 3.201 599.00>80.00 2.5 ~ 125 0.9942 0.1 0.3 21.5 8.4
8 PFTeDA 3.23 712.90>668.90 5 ~ 125 0.9947 1.55 4.7 14.4 10.4
9 PFOS 3.233 499.00>79.90 2.5 ~ 125 0.9881 0.1 0.35 21 9.5
10 PFHpS 3.253 449.00>79.85 2.5 ~ 125 0.9924 0.15 0.45 15.6 9.6
11 PFTrDA 3.257 663.00>619.00 2.5 ~ 125 0.9986 0.75 2.2 23.7 20.7
12 PFDoA 3.265 612.90>569.00 2.5 ~ 125 0.9943 0.5 1.6 13.5 13
13 PF-3,7-DMOA 3.266 469.05>269.00 2.5 ~ 125 0.9918 0.15 0.4 22.3 9.1
14 PFHxS 3.278 399.00>79.90 2.5 ~ 125 0.9923 0.1 0.35 26.9 5.5
15 PFUdA 3.289 563.00>519.00 2.5 ~ 125 0.9975 0.5 1.55 17.7 8.1
16 PFDA 3.307 512.80>468.90 2.5 ~ 125 0.9946 0.75 2.3 25.4 8.3
17 PFBS 3.322 298.80>79.90 2.5 ~ 125 0.9951 0.15 0.45 18.9 8.8
18 PFNA 3.329 462.90>418.90 5 ~ 125 0.9908 1.5 4.55 23.4 12.3
19 PFOA 3.354 413.10>369.10 5 ~ 125 0.9832 1.6 4.8 15 6.1
20 PFHpA 3.38 313.10>269.05 5 ~ 125 0.996 1.25 3.8 14.4 11.8
21 PFHxA 3.399 263.00>219.00 2.5 ~ 125 0.9951 0.3 0.95 18 13.8
22 PFPeA 3.43 212.90>168.95 2.5 ~ 125 0.996 0.35 1.1 18.7 11.4
23 PFBA 3.466 363.10>319.00 2.5 ~ 125 0.9949 0.15 0.5 11.7 8.7
IS1 M-PFOS 3.235 503.00>79.85 10 Not Available
IS2 M-PFOA 3.349 416.90>372.00 10 Not Available
4
Development of on-line SFE-SFC-MS/MS approach

Next, an on-line SFE-SFC-MS/MS approach was developed calibration curves must be established on SFE-SFC-MS/MS
based on the SFC-MS/MS method established. The mixed too (Figure 3(c) & Table 3 (left).
standard samples for calibration curve construction could If we compare the peak areas obtained on SFE-SFC-MS/MS
be introduced into the system only by pre-loading them and SFC-MS/MS, system recovery of the SFE-SFC-MS/MS
onto filter papers. 50 µL of mixed standard solution was could be estimated (see Table 3, columns 8-11). Although
dropped onto half filter paper (recommended by Shimadzu this system recovery may not be highly accurate, it can be
for Nexara UC use) and left it to dryness under N2 flow used as a reference to understand the performance of the
before loading into the SFE vessel (0.2 mL). The results are on-line SFE-SFC-MS/MS for quantitation. The average
shown in Figure 3 and Table 3 (columns 1-7). First, with system recovery measured at the absolute loading amounts
on-line SFE, the elution peaks of the PFCs become broader of 25 pg and 50 pg are 90 % and 74 %, respectively. The
and RTs delay slightly (about 0.2 min) in comparison with average repeatability (RSD%, n=4) of the system with
SFC-MS/MS chromatograms. This peak broadening and loading amounts of 25 pg and 50 pg are 12 % and 14 %,
delay are due to the larger delay volume by the SFE vessel, respectively. It is worth noting that all of the analysis runs
needles and the tubing from SFE to column, which caused shown above are under the condition without splitting of
differences in peak areas and intensity. For direct the flow (sfCO2 and MeOH) from SFE to SFC-MS/MS
quantitation of PFCs using on-line SFE-SFC-MS/MS, (PBR-B was set to zero flow to drain).
(x100,000) (x10,000) Area (x100,000)

5.0
PFOS 2.0
1.25 (a) 23 PFCs and 2 IS 4.5 (b) (c) PFOA
4.0
MPFOS 1.0
1.00
3.5
MPFOA
3.0
0.75 0.0
2.5 0 50 100 pg.
Area (x100,000)
2.0
0.50
PFOA
1.5 (c) PFOS
2.5
0.25 1.0
0.5
0.00 0.0
0.0
0.0 1.0 2.0 3.0 4.0 5.0 min 3.0 3.5 min 0 50 100 pg.
Figure 3: (a) MRM chromatograms of 23 PFC mixed standards with 2 IS on filter paper, 25 pg for each compound.
(b) Separate display of MRM peaks of PFOA, M-PFOA (IS, 10 pg), PFOS and M-PFOS (IS, 10 pg),
(c) Calibration curves of PFOA and PFOS on SFE-SPC-MS/MS.
5
Table 3: Calibration curves of 23 PFCs (on filter paper in 0.2mL SFE vessel) of on-line SFE-SFC-MS/MS method (left table);
System recovery estimated by comparison with SFC-MS/MS method (right Table)
Range Accuracy 25 pg (spiked) 50 pg (spiked)

ID# Name m/z Ret. Time R2
(pg) (%) Recovery % RSD% (n=4) Recovery % RSD% (n=4)
1 N-EtFOSA 526.10>169.00 1.636 25 - 125 0.964 103 137.3 19.4 102.0 12.4
2 N-MeFOSA 512.00>169.15 1.692 25 - 125 0.9668 103.6 89.2 40.1 49.4 33.0
3 H4PFUnA 491.10>367.00 1.694 25 - 125 0.9956 101.2 87.8 24.2 51.6 26.0
4 FOSA 498.00>77.90 1.828 25 - 125 0.9978 100.7 61.0 18.6 40.2 23.8
5 PFODA 913.00>268.95 3.15 25 - 125 0.9946 101.5 91.2 16.9 80.7 20.3
6 PFDHxA 813.00>169.10 3.163 25 - 125 0.988 102.3 83.9 10.7 74.1 14.5
7 PFDS 599.00>98.80 3.3 25 - 125 0.9998 99.7 80.0 11.3 68.9 5.7
8 PFTeDA 712.90>219.20 3.196 25 - 125 0.9968 101.1 104.1 7.7 98.0 13.2
9 PFOS 499.00>98.50 3.344 25 - 125 0.993 101.5 92.6 5.4 75.7 3.5
10 PFHpS 449.00>99.05 3.344 25 - 125 0.9942 101.4 91.9 1.1 77.5 3.6
11 PFTrDA 663.00>169.10 3.198 25 - 125 0.9956 101.3 72.8 10.0 71.5 19.2
12 PFDoA 612.90>319.00 3.22 25 - 125 0.9947 99.9 101.3 12.0 87.1 10.2
13 PF-3,7-DMOA 469.05>68.80 3.229 25 - 125 0.9949 101.5 98.0 8.4 74.4 5.2
14 PFHxS 399.00>79.90 3.401 25 - 125 0.9958 100.7 87.5 1.1 78.0 4.4
15 PFUdA 563.00>268.90 3.265 25 - 125 0.995 98.7 84.4 1.1 76.6 17.7
16 PFDA 512.80>169.00 3.262 25 - 125 0.9905 101.3 106.1 4.9 89.6 14.6
17 PFBS 298.80>98.80 3.357 25 - 125 0.9912 101.6 89.1 7.9 69.0 8.1
18 PFNA 462.90>168.90 3.31 25 - 125 0.9986 100.6 103.6 8.2 78.8 10.4
19 PFOA 413.10>168.85 3.36 25 - 125 0.9972 100.8 97.8 7.7 87.3 15.0
20 PFHpA 363.10>169.05 3.375 25 - 125 0.9904 101.7 95.9 11.0 83.6 4.0
21 PFHxA 313.10>119.00 3.397 25 - 125 0.9776 100.3 76.6 8.5 74.1 11.4
22 PFPeA 263.00>219.00 3.431 25 - 125 0.9991 100.6 87.0 7.1 62.4 12.4
23 PFBA 212.90>168.95 3.446 25 - 125 0.9997 100 53.0 25.7 41.3 32.5
Automated SFE-SFC-MS/MS approach for analysis of PFCs in textiles samples

There are two extraction modes in the on-line SFE stage, sample were detected. The amount of PFCs spiked are
static extraction and dynamic extraction with sfCO2 or a equal to 0.42 ng/g (ppb), where detection sensitivity meets
mixture of sfCO2 and MeOH (modifier). For more effective the requirement for monitoring PFOA and PFOS in
extraction, static extraction is performed first for 4 mins at consumer products [2]. This preliminary finding reveals the
40 ºC, followed by dynamic extraction for 2 mins. Figure 4 potential possibility of using on-line SFE-SFC-MS/MS as a
shows an example of the analysis of un-pretreated clothing new automated screening and quantitation system in
sample and the same sample spiked with mixed PFCs (25 analysis of un-pretreated samples directly. However,
pg each on 60 mg of sample). The results shows that there further studies of this novel approach for PFCs and other
are no PFC detected in the textile sample, which is in targeted analytes in various consumer samples are under
agreement with the offline LC/MS/MS analysis results [4]. investigation for optimizing the SFE conditions and
On the other hand, all of the 23 PFCs spiked into the improving the recovery for samples with complex matrix.
6
(x10,000) (x10,000) (x10,000) (x10,000)

3.0 3.0
7.0
(a) (b) 7.0 (c) (d)
2.5 2.5 PFOS
6.0
6.0
5.0 2.0 2.0

5.0 MPFOS
4.0 1.5 1.5
4.0
MPFOA
3.0
3.0
1.0 1.0 PFOA
2.0 2.0
0.5 0.5
1.0 1.0
0.0 0.0
0.0 0.0
1.0 2.0 3.0 4.0 5.0 min 3.25 3.50 min 0.0 1.0 2.0 3.0 4.0 5.0 min 3.25 3.50 min
Figure 4: MRM chromatograms for screening of 23 PFCs in a textile sample (a-b) and in the same sample spiked
with mixed 23 PFCs standards with 2 IS, 25 pg for each compound (c-d). Only dynamic extraction was used.
Conclusions
In this study, a new analytical approach on the novel analytes in un-pretreated solid samples directly. The
SFE-SFC-MS/MS platform was developed for analysis of 23 detection sensitivity of the 23 PFCs spiked in clothing
PFCs including PFOA and PFOS spiked in textiles. The sample achieved is at the level of 25 pg or 0.42 µg/kg.
results indicate that the new approach is potentially However, validation of the SFE-SFC-MS/MS approach for
possible for screening and quantitation of targeted actual samples are not carried out yet.
References
1. Shimadzu Application News No LAAN-A-LC-E273, Using the Nexera UC Online SFE-SFC-MS System to Analyze
Residual Pesticides in Agricultural Products (2015).
2. I. van der Veen, J. M. Weiss, A. C. Hanning, , J. de Boer and P. E. Leonards, Talanta 147 (2016), 8-15.
3. Wellington Laboratories, “Quick Reference Guide for Perfluoroalkyl Compounds”, http://www.well-labs.com
4. J. X. Lee, Z. Sun, J. Xing, J. Y. Tan and Z. Zhan, ASMS 2016, Poster Session TP 485.
Disclaimer: The products and applications in this poster presentation are intended for Research Use only (RUO).
Not for use in diagnostic procedures.

PO-CON1634E
Determination of Wilfordine
and Wilforine in honey using
Liquid Chromatography with
Tandem Mass Spectrometry
ASMS 2016 ThP 110
Jianli Chen, Jinting Yao, Taohong Huang, Yuki Hashi

Shimadzu Corporation
Determination of Wilfordine and Wilforine in honey using
Liquid Chromatography with Tandem Mass Spectrometry
Introduction
Tripterygium wilfordii, which contains a lot of biological study, a highly sensitive method based on liquid-liquid
toxic compounds such as Wilfordine and Wilforine, is one extraction (LLE) and LC-MS/MS has been developed. The
of the toxic nectar plants. The Wilfordine and Wilforine results showed that the detection limits of Wilfordine and
may be transferred to honey by honey bees. Due to the Wilforine in honey sample were 5.16 and 10.80 ng/kg,
low content and complex matrix, determination of respectively.
Wilfordine and Wilforine in honey is not easy. In this
Wilfordine Wilforine
Figure 1 Structure of Wilfordine and Wilforine
Methods
Preparation of Samples
1.0 g of honey sample was added into 10 mL centrifuge centrifugated at 8000 rpm for 5 minutes. The above
tube, and then diluted with 2 mL of pure water. After solution was withdrawn and filtered (Organic membrane,
adding 2 mL of acetonitrile, 0.3 g of NaCl, and 1.2 g of 0.22 μm) for detection.
MgSO4 in order, the mixture was vortexed for 2 min and
Instrucments
The LC-MS/MS system were Prominence LC-20A and oven, and a DGU-20A3 online degasser. MS/MS detection
triple quadrupole mass spectrometry (Shimadzu was performed by LCMS-8050. Data acquisition and
Corporation, Kyoto, Japan). Shimadzu LC-20A system processing were performed with Labsolution software
consist of a CBM-20A system controller, two LC-20AD Version 5.72. Electrospray ionization was operated in
pumps, a SIL-20AC autosampler, a CTO-20AC column multiple-reaction-monitoring (MRM) mode.
2
High Speed Mass Spectrometer

• 5 msec
Ultra Fast MRM
Result
Method development for Wilforine and Wilfordine
HPLC conditions
Column : InertSustain C8-3 Column（2.1 mm I.D.×150 mm L., 5 μm）

Mobile phase A : 0.1% formic acid aqueous solution
B : Acetonitrile
Elution Mode : Gradient Elute, the initial concentration of MP B was 30%,
Table 1. LC Time Programme
Time Module Command Value

1.00 Pumps Pump B Conc. 30
10.00 Controller Stop
Column temperature : 35 ºC
MS conditions (LCMS-8050)
Ionization : ESI, Positive MRM mode

Nebulizer Flow : 3.0 L/min
Heating Gas Flow : 8.0 L/min
Interface Temperature : 400 ºC
DL Temperature : 150 ºC
Heat block Temperature : 300 ºC
Dry Gas : 12.0 L/min
3
Table 2. MRM transition
Compound MRM transition Q1 Pre Bias (V) CE Q3 Pre Bias (V)

884.30>856.20* -12 -25 -30
Wilfordine
884.30>176.10 -12 -50 -18
868.30>178.10* -12 -60 -18
Wilforine
868.30>206.10 -12 -43 -20
(x1,000)
1.25 1:wilfordine 884.30>856.20(+) CE: -25.0
wilforine
2:wilforine 868.30>178.10(+) CE: -60.0
1.00
wilfordine
0.75
0.50
0.25
0.00
0.0 2.5 5.0 7.5 min
Figure 3 MRM chromatograms of standard solution of Wilforine and Wilfordine

(Concentration of each compound were 0.05 ng/mL)
Analytical Performance
Linearity
The determination of Wilfordine and Wilforine were solution to get final concentrations of Wilfordine and
verified using an external standard method. The external Wilforine at 0.01, 0.02, 0.05, 0.1, 0.5, 1.0, 5.0 and 10
calibration was performed by plotting peak area versus ng/mL.
concentration of Wilfordine and Wilforine (As seen in The detailed calibration curves, ranges, correlation
Figure 4). The sample solutions were spiked with stock coefficients and precisions were shown in Table 2.
Area Area
500000
750000
Wilforine 400000 Wilfordine
500000
300000
200000
250000
100000
0 0
0.0 2.5 5.0 7.5 Conc. 0.0 2.5 5.0 7.5 Conc.
Figure 4 Calibration curve of Wilfordine and Wilforine
4
Table 3. Parameters of Calibration Curves
Compound Calibration Curves Range (ng/mL) Coefficient (r2) Precision (%)

Wilforine Y=(75959.6) X -45.2 0.01~10.0 0.9996 92.1~113.8
Wilfordine Y=(47426.1) X+ 206.6 0.01~10.0 0.9997 87.7~108.3
Sensitivity
Detection and quantification limits were calculated as the concentration corresponding to a signal 3 and 10 times of the
baseline noise, and the detection limits of Wilforine and Wilfordine were 1.3 and 4.3 ng/L, the quantification limits
were 2.7 and 9.0 ng/L, respectively.
Recovery
Preparation of blank honey samples as well as blank honey samples spiked at 0.05 ng/g and 5.0 ng/g. According to the
mentioned method before, each sample was measured three times in parallel. The recovery is calculated by subtracting
the content of Wilfordine and Wilforine in blank honey samples. The recovery results were shown in table 4.
Table 4. Recovery results
No. Compound Spiked at 0.05 ng/g (%) Spiked at 5.0 ng/g (%)
1 Wilfordine 104.0 99.6
2 Wilforine 116.0 98.8
Conclusions
In this paper, a fast and effective method for the sensitive the limit of detection were 1.3 and 4.3 ng/mL, the
and reliable analysis of Wilfordine and Wilforine using quantification limits were 2.7 and 9.0 ng/L, respectively.
LC-MS/MS was established.The method has good The recoveries were between 98.8~116.0%.
linearity, with correlation coefficient greater than 0.999,
Disclaimer: The products and applications in this presentation are intended for Research Use Only (RUO). Not for

PO-CON1636E
Determination of Oleanolic acid

and Ursolic acid in loquat leaf extract
By Liquid Chromatography-Tandem
Mass Spectrometry
ASMS 2016 ThP 335
Dan Luo1, Yuqi Feng2, Jinting Yao1, Taohong Huang1,

Yuki Hashi1
1
Shimadzu Corporation.
2
Department of Chemistry, Wuhan University, China
Determination of Oleanolic acid and Ursolic acid in loquat leaf
extract By Liquid Chromatography-Tandem Mass Spectrometry
Introduction
Ursolic acid (UA) and oleanolic acid (OA) belong to a class After being extracted, a pair of stable isotope probes,
of triterpenic acid, which play significant bioactive roles in 2-dimethylaminoethylamine (DMED) and
anti-inflammatory, antitumor as well as enhancing the d4-2-dimethylaminoethylamine (d4-DMED) were added
cellular immune system. However, due to the lack of for derivatization. The generated heavy labeled triterpenic
suitable chromophoric or ionization groups in molecular acids were used as internal standards for quantification,
structures, both UV and MS responses are far from the detection sensitivities of analytes improved sharply,
satisfaction. In this study, a highly sensitive method based the detection limits of OA and UA were 0.92 and 1.06
on derivatization and LC-MS/MS has been developed. ng/L, respectively.
Ursolic acid (UA) Oleanolic acid (OA)
Figure 1 Structure of Ursolic acid (UA) and oleanolic acid (OA)
Methods
The derivatization of standard solution
Take 200 μL of working standard solution, after adding 1500 rpm for 1 h at 40 ºC. The sample was evaporated to
10 μL 20 μmol/mL of triethylamine (TEA) and 10 μL 20 μ dryness under a mild nitrogen steam at room
mol/mL of 2-chloro-1-methylpyridinium iodide (CMPI), the temperature. The residue was dissolved in 100 µL of
solution was vortexed for 5 min. Then 10 μL 40 μmol/mL mobile phase, and then analyzed by LC-MS/MS.
of DMED was added, and the mixture was vibrated at
Preparation of Samples
0.5 g of sample were added into 50 mL volumetric flask, centrifuged, and the resulting supernatant was used for
and then diluted with saturated salt water to the required derivatization. The procedure of derivatization was the
volume. 1 mL above solution was withdrawn and added same as that of standard solution.
200 μL of ethyl acetate. After being vortexed and
Preparation of Internal Standard

The derivatization process of internal standard was almost the same as that of standard solution except that the DMED
was replaced with d4-DMED. The evaporated residue was dissolved in 1 mL of mobile phase, took 10 μL of that to each
standard solution and real samples, and then used for analysis.
2

• 5 msec
Ultra Fast MRM
The LC-MS/MS system were Prominence LC-20A and oven, and a DGU-20A3 online degasser. MS/MS detection
triple quadrupole mass spectrometry (Shimadzu was performed by LCMS-8050. Data acquisition and
Corporation, Kyoto, Japan). Shimadzu LC-20A system processing were performed with Labsolution software
consists of a CBM-20A system controller, two LC-20AD Version 5.72. Electrospray ionization was operated in
pumps, a SIL-20AC autosampler, a CTO-20AC column multiple-reaction-monitoring (MRM) mode .
Result
Method development for UA and OA
HPLC conditions
Column : InertSustain C18 Column (2.1 mm I.D.×250 mm L., 3 μm)

Mobile phase A : 0.1% formic acid aqueous solution B: Acetonitrile
A:B=47:53 (v/v)
Ionization : ESI, Positive MRM mode

Nebulizer Flow : 2.0 L/min
Heating Gas Flow : 10.0 L/min
Interface Temperature : 300 ºC
DL Temperature : 250 ºC
Heat block Temperature : 400 ºC
Dry Gas : 10.0 L/min
3
Table 1. MRM transition
Compound MRM transition Q1 Pre Bias (V) CE Q3 Pre Bias (V)

527.30>482.30* -20 -34 -23
Oleanolic acid
527.30>203.20 -20 -41 -20
527.30>482.30* -20 -34 -23
Ursolic acid
527.30>203.20 -20 -41 -20
d4-Oleanolic acid 531.30>482.30 -20 -34 -23
d4-Ursolic acid 531.30>482.30 -20 -34 -23
OA and UA are pentacyclic triterpenoids, the structural reported in the literature. Figure 3 shows MRM
difference is merely a methyl group at C-20 on the OA chromatograms of 0.1 μg/mL standard solution of UA
shift to C-19 position. It is very difficult to separate these and OA(a), as well as 0.1 μg/mL standard solution of
two compounds that belong to the geometric isomers. In d4-UA and d4-OA(b). It took 12 minutes per one
this study, 3 μm of InertSustain C18 column was used, LC-MS/MS analysis, and excellent separation and high
the resolution is 1.110, which is better than the results sensitive detection were obtained.
1:OA 527.30>482.30(+) CE: -34.0
OA
2:UA 527.30>482.20(+) CE: -33.0
4000
3000
UA
2000
1000
0
(a)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min
3:OA-d4-DMED 531.30>482.30(+) CE: -34.0

OA-d4-DMED
4:UA-d4-DMED 531.30>482.30(+) CE: -34.0

4000
UA-d4-DMED
3000
2000
1000
0
(b)
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min
Figure 3 MRM chromatograms of standard solution of UA and OA(a), as well as

internal standard solution (Concentration of each compound were 0.1 μg/mL)
4
Analytical Performance
Linearity
The determination of UA and OA was verified using an solutions were spiked with stock solution to get final
internal standard for quantification. The internal concentrations of UA and OA at 0.01, 0.05, 0.1, 0.5, 1.0,
calibration was performed by plotting peak area ratios 5.0 and 10 ng/mL.
versus concentration ratios of DMED derivates and The detailed calibration curves, ranges, correlation
d4-DMED derivates (As seen in Figure 4). The sample coefficients and precisions were shown in Table 2.
Area Ratio Area Ratio

125
100
OA 100
UA
75
75
50
50
25 25
0 0
0.0 2.5 5.0 7.5 Conc. Ratio 0.0 2.5 5.0 7.5 Conc. Ratio
Figure 4 Calibration curve of OA and UA
Table 2. Parameters of Calibration Curves
Compound Calibration Curves Range (ng/mL) Coefficient (r2) Precision (%)

OA Y=(11.230) X + (0.480) 0.01~10.0 0.9995 81.7~117.8
UA Y=(12.197) X+ (0.467) 0.01~10.0 0.9998 94.6~119.7
Sensitivity
Detection and quantification limits were calculated as the concentration corresponding to a signal 3 and 10 times of the
baseline noise, and the limit of detection oleanolic acid and ursolic acid were 0.92 and 1.06 ng/mL, the quantification
limits were 3.07 and 3.53 ng/L, respectively.
Recovery
Preparation of blank loquat leaf extract samples as well as calculated by subtracting the content of OA and UA in
blank loquat leaf extract samples spiked at 100 mg/kg. blank samples. The results showed that the recoveries of
According to the mentioned method before, each sample OA and UA were in the range of 98.7~102.7 % and
was measured three times in parallel. The recovery is 97.2~105.0 %, respectively.
5
Quantitative Analysis of real sample

According to the sample preparation method, loquat leaf extracts from market were analyzed. The sample was
determined parallel twice. The content of oleanolic acid and ursolic acid in this sample were 7.40 and 23.6 mg/Kg,
respectively. The results were shown in Figure 5.
100000 1:OA 527.30>482.30(+) CE: -34.0

2:UA 527.30>482.20(+) CE: -33.0
3:OA-d4-DMED 531.30>482.30(+) CE: -34.0
4:UA-d4-DMED 531.30>482.30(+) CE: -34.0
75000
UA
OA
50000
OA-d4-DMED
UA-d4-DMED
25000
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min
Figure 5 MRM chromatograms of real samples
Conclusions
In this paper, a highly sensitive method based on 0.92 and 1.06 ng/mL, the quantification limits were 3.07
derivatization of oleanolic acid and ursolic acid coupled and 3.53 ng/L, respectively. The recoveries were between
with LC-MS/MSdetection was established. 97.2~105.0 %. This method is simple, rapid, accurate
This method has good linearity, with correlation and can be applied to detect OA and UA in loquat leaf
coefficient greater than 0.999, the limit of detection were extract.
Disclaimer: The products and applications in this presentation are intended for Research Use Only (RUO). Not for

PO-CON1626E
High throughput quantitation

for therapeutic drug monitoring
with Open access LC/MS/MS system
ASMS 2016 ThP 682
Hikaru Shibata1, Miho Kawashima2, Yoshihiro Hayakawa2,

Taku Tsukamoto2, Masafumi Kikuchi3, Masaki Tanaka3,
Shinya Takasaki3, Hiroaki Yamaguchi3, Nariyasu Mano3
1
Shimadzu Scientific Instruments Inc., Columbia, MD, USA,
2
Shimadzu Corporation, Kyoto, Japan,
3
Tohoku University Hospital, Sendai, Japan
High throughput quantitation for therapeutic drug
monitoring with Open access LC/MS/MS system
Introduction
In this paper, we introduce four research use only sensitive analysis by simple sample pretreatment. In the
LC/MS/MS methods for therapeutic drug monitoring field of TDM, it is necessary to measure the specimen
(TDM), mycophenolic acid, sunitinib and axitinib, such as plasma or serum quickly after suitable
voriconazole, itraconazole. TDM is indispensable for pretreatment and report the precise result. LC/MS/MS
managing drug dosage based on the drug concentration system with a simple and user-friendly interface can
in blood in order to conduct a rational and efficient drug provide a streamlined workflow and reduce the load of
therapy. Liquid chromatography coupled with tandem analysts. We developed high-throughput LC/MS/MS
quadrupole mass spectrometry (LC/MS/MS) is increasingly methods for TDM with a new data acquisition and
used in TDM because it can perform selective and processing software.
Method
Instruments and LC/MS/MS analytical conditions
For LC/MS/MS analysis, a LCMS-8050 triple quadrupole column, Shim-pack GIS (Shimadzu corporation, Japan). All
mass spectrometer coupled to a Nexera X2 UHPLC system data acquisition and processing were performed by Open
with mobile phase switching unit (Shimadzu corporation, Solution QuantAnalytics (Shimadzu corporation, Japan), a
Japan) was used. In all the methods, the compounds were software package for acquiring and reviewing quantitative
separated by a reversed phase mode using a common LC/MS/MS data with ease.
2
Table 1 LC/MS/MS consitions
Method 1 Method 2
Mycophenolic acid Voriconazole

Target
(Immuno-suppressant) (Triazole antifungal agent)
Column Shim-pack GIS (2.1 mmI.D. x 75mmL., 3um)

Column Oven 40ºC
Flow rate 0.3 mL/min 0.4 mL/min
Mobile phase A 1% Acetic acid-water 10 mM Ammonium acetate-water
Mobile phase B Acetonitrile Methanol
Bconc. 30% (0 - 0.50min) →

Gradient A/B = 1/1, isocratic
100% (1.50 - 3.00min) → 30% (3.01 - 5.00min)
Injection volum 5 µL 1 µL
Ionization ESI-positive
MRM transition 321.40 > 207.30 350.20 > 281.20
Run time 4 min. 5 min.
Method 3 Method 4
Sunitinib and Axitinib Itraconazole

Target
(Anti-cancer drug) (Triazole antifungal agent)
Column Shim-pack GIS (2.1 mmI.D. x 75mmL., 3um)

Column Oven 40ºC
Flow rate 0.3 mL/min 0.4 mL/min
Mobile phase A 10 mM Ammonium acetate-water 10 mM Ammonium acetate-water
Mobile phase B Acetonitrile Acetonitrile
Bconc. 10% (0 - 0.25min) → Bconc. 65% (0 - 1.00 min) →

Gradient
80% (2.00 - 3.00min) → 10% (3.01 - 5.00min) 95% (1.50 - 2.50min) → 65% (2.51 - 4.50min)
Injection volum 5 µL 3 µL
Ionization ESI-positive
Sunitinib 399.40 > 283.30 Itraconazole 705.40 > 392.40

MRM transition Axitinib 387.40 > 356.30 Hydroxy Itraconazole (Active metabolite of Itraconazole)
SU12662 (Active metabolite of Sunitinib) 371.40 > 283.30 721.40 > 408.40
Run time 5 min. 4.5 min.
3
Shim-pack GIS 2-Position Valve

Pump A
1 2 3 4 Auto Sampler LCMS-8050

Column Oven
(Drain)
Mobile phase with2-position Valve
switching unit
Pump A
1: 1% acetic acid-water
2: 10 mMammonium acetate-water
Pump B Pump B
1: Acetonitrile
1 2 3 4 2: Methanol
Figure 1 Nexera X2 UHPLC system with mobile phase switching unit
Calibration standards and QC samples

For each compound, more than five calibration standards and three QC samples were prepared. Samples were precipitated
in a simple way of deproteination using organic solvent such as methanol or acetonitrile. The resulting supernatant was
diluted and injected into LC/MS/MS without filtration.
Human plasma
Internal standard solution
Organic solvent
Vortex
Centrifuge
LC/MS/MS analysis Directly or diluted
Figure 2 Work flow of the pretreatment
4
Result and discussion

Precision, accuracy and linearity
Table 2 illustrates linearity of all compounds and Table 3 Excellent linearity, accuracy and precision were obtained
illustrates accuracy and precision of the QC samples. within a specific concentration range. Furthermore, All the
Determination coefficient (r2) of all calibration curves was methods took less than 5 minutes per one LC/MS/MS
larger than 0.995, the precision (n=6) was within 15% analysis, including column rinsing.
RSD, and the accuracy (n=6) was within 80-120%.
Table 2 Linear dynamic range for each compound
Compound Linearity (µg/mL) r2

Method 1 Mycophenolic acid 0.2 ~ 20 0.999
Method 2 Voriconazole 0.1 ~ 10 0.999
Sunitinib 3 ~ 300 0.999

Method 3 Axitinib 0.3 ~ 30 0.999
SU12662 3 ~ 300 0.999
Itoraconazole 10 ~ 1000 0.999

Method 4
Hydoroxy Itorazonazole 10 ~ 1000 0.999
Table 3 Precision and accuracy for analysis of QCs
Concentrations of
QC samples (µg/mL) Precision (%) Accuracy (%)
Compound
Low Middle High Low Middle High Low Middle High
Method 1 Mycophenolic acid 0.5 5 15 1.6 3.66 2.55 108.5 104.9 103.5
Method 2 Voriconazole 0.2 4 8 1.74 1.56 1.7 98.6 103.6 99
Sunitinib 5 50 250 4.05 1.36 2.24 100.7 95.5 96

Method 3 Axitinib 0.5 5 25 8.26 4.56 4.43 85.3 88.5 91.2
SU12662 5 50 250 4.07 1.26 3.17 97.2 96.5 97.8
Itoraconazole 0.65 0.82 0.38 98.7 98.8 103.3

Method 4 25 250 750
Hydoroxy Itorazonazole 3.27 4.36 3.1 81.3 90.4 86.9
5
Method 1 Method 3
Area ratio(x10) Area ratio (x10)
1.00
Mycophenolic acid Sunitinib
1.25 0.2~20 µg/mL 3~300 µg/mL
0.75
r2 =0.999 r2 =0.999
1.00
0.50 (x10,000)
0.75 2.5
0.25
0.50 (x1,000) 0.0

2.5
0.00 1.0 2.0
0 100 200 Concentration ratio
0.25
0.0 Area ratio (x0.1)
1.0 2.0
0.00 Axitinib
0.0 5.0 10.0 15.0 Concentration ratio
0.3~30 µg/mL
2.0
r2 =0.999
(x1,000)
Method 2 1.0
1.0
Area raio(x10) 0.0

2.0 3.0
1.00 Voliconazole 0.0
0 10 20 Concentration ratio
0.1~10 µg/mL
Area ratio
r2 =0.999
0.75 SU12662
5.0 3~300 µg/mL
0.50
r2 =0.999
(x10,000) (x10,000)
2.0
2.5
0.25 1.0 1.0
0.0 0.0
2.0 2.5 1.0 2.0
0.00 0.0
0.0 2.5 5.0 7.5 Concentration ratio 0 100 200 Concentration ratio
Method 4
Area ratio Area ratio
2.00 Itoraconazole 2.00 Hydoroxy Itoraconazole
10~1000 µg/mL 10~1000 µg/mL
1.50 r2 =0.999 1.50 r2 =0.999
1.00 1.00
(x10,000) (x10,000)
2.5
2.5
0.50 0.50
0.0 0.0
2.0 2.5 1.0 1.5
0.00 0.00
0 250 500 750 Concentration ratio 0 250 500 750 Concentration ratio
Figure 3 Calibration curves for each compound
6
Easy data acquisition with Open access software

The developed platform was used one common column and placing sample vials in the specified autosampler
for all methods and mobile phase changeover could be plate positions guided by software. The resulting data can
automatically done by just selecting method file. Open be reviewed in office as soon as it becomes available on
Solution QuantAnalytics software enables users to submit the designated data server. This system enables easy and
sample queue and set the LC/MS/MS condition easily and quick data acquisition without tedious manual operation
quickly. Users can intuitively start LC/MS/MS such as replacement of a column and mobile phases.
measurement by just selecting the predefined method
Data server
Open Solution Open Solution Insight

Connect
Laboratory Office/ home/ remote laboratory
(1) Import sample list and select methods. (1) Select batch.
(2) Expert checks data, decides next step.
(2) Confirm sample list then start analysis.
Show analyte
results for
selected sample.
Highlight results
with issues.
Start
Figure 4 User interface for sample queue submission Figure 5 User interface for checking the result
7
Conclusions
• The combination of the Open Solution QuantAnalytics software and LC/MS/MS system with mobile phase
switching unit enables easy sample submission and efficient data acquisition. The user only describes the vial
position of samples, chooses a predefined method and the resulting data can be reviewed in office as soon as it
becomes available on the designated data server.
• Saving time and effort for changing system conditions among each target compounds was achieved with mobile
phase switching system and high through-put methods using a common column.
Disclaimer: Shimadzu LCMS-8050 CL and certain Nexera X2 UHPLC components are registered in the U.S. as a
Class I device and is not specifically cleared for TDM. Other UHPLC components, Shim-pack GIS, and
OpenSolution QuantAnalytics are intended for Research Use Only (RUO). Not for use in diagnostic procedures.

PO-CON1689E
Rapid Screening and Quantitation

of Pesticide Residues in Cannabis
by Modified QuEChERS and LC-MS-MS
ASMS 2016 TP 230
Jeffrey H. Dahl1, Julie Kowalski2, Derek Laine3,

and Jason Zitzer3
1
Shimadzu Scientific Instruments; Columbia, Maryland
2
Restek Corporation; Bellefonte, Pennsylvania
3
Trace Analytics; Spokane, Washington
Rapid Screening and Quantitation of Pesticide Residues
in Cannabis by Modified QuEChERS and LC-MS-MS
Introduction
Medical and recreational use of marijuana (Cannabis spp.) consumers, so highly sensitive and selective methods for
is expanding in the United States at a rapid pace, and their detection in the complex cannabis matrix are
domestic production has increased more than ten-fold in required. We developed a rapid and effective LC-MS-MS
the last 25 years. This extremely high value crop is method with modified QuEChERS sample preparation for
vulnerable to mold and insects so growers frequently detection of nearly 200 chemical residues in dried
apply pesticides and antifungals to protect their plants. cannabis flower and used the method to test a wide
These chemical residues may pose a danger to selection of products offered for commercial sale.
Grind weighed Hydrate 15 mL 15 mL 1% Add AOAC

QuEChERS salts
test portion (1.5 g) water, shake acetic acid in ACN
vortex 2 min
30 min shake 30 min
centrifuge
SPEX GenoGrinder,
2 metal balls, 5 min
at 1500 rpm
Clean up aliquot
of supernatant
by dSPE
Figure 1 Modified QuEChERS Extraction
Method
Test portions of dried cannabis flower were homogenized quadrupole mass spectrometer. Electrospray ionization
and extracted using a modified QuEChERS extraction with was used with continuous polarity switching to measure
dispersive SPE cleanup (Restek cat no. 26237 and 26243). analytes in both modes throughout the run. Pesticide
Grinding was performed with a SPEX GenoGrinder. recovery was determined using spiking experiments and
Detection was carried out by LC-MS-MS using a matrix-matched calibration curves. All analysis was carried
Shimadzu Prominence HPLC with LCMS-8050 triple out in a Washington state certified cannabis testing lab.
Table 1 LCMS-8050 Instrument parameters
LC Column : Restek ARC-18 (2.1×100 mm, 3 µm)

Mobile Phase A : 5 mM NH4OAC + 0.1% Formic Acid
Mobile Phase B : Methanol
LC Flow Rate : 0.5 mL/min
Heater Gas : 10 L/min
Interface Temp : 400 °C
Nebulizing Gas : 3 L/min
Drying Gas : 10 L/min
DL Temp : 250 °C
Heat Block Temp : 400 °C
2
Figure 2 Cannabis samples for pesticide testing
(x10,000,000)
3.5
Number of compounds by recovery from
QuEChERS extraction
3.0
<50% 50–70% 70–120% >120%
2.5
3 6 150 3
2.0
1.5
1.0
0.5
0.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min
160209.FVSETx21_90_FRTMIX L2_011.lcd
Figure 3 Representative chromatogram and number of compounds by recovery (inset)

QuEChERS extraction with dispersive SPE cleanup dependent however the majority were within the range
provided the best combination of pesticide recovery and of 70-120% while outliers above and below the range
cleanup for dried flower cannabis samples. Matrix were observed. The method was validated in three
matched calibration curves were linear within the different cannabis strains using matrix matched
quantitation limits established for each compound, which calibration curves and triplicate QC spikes at three levels.
was compound dependent, but ranged from as low as 20 In a subset of randomly tested cannabis flower samples
ppb or lower to greater than 500 ppb for a few offered for commercial sale, the three most commonly
substances. Detection limits and quantitation limits were detected residues were piperonyl butoxide, myclobutanil,
required to have 3:1 and 10:1 signal to noise respectively, and boscalid. Concentrations for pesticides ranged from
and quantitation limits were required to have less than the detection limit up to the microgram/gram level.
20% RSD in triplicate analyses. Recovery was compound
3
(x10,000) (x100,000) (x100,000)

142.00>94.00(+) 191.95>159.95(+) 1.00 256.10>175.00(+)
4.0
142.00>125.10(+) Methamidophos 4.0
191.95>131.90(+) Carbendazim 256.10>209.00(+) Imidacloprid
3.5
0.75
3.0 3.0
2.5
0.50
2.0 2.0
1.5
1.0 1.0 0.25
0.5
0.0 0.0 0.00

0.9 1.0 1.1 1.2 1.3 3.9 4.0 4.1 4.2 4.3 4.4 4.7 4.8 4.9 5.0 5.1 5.2
(x100,000) (x100,000) (x100,000)

222.20>165.00(+) 1.75 202.10>145.00(+)
1.00 297.10>159.00(+)
6.0 222.20>123.10(+)
Carbofuran 202.10>127.00(+) Carbaryl 297.10>41.10(+) Imazalil
1.50
5.0
1.25 0.75
4.0
1.00
3.0 0.50
0.75
2.0
0.50
0.25
1.0 0.25
0.0 0.00 0.00

6.2 6.3 6.4 6.5 6.6 6.7 6.4 6.5 6.6 6.7 6.8 6.9 6.6 6.7 6.8 6.9 7.0 7.1
(x10,000) (x10,000) (x100,000)

292.15>70.05(+) 343.00>306.80(+) 3.0 732.50>142.10(+)
292.15>125.00(+) Cyproconazole 5.0
343.00>140.00(+) Boscalid 732.50>98.05(+) Spinosyn A
5.0
2.5
4.0
4.0
2.0
3.0
3.0
1.5
2.0 2.0
1.0
1.0 1.0 0.5
0.0 0.0 0.0

8.00 8.25 8.50 7.6 7.7 7.8 7.9 8.0 8.1 9.75 10.00 10.25
(x100,000) (x10,000) (x10,000)

5.0 356.20>177.00(+)
356.20>149.00(+) Piperonyl Butoxide 289.20>70.10(+)
289.20>125.05(+) Myclobutanil 890.50>305.15(+)
1.00 890.50>567.20(+) Abamectin B1a
356.20>119.10(+) 6.0
4.0
5.0
0.75
3.0 4.0
3.0 0.50
2.0
2.0
0.25
1.0
1.0
0.0 0.0 0.00

10.7 10.8 10.9 11.0 11.1 7.9 8.0 8.1 8.2 8.3 8.4 11.1 11.2 11.3 11.4 11.5
Figure 4 Representative individual chromatograms at the 100 ng/g dried cannabis spiking level
(Abamectin chromatogram from 1 mcg/g level)
4
Table 2 Detection of pesticides in 39 dried flower samples offered for retail sale
Survey of pesticides in dried cannabis flower offered for retail sale
Residue Residue
Sample mcg/g Sample mcg/g
detected detected
A P Piperonyl butoxide 0.32

B Q Imidacloprid 0.49
C R
D S Piperonyl butoxide 0.69
E T
F U Myclobutanil 0.02
G Piperonyl butoxide 0.53 V
H Piperonyl butoxide 0.05 W Piperonyl butoxide 12.46
I X Piperonyl butoxide 0.16
J Spinosyn A 0.02 Fipronil 0.04
Spinosyn D 0.02 Y
K Myclobutanil 0.01 Z
L Myclobutanil 0.02 AA
M Dinotefuran 13.44 AB
Boscalid 81.79 AC Piperonyl butoxide 14.99
Pyraclostrobin 0.40 AD Permethrin 0.35
Trifloxystrobin 0.08 AE
Fludioxonil 0.42 AF Piperonyl butoxide 0.08
Myclobutanil 1.21 AG
N Dinotefuran 1.20 AH
Boscalid 5.79 AI
Fludioxonil 0.01 AJ Diuron 0.06
Myclobutanil 0.08 AK Piperonyl butoxide 0.13
O Boscalid 0.15 AL
Myclobutanil 0.05 AM
5
Area Ratio Area Ratio (x10) Area Ratio

5.0
2.5
5.0
Methamidophos Carbendazim 4.0 Imidacloprid
2.0
r2 > 0.996 r2 > 0.995 3.0
r2 > 0.996
1.5
2.5 2.0
1.0
0.5 1.0
0.0 0.0 0.0

0 250 500 750 1000 1250 1500 Conc. Ratio 0 250 500 750 1000 1250 1500 Conc. Ratio 0 250 500 750 1000 1250 1500 Conc. Ratio
Area Ratio (x10) Area Ratio Area Ratio
7.5 4.0
2.0 Carbofuran Carbaryl Imazalil
1.5
r2 > 0.994 5.0
r2 > 0.998 3.0
r2 > 0.988
2.0
1.0
2.5
0.5 1.0
0.0 0.0 0.0

Area Ratio Area Ratio Area Ratio (x10)

3.0 2.5
3.0
2.5
Cyproconazole Boscalid Spinosyn A
2.0 2.5
2.0
r2 > 0.994 r2 > 0.997 r2 > 0.995
2.0
1.5
1.5 1.5
1.0
1.0 1.0
0.5
0.5 0.5
0.0 0.0 0.0

Area Ratio (x10) Area Ratio Area Ratio (x0.01)

4.0
2.0
Piperonyl Butoxide 3.0
Myclobutanil Abamectin B1a
1.5
r2 > 0.996 r2 > 0.996 3.0 r2 > 0.997
2.0
2.0
1.0
1.0 1.0
0.5
0.0 0.0 0.0

Figure 5 Representative calibration curves
6
Dried flower Coarse ground Dispersive SPE

QuEChERS extract
Figure 6 Sample preparation
Table 3 Pesticides detected in 39 dried flower samples
Number of pesticide detections in dried cannabis flower
Residue
Detections Rate
name
Boscalid 3 7.7%
Dinotefuran 2 5.1%
Diuron 1 2.6%
Fipronil 1 2.6%
Fludioxonil 2 5.1%
Imidacloprid 1 2.6%
Myclobutanil 6 15.4%
Permethrin 1 2.6%
Piperonyl butoxide 9 23.1%
Pyraclostrobin 1 2.6%
Spinosyn A 1 2.6%
Spinosyn D 1 2.6%
Trifloxystrobin 1 2.6%
One or more 19 49%
7
70
60
50
40
30
20
10
0
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88 91 94 97 100103106109112115118121124127130133136139142145148151154157
Composite Dutch Treat Orange Kush
Figure 7 RSD for each compound in triplicate QC samples at the 50 ng/g spike level, in three matrices
Conclusion
A validated method for detection of chemical residues in dried cannabis flower samples was developed. Our method
can detect low levels of common pesticides in samples offered for retail sale with excellent selectivity and speed.
Measurements of a larger selection of commercially available cannabis samples are being carried out.

PO-CON1663E
Fully automated platform for

determination of immunosuppressant
drugs in whole blood
ASMS 2016 TP 412
Davide Vecchietti1, Brambilla M.3, Kawakami D.2,

Tsukamoto T.2, Brambilla P.3
1
Shimadzu Corporation, Milan, Italy
2
Shimadzu Corporation, Kyoto, Japan
3
Desio Hospital, University Department of laboratory
medicine, Desio, Italy
Fully automated platform for determination of
immunosuppressant drugs in whole blood
Introduction
Therapeutic drug monitoring of four major same inhibitory target.
immunosuppressant drugs, cyclosporin tacrolimus, Overdosing with these critical dose drugs can cause
sirolimus and everolimus (Fig. 1), is well established. serious toxicity and long term morbidity, while organ
Cyclosporine A, an 11 amino acid metabolite of rejection can occur if a patient is under dosed. These side
Tolypocladium inflatum, is a calcineurin effects include renal toxicity, hypertension,
inhibitor. Tacrolimus, another calcineurin inhibitor, is a hyperlipidemia, and gastrointestinal complaints with
macrolide antibiotic from Streptomyces tsukubaensis. Cyclosporine A and tacrolimus; renal dysfunction,
Sirolimus is a macrocyclic fermentation product of hyperlipidemia, anemia, leukopenia, and
Streptomyces hygroscopicus and a mammalian target of thrombocytopenia with sirolimus, and hyperlipidemia,
rapamycin (mTOR) inhibitor. leukopenia, and thrombocytopenia with everolimus [1]
Lastly, everolimus is a derivative of sirolimus with the [2].
A B
C D
Figure 1. Structure of Immunosuppressants.

A) Cyclosporin, B) Tacrolimus, C) Sirolimus, D) Everolimus
2
As with other monitored drugs, the clinical laboratory has However, current LC–MS/MS platforms demand
two main choices in technologies: immunoassay or personnel expertise and tedious sample preparation and
chromatography based methods. LC–MS/MS's superior sample throughput is generally much lower compared to
specificity makes it the presumptive gold standard in immunoassays [1] [2].
immunosuppressant quantitation. It relieves the method We report a fully automated procedure for the
from common interferences that plague immunoassays; quantitation of four major immunosuppressant in whole
such as metabolites that have structural resemblance and blood samples, increasing data quality/precision,
interfering antibodies. This increased throughput and safety (The work described herein is for
selectivity also allows everolimus and sirolimus to be research use only).
differentiated [1][2].

The quantitative analysis of Immunosuppressant from Table 1) (MRM optimization was performed using Tuning
whole blood samples was performed using reagent mix, ref, 93015). Sample preparation was developed
provided in Chromsystem “MassTox®” Kit (ref, 9300). using Precipitation reagent (ref, 93003), Extraction buffer
Chromatography separation was developed using Mobile (ref, 93005) and Internal standard set (ref, 93046).
Phase A (ref, 93001), Mobile Phase B (ref, 93002), Rinsing Analytical performance of the method was monitored
solution (ref, 93009), trap Column (ref, 93110) and using whole blood calibrators (6PLUS1® ref, 28039) and
Analytical column (ref, 93100). The Immunosuppressant whole blood QC controls (MassCheck®, ref 0081).
and Internal standard were monitored using HPLC-LCMS Automatic sample preparation was performed using
system (Nexera X2 and LCMS-8050, Shimadzu, Kyoto) CLAM-2000 module (Shimadzu, Kyoto).
(see figure 3) following specific MRM transitions (see
Results
LC-MS/MS analysis
The Immunosuppressant standards and the Internal MRM transitions listed in Table 1. Then
standards (see Table 1) were firstly analysed by flow Immunosuppressant standard mix was used to set-up
injection analysis (FIA) to determine the optimum chromatographic separation starting from Chromsystem
ionization polarity and to optimize MRM transitions. All kit (see above) conditions.
compounds were detected as positive ion choosing the
UHPLC conditions (Nexera X2 system)
Flow rate : 1 mL/min Load (0-1 min), 0.6 mL/min Elution (1.76-7min)
Time program : B conc. 0% (0-1 min) – 55% (1.01-1.75 min)
– 98% (1.76-7 min) – 0% (7-8.5 min)
3
Table 1. MRM transition of Immunosuppressant
Compound MRM transition Compound MRM transition

Cyclosporin A 1219.90 > 1202.80 Cyclosporin A-d4 1223.90 > 1206.70
Tacrolimus 821.60 > 768.30 Everolimus-d4 979.60 > 912.40
Sirolimus 931.70 > 864.50 Sirolimus-d3 934.60 > 864.40
Everolimus 975.70 > 908.50 Tacolimus-13Cd2 824.60 > 771.40
Manual sample preparation

Immunosuppressant determination from whole blood add 25 µl of IS+100 µl of Extraction Buffer and mix; after
samples using LC-MS/MS requires a long manual incubation add 250 µl of precipitation reagent and mix.
preparation procedure for drug extraction and protein Finally, after a centrifugation step, it is possible to transfer
precipitation. Briefly, it is necessary to transfer 50 µl of the supernatant into a microvial for HPLC injection
sample from blood collection tube into a 1.5 mL vial and to
Fully Automated sample preparation

With the aim to reduce the operator involvement, to and IS are placed into specific instrument slot (8 °C). The
increase the throughput and the data quality, the manual Fully automated sample preparation procedure (Fig. 2)
sample preparation procedure was substituted by an includes all the steps from sample collection to LCMS/MS
automated procedure using a novel Clinical Laboratory quantitation. The instrument automatically detect the
Automated sample preparation Module (CLAM-2000, Blood collection tubes or the micro-vial and transfer the
Shimadzu) online with the UHPLC-LCMS system (Figure 3). sample into a disposable filtrating-vial, subsequently add a
Blood collection tubes were loaded into the CLAM-2000 specific amount of reagents and proceed with others
together with disposable micro-vial containing calibrators sample preparation steps.
and QC controls. Precipitation Reagent , Extraction Buffer
4
Reagent Sample Reagent Reagent

Stirring Incubation Stirring Filtration
Dispensing Dispensing Dispensing Dispensing
Direct UHPLC
connection for
Extr. Whole Internal Prec. LCMS analysis
Buffer blood STD 2200 Reagent 2200
RT Aspiration
50 ul 25 ul 12.5 ul rpm 125 ul rpm
30 sec 60 sec 30 sec 10 sec 120 sec 30 sec 120 sec 60 sec 8.5 min
Figure 2. Fully Automated Sample preparation procedure.
Figure 3. CLAM-2000 online with Nexera X2 system and LCMS-8050 triple quadrupole mass spectrometer.
Analytical variability and precision

In order to asses the robustness of the fully automated sample preparation protocol, concentration levels of four
Immunosuppressant were evaluated using QC reference materials (see section 2. Methods and Materials), see table 2.
5
Table 2. Repeteability, Reproducibility and Accuracy of Immunosuppressant evaluation
Reference Level CV% BIAS% CV% BIAS%

Compound
(ul/L) (intra-assay) (intra-assay) (inter-assay) (inter-assay)
Low(47.75) 2.7% 3.9% 2.6% 0.6%

Mid-Low(253.2) 3.4% 2.1% 2.1% 0.5%
Cyclosporin
Mid-High(532.3) 4.7% 9.3% 5.2% 5.8%
High(1213.2) 3.4% 10.3% 5.1% 11.8%
Low(2.5) 4.1% 10.1% 6.3% 2.9%
Mid-Low(8.2) 1% 6.1% 3.8% 2.2%
Tacrolimus
Mid-High(17.4) 3.8% 6% 3.7% 2.5%
High(34.7) 2.1% 1% 0.9% 0.4%
Low(2.5) 7.5% 6.6% 9.3% 1.8%
Mid-Low(10.1) 8.3% 2.8% 9.6% 1.1%
Sirolimus
Mid-High(21.3) 4.3% 9.5% 8.9% 4.1%
High(40.4) 4.8% 3.1% 2.7% 1.3%
Low(2.2) 8% 3.1% 8.3% 6.5%
Mid-Low(4.3) 6% 3.7% 5.5% 2.4%
Everolimus
Mid-High(8.9) 8.4% 7.3% 3.7% 3.9%
High(29.3) 9.2% 1% 5.5% 2.1%
Table 3. Linearity of four Immunosuppressant over 6 calibration levels
Compound Accuracy (range) Coefficient (r2)

Cyclosporin 94.3% - 103.9% 0.998
Tacrolimus 96.3% - 103.3% 0.999
Sirolimus 94.3% - 112.8% 0.998
Everolimus 92.8% - 102.3% 0.998
Using reference calibrators (see Method and Material) it was possible to asses the linearity of the method, and the range
(Table 2).
Conclusions
• Fully Automated sample preparation procedure resulted suitable for the quantitation of Immunosuppressant by
elimination of manual preparation steps.
• The automation of the method increases the analytical performance, reduces the risk for human operators and,
due to the reduced reagent consumption, reduces also the cost of the analysis.
• The application of this approach could help to fill the gap between the more reliable LCMS/MS technique and the
traditional Immunoassays by means of full blown lab LCMSMS automation.
6
References
[1] Therapeutic drug monitoring of immunosuppressants by liquid chromatography–mass spectrometry, Clin Chim
Acta 454 (2016),1-5.
[2] The current role of liquid chromatography-tandem mass spectrometry in therapeutic drug monitoring of
immunosuppressant and antiretroviral drugs, Clinical Biochemistry 44 (2011), 14-20.
Disclaimer: Shimadzu LCMS-8050 CL is registered in the U.S. as a Class I device and is not specifically cleared for
TDM. Nexera X2 UHPLC system and CLAM-2000 are intended for Research Use Only (RUO). Not for use in
For Research Use Only. Not for use in diagnostic procedure. Not available in the USA, Canada, and China.
PO-CON1650E
A Fast LC/MS/MS Method

for High Sensitivity Determination
of Twenty-Six Perfluorocompounds
in Textiles
ASMS 2016 TP 485
Jun Xiang Lee1, Zhe Sun1, Jie Xing1, Jia Yi Tan2*

and Zhaoqi Zhan1
1
Singapore,
2
School of Chemical & Life Sciences, Singapore
Polytechnic, Singapore,
* Student
A Fast LC/MS/MS Method for High Sensitivity Determination
of Twenty-Six Perfluorocompounds in Textiles
Introduction
Perfluorocompounds (PFCs) are widely used in different 1 µg/m2 for PFOS while for Norway, a maximum level of
consumer products including textile coatings for their 1 µg/m2 for PFOA. Hence, there is a need for quantitative
excellent oil and water repellent property. However, analysis of extractable PFOS, PFOA and other PFCs in
researches in recent years have shown that PFCs can various skin-contact consumer products like textiles [2-3].
cause adverse effects to organisms and being persistent in Here, we describe a fast LC/MS/MS method for high
both environment and organism. Hence, industries sensitivity screening and quantitation of 26 PFCs in
started to phase out the use of PFCs 2000 [1]. The textiles on Shimadzu LCMS-8050.
European Union (EU) had announced a maximum level of
Experimental
A total of 26 PFCs and M-PFOA as internal standard (See The supernatant was filtered using 0.22 µm Nylon filter. A
Table 2) were obtained from Sigma Aldrich, Wellington calibration series was prepared mixing 490 µL of the
Laboratories [4] and Apollo Scientific. For convenience, filtrate with 5 µL of 26 PFC mix stock solution and 5 µL of
abbreviation names of the PFCs are used in reference of internal standard. An LCMS-8050 triple quadrupole mass
literature [4]. Textile samples obtained from stores was spectrometer coupled with a Nexera UHPLC system was
cut into smaller pieces and 1 gram of sample was used in this study. A Phenomenex, Kinetex UHPLC column
weighed into a 50 mL polypropylene centrifuge tube. 20 (100 x 2.1 mm, 1.7 µm) was used and a gradient elution
mL of pure methanol was subsequently added. The program was adopted for separation of the 26 PFCs and
samples were then left to sonicate at 50 ºC for 2 hours, 1 internal standard. The detailed conditions are as shown
followed by 5 minutes of centrifugation at 10,000 rpm. in Table 1.
Table 1: Analytical conditions of 26 PFCs and 1 internal standard on LCMS-8050
Column : Kinetex C18 100A (100 x 2.1mm, 1.7µm)

Mobile Phase : A : 5mM Ammonium Formate in water
B : Acetonitrile
Oven Temp. : 40ºC
Elution Mode : Gradient elution, LC program 13.50 minutes
20%B (0.00mins to 0.50mins)
→ 80%B (9.00 mins to 12.00mins)
→ 20%B (12.01mins to 13.50mins)
Interface : ESI
MS mode : Negative
DL Temp. : 250ºC
Nebulizing Gas Flow : N2, 1.5 L/min
Heating Gas Flow : 0 Air, 10 L/min
2

Establishment of MRM method for detection and quantitation of 26 PFCs
The MRM optimisation of the 26 PFCs and IS was carried in presence of matrix. The rest four PFCs, i.e., FOEA,
out using mixed standard sample. Two MRM transitions H4PFUnA, N-MeFOSE and N-EtFOSE exhibit relatively
(if available) were used for each PFC, one as quantifier ion lower sensitivity, with their LOQs at 97~476 pg/mL. The
and the other for confirmation as shown in Table 2. A LOQs for PFOA and PFOS of the current method are 17.7
clothing sample free from the 26 PFCs was used as a pg/mL and 16.1 pg/mL, respectively. The MRM peaks of
blank matrix in preparing post-spiked calibrants for PFOA and PFOS of 50 pg/mL (ppt) spiked in matrix are
calibration curves construction. Each post-spiked calibrant displayed in Figure 2, showing both the quantifier MRM
was injected thrice to obtain the average area for reliable and confirmation MRM of each compound. The
results. Calibration curves were built based on the repeatability of the method was evaluated using
internal standard with good linearity (r2>0.997) for all 26 pre-spiked and post-spiked samples at two
PFCs (Table 3). The calibration curves of PFOA and PFOS concentrations, 50 pg/mL & 1,000 pg/mL. The %RSD
are displayed in Figure 2. As can be seen in Table 3, 22 results of the post-spiked samples are tabulated in Table
out of the 26 PFCs exhibit excellent sensitivity with the 3, indicating good repeatability of the method for all the
LOQ at 4.1~50.2 pg/mL while the LOD at 1.4~16.6 pg/mL 26 PFCs studied.
(x100,000) (x10,000)
PFOA
1.75 7.0
PFBA
PFPA
PFOA
1.50 6.0
5.0
MPFOA
1.25
MPFOA
1.00 4.0
PFOS
PFOS
0.75 3.0
N-EtFOSA
0.50 2.0
0.25 1.0
0.00 0.0
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 min 5.0 7.5 min
Figure 1: MRM chromatograms of 26 PFC mixed standards with 13C-PFOA (IS) spiked in textile blank matrix, 1,000 pg/mL for each compound (left).
Separate display of MRM peaks of PFOA, M-PFOA (IS, 500 pg/mL) and PFOS (Right).
R#1 412.90>169.05 9.86e2 R#1 498.90>98.50 3.19e2

Area Ratio
Area Ratio
8 y = 0.802508x + 0.022041 4 y = 0.405275x + 0.017058

1.2e3
R² = 0.9996488 R = 0.9998244 3.0e3 R² = 0.9989661 R = 0.9994829
Curve Fit: Default (Linear) Curve Fit: Default (Linear)
2.5e3 1.0e3
6 Weighting: Default (1/x) 3 Weighting: Default (1/x)
Zero: Default (Not Forced) Zero: Default (Not Forced)
2.0e3 8.0e2
4 1.5e3 2 6.0e2
1.0e3 4.0e2
2 1
5.0e2 2.0e2
0.0e0 0.0e0
0 0
0 2 4 6 8 10 5.0 5.5 6.0 6.5 0 2 4 6 8 10 6.0 6.5 7.0 7.5
Conc.Ratio Conc.Ratio
Figure 2: Calibration curves and MRM peaks (50 pg/mL or ppt) of PFOA (Left) and PFOS (Right) in blank matrix
3
Table 2: Names and MRM transitions of 26 PFCs & M-PFOA (IS) on LCMS-8050
Quantified MRM Reference MRM

No. Abbr. Name
MRM (m/z) CE (V) MRM (m/z) CE (V)
1 PFBA 213.00>169.05 10 Not Available
2 PFPeA 262.90>219.10 8 Not Available
3 PFBS 298.80>79.90 32 298.80>98.80 27
4 PFHxA 312.90>269.05 9 312.90>119.00 21
5 HPFHpA 345.10>281.00 11 345.10>131.15 23
6 PFHpA 362.90>319.10 9 362.90>168.95 17
7 1H,1H,2H,2H-PFOS9 462.90>407.05 23 426.90>81.00 33
8 PFOA 412.90>369.00 10 412.90>169.05 17
9 PFHxS 398.90>79.90 44 398.90>99.00 33
10 FOEA 477.00>392.90 12 477.00>243.05 23
11 PFNA 462.90>418.95 10 462.90>219.10 17
12 PFHpS 448.90>99.05 40 448.90>79.85 51
13 PF-3,7-DMOA 469.00>269.00 22 469.00>219.10 23
14 PFDA 513.00>469.05 10 513.00>218.90 18
15 PFOS 498.90>79.90 53 498.90>98.50 42
16 H4PFUnA 491.10>367.00 20 491.10>387.00 12
17 PFUdA 563.00>519.00 13 563.00>319.05 19
18 PFDoA 612.90>568.90 12 612.90>169.00 25
19 PFDS 598.80>80.00 55 598.80>98.80 48
20 PFTrDA 663.00>619.00 14 663.00>169.10 32
21 PFTeDA 712.90>668.95 13 712.90>168.90 35
22 FOSA 497.90>77.90 40 Not Available
23 PFDHxA 812.90>768.80 15 812.90>169.10 34
24 N-MeFOSA 512.00>169.10 30 512.00>218.9 26
25 PFODA 912.90>868.7 18 912.90>268.95 35
26 N-EtFOSA 526.00>169.00 30 526.00>219.15 26
IS M-PFOA 416.90>372.00 10 416.90>168.90 17
4
Table 3: Calibration curves and key performance values of the MRM method with M-PFOA as IS
for quantitative determination of 26 PFCs on LCMS-8050 with heated ESI interface
RT Range LOD LOQ RSD (%), n=6

No. Name R2
(min) (pg/mL) (pg/mL) (pg/mL) (50 pg/mL) (1,000 pg/mL)
1 PFBA 1.379 50~5,000 1.000 15.6 47.3 3.5 3.7
2 PFPA 3.063 50~5,000 0.9993 11.5 34.8 6.9 1.0
3 PFBS 4.211 20~5,000 0.9991 6.1 18.5 3.5 3.4
4 PFHxA 4.245 50~5,000 0.9995 16.6 50.2 5.7 3.6
5 HPFHpA 4.353 20~5,000 0.9993 2.8 8.5 4.5 2.2
6 PFHpA 5.004 50~5,000 0.9992 13.8 41.8 9.1 3.9
7 1H,1H,2H,2H-PFOS 5.373 50~5,000 0.9992 14.9 45.3 9.3 5.7
8 PFOA 5.594 20~5,000 0.9996 5.8 17.7 5.9 1.3
9 PFHxS 5.733 50~5,000 0.9992 11.8 35.6 10.7 2.1
10 FOEA 5.896 1,000~5,000 0.9988 246.3 746.3 N.A. 12.6
11 PFNA 6.112 50~5,000 0.9996 13.3 40.2 6.3 6.7
12 PFHpS 6.285 50~5,000 0.9991 8.7 26.3 9.6 3.5
13 PF-3,7-DMOA 6.308 50~5,000 0.9996 15.3 46.5 14.4 4.1
14 PFDA 6.592 50~5,000 0.9998 9.6 29.0 7.8 7.2
15 PFOS 6.793 20~5,000 0.9990 5.3 16.1 12.8 6.1
16 H4PFUnA 6.541 500~5,000 0.9973 82.3 249.3 N.A. 12.4
17 PFUdA 7.045 20~5,000 0.9995 5.3 16.0 11.1 4.8
18 PFDoA 7.484 50~5,000 0.9990 11.5 35.0 10.4 3.4
19 PFDS 7.721 50~5,000 0.9991 15.3 46.2 8.0 3.6
20 PFTrDA 7.928 50~5,000 0.9991 15.2 46.0 13.5 8.3
21 PFTeDA 8.372 50~5,000 0.9991 13.4 40.6 7.6 8.8
22 FOSA 8.42 50~5,000 0.9995 11.6 35.0 14 8.2
23 PFDHxA 9.198 20~5,000 0.9985 1.4 4.1 14.7 2.5
24 N-MeFOSA 9.955 200~5,000 0.9986 60.8 184.1 NA 6.2
25 PFODA 9.98 20~5,000 0.9982 5.0 15.1 11 1.7
26 N-EtFOSA 10.369 100~5,000 0.9984 32.1 97.2 N.A. 6.0
IS M-PFOA 5.556 500 Not Available
5
Matrix effect and recovery

The matrix effect and recovery of the method for the 26 samples at two concentrations, 50 pg/mL and 1,000
PFCs were evaluated at two concentrations, 50 pg/mL and pg/mL. As shown in Table 4, the recoveries of the 22 PFCs
1,000 pg/mL. The results are shown in Table 4. Most PFCs which were detectable at 50 pg/mL level were determined
exhibited matrix effect at 60.8 %~143.9 % for 50 pg/mL to be 95.8 %~130.4 %. For 1,000 pg/mL level, good
and 74.0 %~ 142.3 % for 1,000 ng/mL. However, two recoveries of all the PFCs except H4PFUnA were obtained
PFCs, namely FOSA and N-MeFOSE, exhibited strong ion at 96.7 %~125.5 %, respectively. The overly high recovery
enhancement effect (measured matrix effect at 193.3 of H4PFUnA (143.8 %) may be from interferences due to
%~247.9 % for both concentration). The recovery of the its relatively low sensitivity.
method was determined from pre-spiked and post-spiked
Table 4: Matrix effect and recovery of the 26 PFCs compounds spiked in matrix,
MeOH extract of blank textile (n=3)
Matrix effect (%) Recovery (%)

Peak No. Name
(50 pg/mL) (1,000 pg/mL) (50 pg/mL) (1,000 pg/mL)
1 PFBA 143.9 90.1 121.6 102.2
2 PFPA 118.2 95.3 128.6 108.4
3 PFBS 86.2 85.1 124.5 109.5
4 PFHxA 103.6 89.7 128.1 109
5 HPFHpA 105.2 82.8 125.4 114.4
6 PFHpA 92.6 89.8 125.5 113.9
7 1H,1H,2H,2H-PFOS 60.8 99.3 102 120.8
8 PFOA 110.9 87.5 127.5 112.0
9 PFHxS 124.8 80 95.8 107.6
10 FOEA NA 105.3 NA 108.8
11 PFNA 110.9 91.4 105 108.5
12 PFHpS 105.6 80.3 98.2 108.5
13 PF-3,7-DMOA 118.3 86.8 124.3 112.2
14 PFDA 107.8 91.3 98.4 106.7
15 PFOS 135.4 83.1 98.9 105.7
16 H4PFUnA NA 80.2 NA 143.8
17 PFUdA 86.2 88.5 98.2 105.6
18 PFDoA 75.7 75.7 113.1 112.2
19 PFDS 105.7 88.8 120.6 111.6
20 PFTrDA 74.1 74 128.5 111.8
21 PFTeDA 97.2 98.3 130.4 109.7
22 FOSA 247.9 193.3 106.6 125.5
23 PFDHxA 108.4 97.9 115.6 103.3
24 N-MeFOSA-M NA 209.7 NA 102.8
25 PFODA 91.4 99.8 122.6 103.2
26 N-EtFOSA-M NA 142.3 NA 96.7
6
Screening and quantitation of targeted 26 PFCs in textile samples

Five clothing samples were obtained from local stores and surface, the extraction was carried out by sonication at
labelled as BS, GS, YS, WS and BR. Using the method 50 ºC for 2 hours [2-3]. Duplicates of every sample were
established, the samples were analysed for screening and prepared and analysed for accurate and reliable results.
quantitation of the targeted 26 PFCs. Following the sample The analysis results of the five samples reveal that only
extraction method described in experimental section, the IS sample GS showed presence of PFOA of 650 pg/g (0.65
(M-PFOA, equivalent 500 pg/mL in final extract) was spiked ppb). The targeted 26 PFCs were not detected in the other
into the samples at the beginning of the MeOH extraction four samples. The MS chromatogram and the detected
step before pre-treatment. For effective extraction of PFCs PFOA peak with IS peak of Sample GS are displayed in
which may be present in the coating layer on the textile Figure 3.
(x10,000) (x10,000)
MPFOA
3.0
M-PFOA (500 ppt)
4.5
4.0 2.5
3.5
2.0
3.0
PFOA
2.5 1.5
2.0
1.0
1.5
1.0 0.5
0.5 PFOA
0.0
0.0
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 min 5.0 min
Figure 3: MRM chromatograms of textile sample GS with spiked internal standard (M-PFOA, 500 pg/mL) of the MeOH extract
for screening and quantitation of 26 targeted PFCs. Trace level of PFOA was found in the sample.
Conclusions
A sensitive and reliable LC/MS/MS method has been ng/mL for PFOA and PFOS in textile matrix, respectively.
developed for screening and quantitation of 26 PFCs The method performance which includes sensitivity,
including PFOA and PFOS on Shimadzu LCMS-8050 UFMS linearity, repeatability, matrix effect and recovery were
system. The method adopted M-PFOA (13C-PFOA) as evaluated. The results indicate that the method is feasible
internal standard to enhance the reliability for screening and reliable for determination of 26 targeted PFCs,
analysis of unknown sample. The LOQs of the MRM especially PFOA and PFOS in different textile samples.
method achieve extremely low level, e.g., 17.7 and 16.1
7
References
1. M. M. Schultz, D. F. Barofsky, J. A.. Field, Fluorinated Alkyl substances, Environ. Eng. Sci. 20 (2003) 487-501.
2. M. P. Mawn, R. G. McKay, T. W. Ryan, B. Szostek, C. R. Powley and R. C. Buck, The Analyst, 130 (2005) 670-678.
3. I. van der Veen, J. M. Weiss, A. C. Hanning, , J. de Boer and P. E. Leonards, Talanta 147 (2016), 8-15.
4. Wellington Laboratories, “Quick Reference Guide for Perfluoroalkyl Compounds”,
http://www.well-labs.com/docs/pfc_reference_handling_guide.pdf

PO-CON1681E
MRM-based assay for identification

of potential protein biomarkers
in Meningioma patients
ASMS 2016 WP 094
Shuvolina Mukherjee(1), Ajit Datar(2), Rashi Kochhar(2),

Vedita Anand Singh(1), Nikita Gahoi(1),
Saicharan Ghantasala(1), Aliasgar Moiyadi(3),
Epari Sridhar(3), Sanjeeva Srivastava(1)
(1) Department of Biosciences and Bioengineering,
Indian Institute of Technology Bombay, Powai,
Mumbai 400076, India.
(2) Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh
(3) Department of Neurosurgery, TMH, Parel,
Mumbai - 400 012, Maharashtra, India.
MRM-based assay for identification of potential protein
biomarkers in Meningioma patients
Introduction
Meningiomas are tumors originating from meningeal for meningioma development and progression has
layers of brain and spinal cord, comprising nearly 30 % of pointed out involvement of tumor suppressors:
the primary Central Nervous System (CNS) tumors. The TIMP1,TIMP3 among several others [1,2].
2007 WHO classification showed 15 histopathological Subsequently, proteomic alterations in meningiomas have
subtypes of meningioma and 3 different histological also been looked into to identify the exact players that
grades namely the grade I, II and III. However, there is an are involved in meningioma pathobiology[3,4]. Hence,
enormous amount of heterogeneity among the grades MRM based methodology was developed to study these
thus pointing out to a need of grade specific biomarkers potential protein biomarkers from meningioma patients
for diagnosis and better prognosis of the tumor. post surgical resection so as to gain mechanistic insights
Molecular studies to decipher the molecular mechanisms for therapeutic intervention.
MGI MGII MGIII
Figure 1: Radiological image of Meningioma grades.

(Image obtained from Johns Hopkins Medicine,
Department of Neurology and Neurosurgery)
Workflow and key findings of the discovery phase

In the discovery phase global tissue proteomic profiling of the samples was done using QTOF and Q-Exactive mass
spectrometric platform which yielded 4308 proteins (1% FDR) among which 2367 were found to be differentially
expressed (>2 peptides and 1.5 fold in at least one grade).
Figure 2: Schematic depicting the discovery based workflow for global tissue proteomic profiling in meningiomas [5]
2
The differentially expressed proteins were part of several signaling cascades namely Integrin, Wnt and EGF receptor
signaling. Many of the differentially expressed proteins were also found to be part of interaction networks. Looking into
these abberated proteins could yield novel insights into mechanistics of the tumor progression.
Panel of protein selected from discovery phase

Based on the initial i-TRAQ tissue proteomic analysis, few candidates were selected for MRM based assay to validate
their presence in different patients of meningioma (Table 1)
Table 1. Panel of differentially expressed proteins in meningiomas that were selected from discovery phase[5]
Fold Change Fold Change Fold Change Up/Down

Accession Description
(MGI) (MGII) (MGIII) regulated
P08670 Vimentin 3.284 1.95 3.508 Up

P07355 Annexin A2 4.883 2.76 3.746 Up
Q13228 Selenium Binding Protein-1 1.54 2.14 3.62 Up
P29762 Cellular Retinoic Acid Binding Protein-1 1.724 4.455 2.815 Up
Method of Analysis
Figure 3. LCMS-8050 triple quadrupole mass spectrometer by Shimadzu Figure 4. Heated ESI probe
LCMS-8050 triple quadrupole mass spectrometer by In order to improve ionization efficiency, the newly
Shimadzu (shown in Figure 3), sets a new benchmark in developed heated ESI probe (shown in Figure 4) combines
triple quadrupole technology with an unsurpassed high-temperature gas with the nebulizer spray, assisting
sensitivity (UFsensitivity), ultra fast scanning speed of in the desolvation of large droplets and enhancing
30,000 u/sec (UFscanning) and polarity switching speed ionization. This development allows high-sensitivity
of 5 msec (UFswitching). This system ensures highest analysis of a wide range of target compounds with
quality of data, with very high degree of reliability. considerable reduction in background.
3
Figure 5. Schematic of experimental strategy from discovery phase to validation using

LCMS-8050 triple quadrupole mass spectrometer (Shimadzu)
Tissue samples were procured from post surgical resection of meningioma patients. These tissues were subjected to
protein extraction in a mass spectrometry compatible buffer (0.5 M TEAB). Protein concentration was measured using
Bradford Assay with BSA (Bovine Serum Albumin) as standard.
LC/MS/MS analysis
Column : Shim-pack XR-ODS II (75 mm L x 3 mm I.D.; 2.2 µm)

Guard column : Phenomenex SecurityGuard ULTRA cartridge
Mobile phase : A: 0.1 % formic acid in water
Gradient program (B %) : 0–1.5 min → 3 (%); 1.5–30.0 min → 3-50 (%); 30.0–31.0 min →
50-95 (%); 31.0–35.0 min → 95 (%); 35.0–36.0 min → 95-3 (%);
36.0–40.0 min → 3 (%)
4
Results
The selected peptides from the discovery phase (Table 1) peptides with respect to ACTB.
were screened in samples from meningioma patients Furthermore, based on the initial screening good
using MRM based approach. Along with the selected transitions of several clinically relevant peptides like
peptides, the method was also optimized for monitoring vimentin, cellular retinoic acid binding protein and
transitions for beta-actin (ACTB) which is an endogenous selenium binding protein were successfully optimized and
peptide. This would help in development of an assay to monitored (Figure 6)
monitor differential expression of clinically significant
Table 2. List of peptides that were screened via MRM assay and monitored across patients
Protein Name/ Abbreviations Sequences monitored Retantion Time Monitored (min)
• R.SLYASSPGGVYATR.S [50, 63] 9.8

Vimentin (VIM) • R.QDVDNASLAR.L [207, 216] 6.8
• R.QQYESVAAK.N [273, 281] 5.7
Cellular Retinoic Acid Binding Protein 1 • R.SSENFDELLK.A [11, 20] 12.1-12.2

(RABP-1) • R.QDGDQFYIK.T [45, 53] 14.1
Selenium Binding Protein-1 • R.QYDISDPQRPR.L [333, 343] 7.3

(SBP1) • R.LTGQLFLGGSIVK.G [344, 356] 14.2-14.3
Arginino succinate synthase • R.IDIVENR.F [265,271] 9.3-9.5

(ASSY) • KGQVYILGT.E[355,362] 10.2-10.9
• KAYTNFDAER.D[28,36] 8.1-8.2
Annexin A2 (ANXA2) • R.DALNIETAIK[37,46] 12.2-12.3
• K.TPAQYDASELK.A [104, 114] 8.1-8.3
Beta-Actin (ACTB) • KR.GYSFTTTAER[196,205] 8.7-8.8
• HLVDEPQNLIK 9.9-10
Bovine Serum Albumin (BSA)
• AEFVEVTK 8.7-8.8
(Was used to spike the samples)
• GACLLPK 8.3-8.4
5
Beta-actin taken as endogenous control across samples R.GYSFTTTAER.E [196, 205]
Cellular Retinoic Acid Binding Protein 1 (RABP-1) R.SSENFDELLK.A [11, 20]
Selenium Binding Protein-1 R.LTGQLFLGGSIVK.G [344, 356]
6
Vimentin R.QQYESVAAK.N [273, 281]
Annexin A2 R.DALNIETAIK.T [37, 46]
Figure 6. Transitions for various peptides that were screened from meningioma patients also including beta-actin and
BSA which were used as endogenous control and internal standard respectively.
7
Conclusion
• The current study attempts to establish a method to screen potential protein biomarkers to analytically distinguish
and confirm various grades of meningioma patients. This can help in constructing a panel to get insight into the
tumor pathobiology and recurrence rates.
• Several peptides that were found to be differentially expressed in the discovery phase were detected using the
MRM assay. These peptides when screened in larger cohort with synthetically labelled peptides would enable
detection of absolute levels in the patient samples and substantiate the clinical relevance of these findings.
• The MRM based assays along with parallel proteomic profiling and histopathological studies can lead to accurate
determination of grades of meningioma thereby limiting diagnostic dilemma
References
[1] Norden, A.D., J. Drappatz, and P.Y. Wen, Advances in meningioma therapy. Curr Neurol Neurosci Rep 9(3)
(2009), 231-240.
[2] Miller, R., Jr., et al., Molecular Targets and Treatment of Meningioma. J Neurol Neurosurg (2014).
[3] Kocevar, N., P. Hudler, and R. Komel, The progress of proteomic approaches in searching for cancer biomarkers.
N Biotechnol, (2013), 319-326.
[4] Okamoto, H., et al., Comparative proteomic profiles of meningioma subtypes. Cancer Res 66(20) (2006),
10199-204.
[5] Sharma, S et al., Multipronged quantitative proteomic analyses indicate modulation of various signal
transduction pathways in human meningiomas. Proteomics 15(2-3) (2015), 394-407.
Disclaimer : LCMS-8050 is intended for Research Use Only (RUO). Not for use in diagnostic procedures.
PO-CON1683E
Development of MRM assay for

the determination of potential
biomarkers in case of severe
vivax malaria
ASMS 2016 WP 096
Sandip Kumar Patel(1), Shailendra Rane(2), Ajit Datar(2),

Swati Patankar(1) and Sanjeeva Srivastava(1)*
Indian Institute of Technology Bombay, Powai,
(2) Shimadzu Analytical (India) Pvt. Ltd.,
1 A/B Rushabh Chambers, Makwana Road,
Marol, Andheri (E), Mumbai-400059,
Maharashtra, India.
Development of MRM assay for the determination of
potential biomarkers in case of severe vivax malaria
Introduction
Plasmodium vivax malaria has the most widespread global is no more considered benign; causes of its severity and
incidence (Figure 1A) and its diagnosis, treatment and the associated mechanism still remains obscure. In this
control is much more challenging than P. falciparum study, serum samples from patients diagnosed with
malaria[1]. P. vivax can cause severe and fatal Severe Vivax Malaria (SVM) and Healthy Controls (HC)
manifestations (Figure 1B) even at a very low parasite from different endemic regions of India were investigated
biomass[2]. However, there is no precise, systematic global using quantitative MRM approach.
assessment of endemicity for vivax malaria. Vivax malaria
Figure 1A. Vivax malaria scenario worldwide and India Figure 1B. Life cycle of the malaria parasite
Mechanisms
triggering
transition
Non-severe vivax malaria Severe vivax malaria
Fever with chill, shivering, Coma, acidotic breathing,
headache circulatory collapse or shock,
acute kidney injury and
abnormal bleeding
2
Method of analysis
Sample collection and diagnosis
Healthy Thick
NSVM Ts)
(RD
cTests
Thin smear osti
iagn
id D
Rap
SVM
Blood
collection
Figure 2A. Representation of sample types, sampling strategy and diagnostic tools employed to identify and stratify vivax malaria severity
Discovery phase analysis using quantitative proteomics

Comparative proteomic analysis of vivax malaria to healthy controls was performed using two complementary
quantitative proteomic approaches; 2D-DIGE along with MALDI-TOF/TOF and iTRAQ based quantitative proteomics in
combination with ESI-Q-TOF LC/MS/MS as shown in Figure 2B.
SVM NSVM Healthy

Cy5
Healthy Trypsin digestion

Cy 3
Overlay image
MS/MS Analysis
Cy3
NSVM/SVM Cy 5 iTRAQ Labelling

Liquid chromatography
Mix
Cy2
Data analysis
Cy 2 Mix
Internal control
Off-gel fractionation
2D-DIGE iTRAQ
Figure 2B. Strategy employed to determine altered proteome in vivax malaria
3
LC/MS/MS analysis
Samples were analyzed using Ultra High Performance Liquid Chromatography (UHPLC) Nexera coupled with LCMS-8050
triple quadrupole system (Shimadzu Corporation, Japan) as shown in Figure 2C.
Figure 2C. LCMS-8050 triple quadrupole mass spectrometer by Shimadzu Figure 2D. Heated ESI probe
LCMS-8050, sets a new benchmark in triple quadrupole In order to improve ionization efficiency, the newly
technology with an unsurpassed sensitivity (UFsensitivity), developed heated ESI probe (shown in Figure 2D)
ultra fast scanning speed of 30,000 u/sec (UFscanning) combines high-temperature gas with the nebulizer spray,
and polarity switching speed of 5 msec (UFswitching). assisting in the desolvation of large droplets and
This system ensures highest quality of data, with very high enhancing ionization. The details of analytical conditions
degree of reliability. are given in Table 1.
Table 1. LC/MS/MS conditions

Gradient program (B %) : 0.0-1.5 min → 3 (%); 1.5-30.0 min → 3-40 (%); 30.0-32.0 min →
40-95 (%); 32.0-36.0 min → 95 (%); 36.0-37.0 min → 95-3 (%);
37.0-40.0 min → 3 (%)
Zero air flow : Heating gas 15 L/min
Interface 400 °C
4
Results
Discovery phase
2D-DIGE (gel-based) and 4-plex iTRAQ-based quantitative C4-B, C-reactive protein, hemopexin, plasminogen and
proteomics analysis (gel free) of HC, Non Severe Vivax vitronectin were significantly modulated (fold change <
Malaria (NSVM) and SVM identified 279 proteins with 95 1.5, with < 2 peptide and 1 % FDR) in malaria patients.
% confidence interval and 1 % FDR. The overall Representative spectra along with the respective label
distribution patterns of proteins expressed differentially in intensity of a few selected proteins is shown in Figure 3C.
SVM and NSVM compared to HC is displayed in Figures Interestingly, superoxide dismutase, vitronectin, titin,
3A and 3B respectively. Alpha-2-macroglobulin, carbonic anhydride and nebulin may be considered as
apolipoprotein A-I, apolipoprotein B, apolipoprotein E, potential prognostic markers to assess severity in vivax
ceruloplasmin, clusterin, complement C3, complement malaria.
A C
Vitronectin Haptoglobin
CRP
(DWHGVPGQVDAA) (DIAPTLTLYVGK)
Adjusted p-value (-Log10)
APO A-I
SAA1
ALB APO E
HP HPX
SOD1 TTN
PLS
VTN CP
B SVM:HC (Log 2)
SAA1 Apolipoprotein E Serum amyloid A

Adjusted p-value (-Log10)
(SWFEPLVED) (SGKDPNHF)
APO E
HP HPX
APO A-I
CP
ALB PLS
SAA2
VTN
NSVM:HC (Log 2)
Figure 3. A) Volcano plot of SVM vs. HC; B) Volcano plot of NSVM vs HC; C) iTRAQ reporter ion intensities
for representative peptides in HC, NSVM and SVM cohort of malaria patients
Validation of selected protein candidates

Novel biomarker candidates were selected for validation LabSolutions software (Shimadzu Corporation, Japan) for
using MRM based assay for relative quantitation. The screening. After screening, for each protein, 2-3 unique
FASTA sequences were fed into the Skyline (MacCoss Lab peptides with 3-4 MRM transitions were selected for
Software- University of Washington) software to generate further studies (Figure 4). Data analysis was performed
in silico trypsin digested peptides and respective MRM using skyline to identify significantly altered protein which
transitions. Precursor ions of +2 and+3 charge and discriminated HC with SVM. Standards of few proteins
product ions of +1 and +2 charge were taken into were also analysed to get further confidence in retention
consideration. The transition lists were imported into time and transition selection.
5
(x100,000)
3.5
3.0
2.5
Intensity
2.0
1.5
1.0
0.5
0.0
5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min
Retention time
Figure 4. MRM chromatogram of 35 peptides representing 12 proteins
Representative MRM chromatograms of standard ceruloplasmin and samples (SVM and HC) are shown in Figure 5A.
Disease Healthy
STD
Library Disease Healthy STD Disease Healthy STD
Figure 5A. Up-regulation of DIFTGLIGPMK peptide unique to ceruloplasmin
6
Figure 5B. Trends of a 12 selected differentially expressed proteins in severe vivax malaria patients using MRM
Figure 5C. Trends of a 6 selected differentially expressed proteins in severe vivax malaria patients using ELISA
7
Conclusion
• This is first systemic report to distinguish the severity in vivax malaria.
• MRM assay was developed for few proteins as a predictive markers for vivax malaria.
• Lipid metabolism related proteins like apolipoprotein A-I, apolipoprotein B, apolipoprotein E were found to be
altered in malaria patients.
References
[1] P L Alonso et al., Nature Medicine, Volume 19, (2013), 150–155.
[2] Anstey N M, Cell Press, Volume 25, (2009), 220–227.
* Correspondence: Prof. Sanjeeva Srivastava, Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai
400076, India E-mail: sanjeeva@iitb.ac.in ; Phone: +91-22-2576-7779; Fax: +91-22-2572-3480
PO-CON1635E
Simultaneous analysis of supernatant

components of cell culture using
triple quadrupole LC/MS/MS
ASMS 2016 WP 160
Qiaoxia Liu1, Hongyuan Hao1, Yuling Song1 , Qiang Li1,

Zhao Liu1, Taohong Huang1 , Yuki Hashi1
1
Shimadzu (China) Co., LTD. Shanghai Branch 1,
West Huaihai Road, Shanghai 200052, China
Simultaneous analysis of supernatant components of cell
culture using triple quadrupole LC/MS/MS
Introduction
Fermentation for the production of biofuels, altogether. To meet the demand for comprehensive
biopharmaceutics or other compounds requires routine analysis of medium components, we optimized analytical
monitoring of medium conditions such as pH, dissolved conditions and developed “Method Package for Cell
gas, carbon source and nitrogen source for optimization Culture Profiling” for monitoring relative abundance of
and control of the fermentation process. However, 95 compounds. Using this method package, we
culture media also consist of various other biologically demonstrated the change in abundance of culture
important compounds such as vitamins, nucleic acids and supernatant (bacteria and streptomyces cells) components
other primary metabolites, which would lead to more for over the culture periods.
detailed understanding of the bioprocess if monitored
Table 1. List of 96 compounds
Internal Standard Amino Acid Ornithine Others

2-Isopropylmalic acid 2-Aminoadipic acid Oxidized glutathione 2-Aminoethanol
Sugars 4-Aminobutyric acid Phenylalanine 2-Ketoisovaleric acid
Gluconic acid 4-Hydroxyproline Pipecolic acid 3-Methyl-2-oxovaleric acid
Glucosamine 5-Glutamylcysteine Proline 4-Hydroxyphenyllactic acid
Hexose (Glucose) 5-Oxoproline Serine Citric acid
Sucrose Alanine Threonine Ethylenediamine
Threonic acid Alanyl-glutamine Tryptophan Fumaric acid
Nucleic acid Arginine Tyrosine Glyceric acid
Adenine Asparagine Valine Histamine
Adenosine Aspartic acid Vitamins Isocitric acid
Adenosine monophosphate Citrulline 4-Aminobenzoic acid Lactic acid
Cytidine Cystathionine Ascorbic acid Malic acid
Cytidine monophosphate Cysteine Ascorbic acid 2-phosphate O-Phosphoethanolamine
Deoxycytidine Cystine Biotin Putrescine
Guanine Glutamic acid Choline Pyruvic acid
Guanosine Glutamine Cyanocobalamin Succinic acid
Guanosine monophosphate Glutathione Ergocalciferol
Hypoxanthine Glycine Folic acid
Inosine Glycyl-glutamine Folinic acid
Thymidine Histidine Lipoic acid
Thymine Isoleucine Niacinamide
Uracil Kynurenine Nicotinic acid
Uric acid Leucine Pantothenic acid
Uridine Lysine Pyridoxal
Xanthine Methionine Pyridoxine
Xanthosine Methionine sulfoxide Riboflavin
Antibiotics N-Acetylaspartic acid Tocopherol acetate
Penicillin G N-Acetylcysteine
2

Pretreatment: The culture solution was taken from a was thoroughly agitated. and then analyzed by
culture dish or flask, and then was separated by LC-MS/MS.
centrifugation. The culture supernatant was transferred to Instrument: As an LC-MS/MS system, UHPLC was coupled
a new tube and then the internal standard solution was to triple quadrupole mass spectrometer (Nexera MP with
added into the tube. After that acetonitrile was added LCMS-8050, Shimadzu Corporation, Kyoto, Japan).
into the tube and the solution was thoroughly agitated. LC-MS/MS with electrospray ionization was operated in
After centrifugal separation centrifuged supernatant was multiple-reaction-monitoring (MRM) mode with ultra-fast
transfer to a new tube with water and then the solution polarity switching.

• 5 msec
Ultra Fast MRM
Figure 1. LCMS-8050 triple quadrupole mass spectrometer
Result
In order to achieve appropriate signals for 95 components performed at a rate of 17 minutes per cycle. Bacteria and
in high concentration (glutamine, glucose, etc.) and in streptomyces samples were investigated.
trace amounts(such as vitamins), ion source parameters, Figure 2 showed that it was possible to measure a wide
voltage, data acquisition time and LC condition were concentration range components in culture supernatant
optimized. Under optimized condition, all the 95 using single method.
compounds had good signals, and the analysis can be
UHPLC conditions (Nexera MP system)
Column : see “Method Package for Cell Culture Profiling”

Mobile phase A : 0.1% formic acid in H2O
B : 0.1% formic acid in acetonitrile
Time program : see “Method Package for Cell Culture Profiling”
Injection vol. : 1 μL
Ionization : ESI, Positive/Negative MRM mode

MRM parameters : see “Method Package for Cell Culture Profiling”
3
(x10,000,000)
2.00
(x1,000,000)
1.3
1.2
1.75 1.1
1.0
0.9
0.8
1.50 0.7
0.6
0.5
0.4
1.25 0.3
0.2
0.1
0.0
1.00
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 min
0.75
0.50
0.25
0.00
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 min
Figure 2. Mass chromatogram of bacteria culture supernatant
4
The change trend of compounds

in the bacteria culture supernatant over time
The results of culture supernatant of the bacteria showed of 2-aminoethanol increased with time, the amount of
the change trend of compounds in the supernatant deoxycytidine increased from 0 hour to 10 hours, and
during the culture period of 12 hours. Such as the began to decrease after 10 hours; the amount of biotin
amount of succinic acid decreased with time; the amount showed no change (Fig.3-6).
1.0 0.8
Succinic acid 2-Aminoethanol
0.7
0.8
0.6
0.6 0.5
Area ratio
Area ratio
0.4
0.4
0.3
0.2
0.2
0.0 0.1
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Time (h) Time (h)
Figure 3. Area ratio of succinic acid change trend Figure 4. Area ratio of 2-Aminoethanol change trend
1.0 2.0
Deoxycytidine Biotin
0.8 1.6
0.6
1.2
Area ratio
Area ratio
0.4
0.8
0.2
0.4
0.0
0.0
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Time (h) Time (h)
Figure 5. Area ratio of Deoxycytidine change trend Figure 6. Area ratio of Biotin change trend
5
The change trend of compounds

in the streptomyces culture supernatant over time
The results of culture supernatant of Streptomyces the amount of citrulline sharply decreased from 0 hour to
showed the change trend of compounds in the 72 hours, and began to increase after 72 hours; the
supernatant during the culture period of 120 hours. Such amount of cytidine increased from 0 hour to 86 hours,
as the amount of alanine sharply decreased from 0 hour and began to decrease after 86 hours; the amount of
to 72 hours, and stayed almost the same after 72 hours; gluconic acid increased with time; (Fig.7-10).
10 30
Alanine Citrulline
8 25
20
6
Area ratio
Area ratio
15
4
10
2
5
0
0
50 60 70 80 90 100 110 50 60 70 80 90 100 110
Time (h) Time (h)
Figure 7. Area ratio of Alanine change trend Figure 8. Area ratio of Citrulline change trend
35
Cytidine 0.24 Gluconic acid
30
25 0.21
20 0.18
Area ratio
Area ratio
15 0.15
10
0.12
5
0.09
0
0.06
50 60 70 80 90 100 110 50 60 70 80 90 100 110
Time (h) Time (h)
Figure 9. Area ratio of Cytidine change trend Figure 10. Area ratio of Gluconic acid change trend
6
Conclusions
“Method Package for Cell Culture Profiling” enables cells. And the optimized method realizes simultaneous
users to analyze simultaneously multicomponents of analysis in 17 minutes that include chromatographic time
culture supernatant using UHPLC coupled with triple and column equilibration time. This package supports
quadrupole mass spectrometry. simultaneous analysis of high concentration components
This package covers analysis method not only for basal and trace components in single analysis.
medium of cell culture but also metabolites secreted by

PO-CON1684E
Development of GeLC-MRM approach

to study carbon concentrating
mechanism during nitrogen
starvation in Chlorella sp FC2 IITG
ASMS 2016 WP 189
Vineeta Rai(1), Deepti Bhandarkar(2), Ajit Datar(2),

Sanjeeva Srivastava(1)
Indian Institute of Technology Bombay, Mumbai-400076,
Maharashtra, India.
(2) Shimadzu Analytical (India) Pvt. Ltd.,
1 A/B Rushabh Chambers, Makwana Road,
Marol, Andheri (E), Mumbai-400059,
Maharashtra, India.
Development of GeLC-MRM approach to study carbon
concentrating mechanism during nitrogen starvation
in Chlorella sp FC2 IITG
Introduction
The need for sustainable fuel resource has recurred the signals for metabolic pathway re-organization including
interest of biotechnologists and industrialists in nitrogen assimilation, altered photosynthetic activity,
generating economically viable biofuels. Algae is cellular division, carbon fixation, and lipid biosynthesis
preferred to other biofuel source viz. first-generation and (TAG) in algae. However the precise molecular
second-generation due to several rationales (Figure 1)[1]. mechanism is only partially known. Therefore the present
Algal lipids consisting of triacylglycerol (TAG) has the study was undertaken to confirm proteins involved in
potential to be converted into biodiesel[2]. Globally, distinct temporal TAG accumulation toward N-starvation
nitrogen starvation (N-starvation) leads to disproportion in using shotgun and targeted proteomics.
carbon-to-nitrogen ratio, thereby inducing alarming
Figure 1. Evolutionary trends in biofuel production
Method of analysis
Shotgun proteomics: DIGE and iTRAQ-coupled with LC/MS/MS
Chlorella sp FC2 IITG (FC2) was grown under N-starvation 2016; containing 9831 sequence for C. variabilis and
for three different time-points viz. 40 h, 88 h and 120 h 7001 sequence for C. protothecoides) downloaded from
whereas 0 h sample was used as control. Difference In Uniprot and integrated into the Spectrum Mill search
Gel Electrophoresis (DIGE) and Isobaric Tags for Relative engine (version Rev B.04.00). In total 137 significant
and Absolute Quantitation (iTRAQ) coupled with high proteins were filtered using 1 % False Discovery Rate
resolution mass spectrometry was employed for shotgun (FDR) and consistent in at least two biological replicates
proteomics as shown in Figure 2. Obtained spectra were with ≥ 1.2-fold change. Few selected proteins were
matched against Uniprot_Chlorella sp. (dated 30th April further validated using immuno-blotting and MRM assays.
2
GeLC-MRM assay
At least three unique peptides for each of the six selected processed similarly and used as an internal standard. 25
proteins ranging in length from 8 to 20 amino acids and peptides were optimized for six selected proteins and
containing K/R tryptic ends with no miss-cleavages were BSA. Equal amount of samples (spiked with BSA) were
chosen using Skyline v3.5. Unique peptides which were loaded on SDS-PAGE, gel-slices were cut, followed by
observed in iTRAQ data were prioritized during the in-gel digestion and analysed on LCMS-8050 a triple
peptide selection. Bovine Serum Albumin (BSA) was quadrupole from Shimadzu Corporation, Japan.
Figure 2. Schematic representation of the experimental strategy used for comparative

analysis of differentially expressed Chlorella sp FC2 IITG proteome
LC/MS/MS analysis
FC2 grown under N-starvation for three different time-points (40 h, 88 h and 120 h) and 0 h control samples were
analyzed in Electro Spray Ionization (ESI) mode using Ultra High Performance Liquid Chromatography (UHPLC) Nexera
coupled with LCMS-8050. The details of analytical conditions are given in Table 1.
Figure 3. LCMS-8050 triple quadrupole mass spectrometer by Shimadzu Figure 4. Heated ESI probe
3
LCMS-8050 triple quadrupole mass spectrometer by In order to improve ionization efficiency, the newly
Shimadzu (shown in Figure 3), sets a new benchmark in developed heated ESI probe (shown in Figure 4) combines
triple quadrupole technology with an unsurpassed high-temperature gas with the nebulizer spray, assisting
sensitivity (UFsensitivity), ultra fast scanning speed of in the desolvation of large droplets and enhancing
30,000 u/sec (UFscanning) and polarity switching speed ionization. This development allows high-sensitivity
of 5 msec (UFswitching). This system ensures highest analysis of a wide range of target compounds with
quality of data, with very high degree of reliability. considerable reduction in background.
Table 1. LC/MS/MS analysis conditions

Gradient program (B %) : 0–1.5 min → 3 (%); 1.5–33.0 min → 3-60 (%); 33.0–34.0 min →
60-95 (%); 34.0–36.0 min → 95 (%); 36.0–36.1 min → 95-3 (%);
36.1–40.0 min → 3 (%)
Zero air flow : Heating gas 15 L/min
Interface 400 °C
Results
FC2 subjected to N-starvation for different time points; 40 complement iTRAQ data, in which 7 out of 13 proteins
h, 88 h and 120 h was compared to unstressed controls identified were consistent.
(0 h) for proteomics analysis. Of approximately 600 Based on the observations from the shotgun proteomic
proteins identified, 137 proteins were significantly studies, 6 proteins were selected for MRM assays as
modulated under N-starvation. 66 proteins were mentioned in Table 2. Representative MRM results
expressed globally in a time-independent manner, while highlighting up or down regulation of some of the
others served as a linker for adaptive transition from one proteins are shown in Figures 6 and 7. Further, western
time-point to other. For preliminary study, 59 proteins blotting of 3 proteins were performed to complement
including well-known and novel N-starvation responsive MRM results.
proteins could be annotated. DIGE was used to
4
Table 2. List of 6 proteins selected for MRM assay based on iTRAQ study, respective expression levels during different N-starvation
time-points (40 h, 88 h and 120 h) as compared to unstressed control (0 h) and the optimized peptides
No of Unique Uniprot iTRAQ Peptide sequence

Protein name
Peptides Accession No 40 h vs 0 h 88 h vs 0 h 120 h vs 0 h optimized
DQAAAGMGIYGPR;
Sedoheptulose 1,7-
2 E1Z6L2 0.437 ± 0.19 0.638 ± 0.15 0.723 ± 0.09 ILFEVAPLALLVEK;
bisphosphate (SBP)
GVFTNVTSPSTK
NPSGEDINALEQHIK;
Reversibly glycosylated
3 E1ZRS1 1.736 ± 0.11 3.077 ± 0.15 3.329 ± 0.20 GTLFPMCGMNLAFDR;
protein (RGP)
TGLPYIWHSK
HIIGESDEFIADK;
Triosephosphate isomerase VVVAYEPVWAIGTGK;
2 E1ZKB3 1.223 ± 0.69 2.184 ± 1.52 2.232 ± 0.96
(TPI) VATPEQAQEVHDAVR;
IIYGGSVTAK
KPDFDAYIDPQK;
Phosphoribulose kinase
3 E1ZF27 1.280 ± 0.05 1.363 ± 0.22 1.137 ± 0.17 IYLDISDEIK;
(PRK)
FYGEITQQMLK
IIGVTTLDVVR;
Malate dehydrogenase 3 E1ZRS4 1.539 ± 0.28 2.308 ± 0.47 3.312 ± 0.17 VAVLGAAGGIGQPLSLLMK;
(MDH) GYAGEDQLGEALK;
TQDGGTEVVQAK; LISYYLGEK
DLESLSIEEVMLK;
Superoxide dismutase
4 E1Z580 1.000 ± 0.57 1.928 ± 0.89 2.152 ± 1.18 AAGATQFGSGWAWLSVNK;
(SOD)
EGKLEVTK
Representative MRM chromatogram of 6 selected proteins and BSA is shown in Figure 5. % RSD of the BSA in each
sample was < 20, suggesting that samples were uniform.
Figure 5. MRM chromatograms of 25 peptides representing six differentially expressed proteins and BSA
5
Figure 6. Up-regulation of HIIGESDEFIADK representing triosephosphate isomerase
Figure 7. Down-regulation of ILFEVAPLALLVEK representing sedoheptulose 1,7-bisphosphate
Comparisons of differential expression levels obtained from iTRAQ and MRM analysis of SBP, SOD, RGP, TPI, PRK and
MDH are presented in Figure 8. Western blotting results of three proteins viz. TPI, PRK and MDH are also presented.
6
Figure 8. MRM and immuno assay based validation of selected differentially expressed proteins
Conclusion
• MRM assay for 6 different proteins from Chlorella sp FC2 IITG and BSA were optimized.
• Current study revealed interesting molecular mechanism underlying oil accumulation, which includes homeostasis
of chromatic remodeling via histone proteins, carbon concentrating mechanisms (CCMs) including photosynthesis,
glycolysis/gluconeogenesis, reductive pentose phosphate pathway, lipid accumulation and nitrogen assimilation
via proteolysis.
• The knowledge of proteins regulated during N-starvation has improved understanding of inherent modulation of
the intracellular protein repository and proteome re-adjustment, which can be implied for raising transgenic high
oil-yielding algae.
7
References
[1] R. H. Wijffels and M. J. Barbosa, Science, Volume 329, (2010), 796-799.
[2] V. L. Colin et al., (2011). J Biomed Biotechnol, Volume 2011, (2011), 601834.
[3] H. Jaliliannosrati et al., (2013). Bioresour Technol, Volume 136, (2013), 565-573.
Acknowledgements: VR is grateful to DBT for proving post-doctoral fellowship. This work is funded by Department of Biotechnology grants
(BT/PR484/PBD/26/259/2011) and DBT PAN IIT Centre for Bioenergy grant (BT/EB/PANIIT/2012) to SS.

PO-CON1649E
A High Sensitivity LC/MS/MS

Method with QuEChERS Sample
Pre-treatment for Analysis of
Aflatoxins in Milk Powder Samples
ASMS 2016 WP230
Yin Ling Chew1; Jie Xing1; Leonard Guan Seng Lim*2;

Zhaoqi Zhan1
1
Shimadzu (Asia Pacific) Pte Ltd, Singapore;
2
School of Physical & Mathematical Sciences,
Nanyang Technological University, Singapore,
*Student
A High Sensitivity LC/MS/MS Method with QuEChERS Sample
Pre-treatment for Analysis of Aflatoxins in Milk Powder Samples
Introduction
Aflatoxins (B1, B2, G1 and G2) are metabolites produced dairy products have been set. For example, European
by fungi (Aspergillus favus and Aspergillus parasiticus) in Union (EU) limits the level of aflatoxin M1 to no more
crops, animal feed and dairy products. Aflatoxins are than 0.05 µg/kg in milk and dairy products and 0.025
highly toxic contaminants in food and feed and their µg/kg in infant food. We present a high sensitivity
amounts increase under bad storage conditions favourite LC/MS/MS method for quantitative analysis of the five
for fungal growth. Aflatoxin M1 is a hydroxylated aflatoxins (B1, B2, G2, G2 and M1) in milk powder
metabolite of aflatoxin B1 found in milk of cow fed with incorporating QuEChERS sample pre-treatment
a diet contaminated with aflatoxin B1[1]. Aflatoxin B1 is procedure, which is more cost effective as compared to
known the most carcinogenic among all the aflatoxins, the traditional procedure using immunoaffinity column
and hence its metabolite aflatoxin M1 is given critical (IAC)[2]. High sensitivity and good recoveries were
attention. Strict regulations for aflatoxin M1 in milk and achieved using this LC/MS/MS method.
Experimental
A mixed standard of aflatoxin B1, B2, G1 and G2 was dSPE tubes. A LCMS-8060 triple quadrupole LC/MS/MS
obtained from Supelco. Aflatoxin M1 was obtained from (Shimadzu Corporation, Japan) was used in this work. A
Romer Labs. A stock solution of the mixture of 5 C18 column (Kinetex, 2.1 x 100mm, 1.7um) was used for
aflatoxins was prepared using methanol as the diluent, fast separation of aflatoxins using a gradient elution
from which calibrant series and spiked samples were program. Method performance evaluation were carried
prepared. The QuEChERS kits were purchased from out using spiked aflatoxins in milk powder samples. Table
RESTEK. Two grams of milk powder was first extracted 1 shows the analytical conditions on LCMS-8060.
with the extraction kits followed by cleaning up using
Shimadzu LCMS-8060, an UFMS triple quadrupole system with a heated ESI interface
2
Table 1: LC/MS/MS analytical conditions of aflatoxins on LCMS-8060
Column : Kinetex C18 (2.1mmI.D x 100mmL., 1.7 µm)

Mobile phase : A: 5 mM ammonium acetate in water with 0.1% FA
B: 5 mM ammonium acetate in MeOH
Oven temp. : 40ºC
Elution mode : Gradent elution, B%: 5% (0-5 min) 50% (4- 5.5 min)
85% (6-7.5 min) 5% (8.1-10 min)
Interface : ESI (Heated)
MS mode : Positive, MRM, 2 transitions each compound
Interface temp. : 350ºC
Block temp. : 400ºC
DL temp. : 250ºC
CID gas : Ar (350 kPa)
Nebulizing gas flow : 3.0 L/min
Drying gas flow : 10.0 L/min
Heating gas flow : 10.0 L/min

QuEChERS sample pre-treatment
Hexane was used in the procedure to remove fats, oils citrate). Dispersive SPE tube containing MgSO4, PSA and
and non-polar components from the milk powder C18 was used in the clean-up process to remove
samples. The extraction step was completed using Q-sep remaining water, organic acid and non-polar components
QuEChERS extraction salt packet (4 g MgSO4, 1 g NaCl, 1 respectively. The process of the sample preparation is
g trisodium citrate dehydrate, 0.5 g disodium hydrogen illustrated in Figure 1.
Method Development
Automated MRM optimisation of the five aflatoxins was A milk powder matrix free from aflatoxins was used as a
carried out using the LabSolutions workstation. Two “blank” and matrix for the preparation of post-spiked
MRM transitions for every aflatoxin were chosen as calibrants to build calibration curves. The blank and every
quantifier and confirmation ion (Table 2). post-spiked calibrant was injected thrice and the average
area was calculated to obtain reliable results.
3
Weigh 2 g of milk powder sample into a

50 mL centrifuge tube
Add CAN/H2O (ratio of 3:1) and
20 mL of hexane, with formic
acid of final conc. of 0.1%
Shake and vortex for 20 minutes

Add the Q-sep Extraction salt
Shake and vortex for 20 minutes and

centrifuge at 10,000 rpm for 10 minutes
Discard the hexane layer
Transfer the solution into a 20 mL flask
Top-up to the mark of the flask using ACN
Transfer 1mL solution into a 2mL dSPE tube
Vortex for 1 minute and centrifuge at

13,000 rpm for 10 minutes
N2 blow to dryness the supernatant and

reconstitute with water/MeOH (95:5)
Transfer the supernatant into a sample vial

for injection into LCMS-8060
Figure 1: Flowchart of sample pre-treatment for aflatoxins in milk powders by modified QuEChERS method.
Table 2: LC/MS/MS analytical conditions of LCMS-8050 for aflatoxins
CID Voltage (V)

Compound MRM (m/z)
Q1 CE Q3
313.1>241.0* -12 -40 -17
Aflatoxin B1
313.1>213.0 -21 -44 -15
315.1>287.0* -22 -27 -20
Aflatoxin B2
315.1>259.1 -11 -30 -18
329.1>243.0* -12 -28 -17
Aflatoxin G1
329.1>200.0 -12 -40 -22
331.1>189.0* -24 -43 -19
Aflatoxin G2
331.1>245.0 -12 -31 -18
329.0>273.0* -12 -23 -18
Aflatoxin M1
329.0>259.0 -23 -24 -29
* MRM transitions used as quantifiers.

4
A chromatogram of spiked sample is shown in Figure 2. Linear calibration curves were obtained for all five aflatoxin
compounds with good linearity (r2 >0.999). The calibration curves of aflatoxins spiked in milk powder matrix are shown in
Figure 4.
Method Performance Evaluation

The LOD and LOQ of aflatoxins in milk powder matrix are lower than 0.83 pg/mL and 2.50 pg/mL respectively (Table 3).
The repeatability of the method was evaluated using spiked samples at two concentrations. The peak area %RSD of
aflatoxins were found to be lower than 7.46%.
(x10,000)
3.50
3.25
B1
3.00
2.75
2.50
2.25
G1
B2
2.00
1.75
1.50
G2
1.25
1.00
0.75
M1
0.50
0.25
0.00
4.0 4.5 5.0 5.5 6.0 min
Figure 2: Total ion chromatogram of 5 aflatoxins (Concentrations of B1,G1 and M1 at 10 pg/mL; B2 and G2 at 3 pg/mL)
B1 B2 G1 G2 M1
5.0e3 1.5e3
2.5e3 4.0e3
1.3e3
4.0e3
2.0e3
1.0e3
3.0e3
1.0e3
3.0e3
1.5e3
7.5e2
2.0e3
2.0e3 1.0e3 5.0e2
5.0e2
1.0e3
1.0e3 5.0e2 2.5e2
0.0e0 0.0e0 0.0e0 0.0e0 0.0e0
6 5 6 5 4 5 4.5 5.0 5.5
Figure 3: Single LOQ MRM chromatograms of 5 aflatoxins

(Concentrations of B1, G1 and M1 (5 pg/mL); B2 and G2 (3 pg/mL)
5
Area (x10,000,000) Area (x1,000,000) Area (x10,000,000) Area (x1,000,000) Area (x1,000,000)
2.5 1.75 4.0
4.0
1.50 2.0
2.0 AFB1 AFB2 AFG1 AFG2 3.0 AFM1
3.0 1.25
1.5
1.5 1.00
2.0 2.0
0.75 1.0
1.0
0.50
1.0 0.5 1.0
0.5
0.25
0.0 0.0 0.00 0.0 0.0

0 1000 2000 3000 4000 Conc. 0 250 500 750 1000 1250 Conc. 0 1000 2000 3000 4000 Conc. 0 250 500 750 1000 1250 Conc. 0 1000 2000 3000 4000 Conc.
Figure 4: Calibration curves of aflatoxins B1, B2, G1, G2 and M1 in milk powder matrix.
Table 3: LOD, LOQ and repeatability of aflatoxin spiked samples at different concentrations
Range LOD LOQ %RSD (n=6)

Aflatoxin Linearity
(pg/mL) (pg/mL) (pg/mL) 5 pg/mL 6 pg/mL 30 pg/mL 50 pg/mL
B1 1-5000 0.9999 0.14 0.44 3.1 2.3
B2 3-1500 0.9999 0.36 1.09 6.4 2.4
G1 3-5000 0.9998 0.71 2.16 4.0 2.44
G2 3-1500 0.9999 0.41 1.22 5.8 3.5
M1 3-5000 0.9999 0.83 2.50 7.5 2.7
Both the matrix effect and recoveries of aflatoxins were evaluated by using a duplicate set of samples at different
concentrations. Each duplicate was obtained from the average of three injections. The results are shown in Table 4 and
Table 5.
Table 4: Matrix effects of the MRM method for aflatoxins in spiked milk powder samples
Concentration Matrix effect (%) Concentration Matrix effect (%)

(pg/mL) B1 G1 M1 (pg/mL) B2 G2
5.0 105.1 116.0 99.4 6.0 105.8 116.3
50.0 105.3 107.9 105.4 30.0 110.2 109.3
Table 5: Recoveries of aflatoxins in spiked milk powder samples
Concentration Recovery (%) Concentration Recovery (%)

(pg/mL) B1 G1 M1 (pg/mL) B2 G2
5.0 76.6 87.3 83.8 6.0 71.6 70.8
50.0 73.8 76.5 75.6 30.0 73.9 75.6
Analysis of aflatoxins in actual milk powder samples

Three milk powder samples from local supermarket were analysed using the established MRM method. The results showed
that no aflatoxin was detected in all three samples.
6
(x10,000) (x10,000) (x10,000)

3.25
3.50
3.25 Sample LF01 1.50

Sample SL05 3.00
Sample CL03
3.00 2.75
2.75 2.50
1.25
2.50 2.25
2.25
2.00
1.00
2.00
1.75
1.75
1.50
0.75
1.50
1.25
1.25
0.50 1.00
1.00
0.75 0.75
0.50 0.25 0.50
0.25 0.25
0.00 0.00 0.00
4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 min 4.0 4.5 5.0 5.5 6.0 min 4.5 5.0 5.5 6.0 min
Figure 5: MRM Chromatograms for Aflatoxin B2, B1, G2, G1 and M1 (top to bottom) of three milk powder samples
from local supermarket. Targets were not detected in all samples.
Conclusions
A high sensitivity LC/MS/MS method with QuEChERS for repeatability, matrix effect and recovery were carried out
sample pre-treatment was established using Shimadzu and the results confirm that the method is feasible and
LCMS-8060 system. The QuEChERS sample preparation reliable for determination of aflatoxins in milk powder
method was proven effective and easy to operate. The samples.
method performance including sensitivity, linearity,
References
1. Iqbal, S. Z.; Jinap, S.; Pirouz, A. A.; Ahmad Faizal, A.R. Trends in Food Science & Technology 2015, 46, 110-119.
2. Cherkani-Hassani, A.; Mojemmi, B.; Mouane, N. Trends in Food Science & Technology 2016, 50, 56-69.
Disclaimer: The products and application in this presentation are intended for Research Use Only (RUO). Not for

PO-CON1651E
Development of Automated Screening

and Quantitation Approach on Novel
On-Line SFE-SFC-MS/MS Platform –
(II) For Five Aflatoxins in Powdered
Food Samples
ASMS 2016 WP 231
Zhaoqi Zhan, Jun Xiang Lee; Yin Ling Chew,

Wan Tung Liw and Jie Xing
Singapore
(II) For Five Aflatoxins in Powdered Food Samples
Introduction
Supercritical fluid chromatography (SFC) is one of the pre-treatment and separation to superior MS/MS
trendy analytical techniques in recent years. By using detection and quantitation in a fully automated manner.
carbon dioxide (CO2) as eluent, the SFC has a number of The novel SFE-SFC-MS/MS system has been used
advantages like environmental friendly, cost effective and successfully for analysis of 510 residual pesticides in
suitable for a wide range of analytes’ polarities. The agricultural products [1]. Here we describe the
Nexera UC system (Shimadzu) is a further developed applications and advantages of the Nexera UC system in
platform combining SFE (extraction) and SFC with MS/MS analysis of aflatoxins (B1, B2, G1, G2 and M1) in
into a complete system to handle from sample powdered food such as corn flour and wheat flour.
Experimental
Five aflatoxins (B1, B2, G1, G2 and M1) were obtained standard solution and leaving it to dryness under N2 flow.
from Supelco and Romer Labs. Sixty mg of powdered A flow chart of the Nexera UC coupled with LCMS-8050
sample (corn and wheat) were loaded into a 0.2 mL used in this study is shown schematically in Figure 1. A
stainless steel vessel tightly before proceeding to on-line Shim-pack UC-X Sil column was used and a gradient
SFE-SFC-MS/MS. Spiked samples were prepared by elution program was adopted for analysis of the 5
soaking the powders in a desired amount of mixed aflatoxins. The detailed conditions are as shown in Table 1.
Make up solvent pump
SFE Unit Make up

solvent
sfCO2 pump Splitter

CO2 Column
Cylinder MS/MS
Auto-
sampler Column oven BPR-A
BPR-B
SFC Unit with MS/MS
Extraction vessel
Modifier Modifier
solvent pump drain
Figure 1: Schematic diagram of Nexera UC system for SFE-SFC-MS/MS analysis of un-pretreated samples
2
Table 1: Analytical conditions of five aflatoxins on Nexera UC with LCMS-8050
Column : Shim-pack UC-X Sil (250 mmL. x 2.1mm I.D., 3µm)

0.2 mL/min (make up pump)
Mobile Phase : A : Carbon dioxide
B : Methanol with 5 mM ammonium formate
C : Methanol with 0.1% formic acid
Oven Temp. : Column oven: 40ºC; SFE unit: RT
Injection vol. : SFC: 5 µL; SFE-SFC: 200 µL
Elution Mode : Gradient elution, LC program 5 minute
0% B (0.00 mins to 0.50 mins)
→ 40% B (3.00 mins to 3.50 mins)
→ 0% B (3.60 mins to 5.00 mins)
Interface : ESI
MS mode : Positive, MRM
DL Temp. : 250ºC
Nebulizing Gas Flow : N2, 1.5 L/min
Drying Gas Flow : N2, 5.0 L/min
Heating Gas Flow : 0 Air, 15.0 L/min

Establishment of SFC-MS/MS method for five aflatoxins
A SFC-MS/MS method for aflatoxin B1, B2, G1 G2 and 0.325 pg. The repeatability of the method was evaluated
M1 was established using mixed standard samples. Two at two absolute amounts, 1.25 and 2.5 pg (B1, G1 and
MRM transitions were used for each aflatoxin compound, M1); 0.375 and 0.75 pg (B2 and G2). Six injections were
one as quantifier ion and the other for confirmation. made at every level of absolute amount for reliable
Calibration curves with linearity (r2>0.995) were obtained results. The %RSD results of the mixed standards ranges
for all 5 aflatoxins (Table 2). Instead of using from 4.0 ~ 9.5%, except for G2 at absolute amount of
concentration for construction of the calibration curves, 0.375 pg where %RSD of 17.8% was obtained. The
absolute amount (pg) was used (injection volume: 5uL). results of method performance are tabulated in Table 2.
The LOQ of the SFC-MS/MS method ranges at 0.125 ~
3
(x10,000) Area (x100,000) Area (x100,000) Area (x100,000)
B1
1.75
(a) 5.0 (b) B1 1.00 B2 3.0 G1
1.50 0.75
G1 2.0
2.5 0.50
1.25
1.0
B2
0.25
1.00 0.0 0.00 0.0

0.0 5.0 10.0 Conc. 0.0 1.0 2.0 3.0 Conc. 0.0 5.0 10.0 Conc.
Area (x10,000) Area (x10,000)
0.75
5.0
G2 M1
G2
4.0
7.5
B1, G1 and M1:
M1
0.50
3.0
5.0
0.5~12.5 pg
0.25
2.0
B2 and G2:
2.5 0.15~3.75 pg
1.0
0.00
0.0 0.0
0.0 1.0 2.0 3.0 4.0 min 0.0 1.0 2.0 3.0 Conc. 0.0 5.0 10.0 Conc.
Figure 2: (a) MRM chromatograms of aflatoxins mixed standards B1, G1 and M1 at 1.25 pg; B2 and G2 at 0.375 pg.
(b) Calibration curves for aflatoxins B1, B2, G1, G2 and M1 in neat solution.
Table 2: Calibration curves and performance values of the MRM method for quantitation of five aflatoxins on SFC-MS/MS.
Absolute amounts (in pg) of analytes are used for convenience (inj. volume: 5 µL)
Ret. MRM Cali. Range Ave. LOQ RSD (%), n=6

Aflatoxin r2
Time transition (pg) Accuracy (pg) 0.38 pg 0.75 pg 1.25 pg 2.5 pg
B1 2.452 313.1>241.1 0.5 - 12.5 0.998 100.2% 0.175 N.A. N.A. 8.9 4.0
B2 2.473 315.1>287.0 0.15 - 3.75 0.998 102.6% 0.125 6.7 6.8 N.A. N.A.
G1 2.698 329.1>243.1 0.5 - 12.5 0.998 100.5% 0.225 N.A. N.A. 9.4 4.1
G2 2.693 331.1>245.1 0.15 - 3.75 0.995 99.8% 0.15 17.8 9.5 N.A. N.A.
M1 2.637 329.2>229.1 0.5 - 12.5 0.997 101.9% 0.325 N.A. N.A. 6.1 6.3
SFE-SFC-MS/MS method and system recovery

Based on the SFC-MS/MS method established above, aflatoxins become broader and RTs delay slightly in
on-line SFE-SFC-MS/MS was developed and evaluated. The comparison with the SFC-MS/MS chromatograms. This
mixed aflatoxin standard samples can be introduced into peak broadening and delay (~0.5 mins) are due to the
the system only by pre-loading them onto filter papers: larger delay volume contributed by the SFE vessel, needles
50 µL of mixed standard solution was dropped onto an and the tubing from SFE to column, which caused
half filter paper (recommended by Shimadzu) and left it to differences in peak shape and intensity. For direct
dryness under N2 flow before loading into the SFE vessel quantitation of PFCs using on-line SFE-SFC-MS/MS,
(0.2 mL). The results are shown in Figure 3 and Table 3. It calibration curves must be established on SFE-SFC-MS/MS
can be seen that, with on-line SFE, the elution peaks of the too (Figure 3(b) & Table 3).
4
(x10,000) Area (x100,000) Area (x10,000) Area (x100,000)
B1
5.0 2.5
2.25
(a) (b) B1 7.5 B2 G1
4.0 2.0
G1
2.00
3.0 5.0 1.5
1.75
2.0 1.0
B2
2.5
1.50 1.0 0.5
1.25 0.0 0.0 0.0

0.0 2.5 5.0 7.5 10.0 Conc. 0.0 1.0 2.0 3.0 Conc. 0.0 2.5 5.0 7.5 10.0 Conc.
1.00 Area (x10,000) Area (x10,000)

G2
4.0
M1
0.75 G2 7.5 M1 B1, G1 and M1:

3.0
0.50
5.0
2.5, 5, 12.5 pg
0.25
2.0
B2 and G2:
1.0 2.5 0.75, 1.5, 3.75 pg
0.00
0.0 0.0
0.0 1.0 2.0 3.0 4.0 min 0.0 1.0 2.0 3.0 Conc. 0.0 2.5 5.0 7.5 10.0 Conc.
Figure 3: (a) MRM chromatograms of 5 aflatoxins on filter paper (B1, G1 and M1 at 5 pg; B2 and G2 at 1.5 pg).
(b) Calibration curves of aflatoxin B1, B2, G1, G2 and M1 on filter paper in SFE
Table 3: Calibration curves and performance values of MRM method for quantitation of five aflatoxins on SFE-SFC-MS/MS.
Absolute amounts (pg) of analytes are used for convenience.
Ret. MRM Cali. Range Ave. LOQ RSD (%), n=6

Aflatoxin r2
Time transition (pg) Accuracy (pg) 0.75 pg 1.5 pg 2.5 pg 5 pg
B1 2.922 313.1>241.1 2.5 - 12.5 0.9963 98.6% 0.68 N.A. N.A. 17.6 8.2
B2 2.949 315.1>287.0 0.75 - 3.75 0.9961 98.6% 0.59 11.0 5.7 N.A. N.A.
G1 3.170 329.1>243.1 2.5 - 12.5 0.9920 97.9% 1.1 N.A. N.A. 11.9 6.9
G2 3.169 331.1>245.1 0.75 - 3.75 0.9993 99.4% 2.5 17.5 11.8 N.A. N.A.
M1 3.168 329.2>229.1 2.5 - 12.5 0.9989 99.2% 1.3 N.A. N.A. 12.3 7.9
If we compare the peak areas obtained on SFE-SFC-MS/MS for quantitation. The system recovery
SFE-SFC-MS/MS and SFC-MS/MS, system recovery of the measured with specific loading amounts are shown in
SFE-SFC-MS/MS could be estimated. Although this system Table 4. It is worth to note that all of the analysis runs
recovery may not be highly accurate, it can be used as a shown above are under the condition without splitting of
reference to understand the performance of the on-line the flow (sfCO2 and MeOH) from SFE to SFC-MS/MS.
Table 4: System recovery results of on-line SFE for aflatoxins
Ret. MRM Loaded Measured System

Aflatoxin
Time transition (pg) (pg), n=3 Recovery (%)
B1 2.452 313.1>241.1 5.0 4.30 86.0

B2 2.473 315.1>287.0 1.5 1.20 80.0
G1 2.698 329.1>243.1 5.0 3.80 76.0
G2 2.693 331.1>245.1 1.5 1.12 74.7
M1 2.637 329.2>229.1 5.0 4.45 89.0
5
On-line SFE-SFC-MS/MS for analysis of aflatoxins in powdered food samples

There are two extraction modes in the on-line SFE stage, expected to be obtained if a static extraction step is used
static extraction and dynamic extraction with sfCO2 or a before the dynamic extraction.
mixture of sfCO2 and MeOH (modifier) [2]. In this work, The results of wheat flour (free of aflatoxins) samples
only dynamic extraction of 2 mins was applied. A spiked with aflatoxins are shown in Figures 5. It can be
powdered corn flour sample (free of aflatoxins) was used seen that more background peaks appeared in the
to test the on-line SFE-SFC-MS/MS approach. The results of chromatograms, which indicates that the matrix of wheat
a representative analysis are shown in Figure 4 and Table 5. flour extracted by the on-line SFE is more complicated than
The spiked sample was prepared by dropping 50 µL of an the corn flour. This high backgrounds (relative) caused
aflatoxin stock solution onto 60 mg of the corn flour and more difficulties to detect very low contents of aflatoxins in
left to dryness under N2 flow. Then, the sample was the sample. The results shown in Figure 5 suggest that the
loaded into the 0.2 mL SFE vessel. It is noted that the amounts of aflatoxins at 60~72 pg could be detected,
measured amounts of aflatoxins in Table 5 are from the which correspond to aflatoxin contents of 1.0~1.2 µg/kg in
sum of two consecutive runs of the same sample. This is the spiked sample. Further studies on optimizing the SFE
because the first run in a dynamic extraction mode could conditions to improve the on-line SFE recovery and reduce
extract only less than 20 % of the spiked aflatoxins under interference are on-going.
the conditions. However, higher SFE extraction recovery is
(x10,000) (x1,000) (x1,000) (x1,000) (x1,000)

1.25 313.05>241.10(+) 315.10>287.00(+) 329.10>243.05(+) 2.0 331.10>245.05(+) 329.20>229.15(+)
313.05>213.10(+) 315.10>259.10(+) 3.0 329.10>200.00(+) 331.10>189.00(+) 329.20>259.10(+)
2.0 2.0
329.10>200.00(+)
G1
M1
B2
B1
1.00 2.5
B1 B2 G1 G2 M1
G2
1.5
1.5 1.5
2.0
0.75
1.5 1.0
1.0 1.0
0.50
1.0
0.5 0.5
0.25 0.5
0.5
0.00 0.0 0.0

0.0 0.0
2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0
Figure 4: MRM peaks of aflatoxin detected in spiked corn flour sample detected in the 2nd run of the sample
on SFE-SFC-MS/MS system. The spiked amounts of aflatoxins are shown in Table 5.
Table 5: Analysis results of aflatoxins spiked in powdered corn sample

by on-line SFE-SFC-MS/MS approached
Spiked Measured Recovery

Aflatoxin
Abs (pg) Cont (ug/kg) Abs (pg) Cont (ug/kg) (%)
B1 10.0 0.167 6.00 0.100 60.0

B2 3.0 0.050 1.84 0.031 61.3
G1 10.0 0.167 2.61 0.044 26.1
G2 3.0 0.050 0.71 0.012 23.7
M1 10.0 0.167 3.42 0.057 34.2
6
(x100) (x100) (x100) (x100) (x100)

2:313.05>241.10(+) 6.0 1:315.10>287.00(+) 4:329.10>200.00(+) 3.0 3:331.10>245.05(+) 5:329.20>229.15(+)
7.5 2:313.05>213.10(+) 1:315.10>259.10(+) 4:329.10>243.05(+) 3:331.10>189.00(+) 1.5 5:329.20>259.10(+)
B1
B2
5.0 2.0 2.5
G1
B1 B2 G1 G2 M1
M1
2.0
4.0
1.5 1.0
5.0
G2
1.5
3.0
1.0
1.0
2.0 0.5
2.5
0.5 0.5
1.0
0.0 0.0
0.0 0.0
0.0
2.5 4.5 2.5 4.5 2.5 4.6 2.5 4.6 2.5 4.5
Figure 5: MRM peaks of aflatoxins spiked in wheat flour sample of 60 mg

(B1, 60 pg, B2, 72 pg, G1, 60 pg, G2, 72 pg and M1, 60 pg) by on-line SFE-SFC-MS/MS.
Conclusions
A new analytical approach was developed on Shimadzu novel SFE-SFC-MS/MS platform for direct analysis of Aflatoxin B1,
B2, G1, G2 and M1 in un-pretreated flour samples. The preliminary results indicate that this new approach is potentially
applicable for screening and quantitation of aflatoxins in powdered food samples without any sample pre-treatment.
References
1. Shimadzu Application News No LAAN-A-LC-E273, Using the Nexera UC Online SFE-SFC-MS System to Analyze
Residual Pesticides in Agricultural Products (2015).
2. J. Xing, , J. X. Lee, P. Zeng and Z. Zhan, Development of Automated Screening and Quantitation Approach on Novel
On-line SFE-SFC-MS/MS Platform – (I) For 23 Restricted PFCs in Textiles; ASMS 2016, Poster Session MP 283.

PO-CON1661E
Simultaneous Assay of Multiple

Synthetic Contraceptive Hormones
in Human Serum by LCMS-MS
ASMS 2016 WP718
Steven W. Blue1, Rachel A. Lieberman2, David W. Erikson1,

Christopher Gilles2,
1
Endocrine Technologies Support Core, Oregon National
Primate Research Center, Beaverton, OR
2
Shimadzu Scientific Instruments, Columbia, MD, USA
Simultaneous Assay of Multiple Synthetic Contraceptive
Hormones in Human Serum by LCMS-MS
Novel Aspect
Ultra-fast switching eight minute method to analyze and quantifiy low levels of synthetic contraceptive hormones in
serum by LCMS-MS.
Introduction
Immunoassay is the conventional method for measuring with synthetic steroid hormones used for female
steroid hormone levels in the clinical laboratory. Recently, contraceptive hormones, there are no reliable
the questionable sensitivity of some immunoassays and immunoassays available. This poster describes a
the desire for more sensitive methods has led to the need comprehensive method for evaluating multiple synthetic
to develop alternative methods for measuring steroid steroids in serum using ultra-high performance liquid
hormones in human serum samples. In some cases, as chromatography-tandem mass spectrometry (LCMS-MS).
Methods
Commonly used contraceptive hormones, ethinyl estradiol quality controls (QCs) and samples were subjected to the
(EE2), etonogestrel (ENG), levonorgestrel (LNG), same extraction procedure using a Biotage Supported
medroxyprogesterone acetate (MPA), norethindrone (Net) Liquid Extraction (SLE+) plate. All hormones were eluted
in addition to estradiol-17b (E2) and progesterone (P4) with dichloromethane, dried and then reconstituted in
were simultaneously measured using ultra-high 25:75 methanol:water (v/v). MRM transitions for each
performance liquid chromatography tandem mass analyte were optimized and separated using reversed
spectrometry. Heated electrospray ionization (hESI) in phase chromatography in a single sub 8 minute method.
both positive and negative modes with polarity switching
was used for ionization of the compounds. All calibrators,
Chromatography Conditions Calibration levels

Mobile Phase A : Water with 2mM NH4F
Level ng/mL (ppb)
Mobile Phase B : Methanol
Column : Restek Raptor Biphenyl (2.7 µm x 50 x 2.7mm) 1 10
Gradient : Linear from 34% - 100% B. 2 5

Flow Rate : 0.2 mL/min 3 2.5
Column oven : 40°C 4 1.25
6 0.3125
7 0.15625
8 0.078125
9 0.0390625
10 0.0195313
11 0.0097656
12 0
2
Results - Positive Control Sample

A representative chromatogram for a positive control of Both positive and negative mode hESI was used in this
the analyte mix is shown. LNG has the highest response fast and sensitive method. The retention times for each of
as shown in the figure above. Each synthetic hormone the analytes are listed in the table below.
has a representative ISTD as a part of the research assay.
90000
LNG
80000
70000
60000
Net
50000
40000 MPA P4
ENG
30000
EE2 E2 LNG-d6
20000
Net-d6 ENG-d7
MPA-d6 P4-d9
10000
1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 min
Chromatogram for Synthetic Hormone Analysis

Positive control sample chromatogram of all synthetic hormones tested.
Analyte Rt. Time Analyte Rt. Time

EE2-d7 1.696 LNG-d6 4.422
EE2 1.727 LNG 4.48
E2-d5 1.742 MPA-d6 5.071
E2 1.764 MPA 5.084
Net-d6 3.59 P4-d9 5.424
Net 3.669 P4 5.452
ENG-d7 4.349
ENG 4.424
3
LCMS-8050 Parameters
• Ultrafast MRM methods

• Up to 555 MRM transitions per sec
• Heated electrospray source
• Scan speeds up to 30,000 u/sec
• Polarity switching 5 msec
LCMS-8050 Parameters
Nebulizing Gas Flow : 3 L/min

Heating Gas Flow : 20 L/min
Drying Gas Flow : 10 L/min
Interface Temperature : 400 °C
DL Temperature : 150 °C
Heat Block Temperature : 500 °C
Analyte Type m/z Ref. Ion Rt. Time

E2 Target 271.00>183.20 271.00>144.90 1.764
E2-d5 ISTD 275.90>147.25 275.90>187.20 1.742
P4 Target 315.30>97.25 315.30>109.00 5.452
P4-d9 ISTD 323.85>100.10 323.85>113.10 5.424
EE2-d7 ISTD 302.20>150.25 302.20>272.10 1.696
295.00>143.30
EE2 Target 295.00>145.35 1.727
295.00>267.35
MPA-d6 ISTD 393.20>330.20 393.20>288.35 5.071
MPA Target 387.30>327.00 387.30>123.20 5.084
LNG Target 313.10>109.20 313.10>245.25 4.48
LNG-d6 ISTD 319.10>251.35 319.10>113.15 4.422
ENG-d7 ISTD 332.30>263.35 332.30>115.20 4.349
Etonogestrel Target 324.70>109.20 324.70>91.25 4.424
Norethindrone Target 299.10>109.30 299.10>91.00 3.669
Norethindrone-d6 ISTD 305.15>237.35 305.15>113.30 3.59
4
MPA Net E2 P4
O O
OH
O O OH
H
H H H
H H
H H H H
H H H H
O HO
O O
ENG EE2 LNG

OH
OH
OH
H
H
H H
H H
H H
H H
HO
O
O
Results - Human Serum Assay

LNG is a synthetic progestin usually administered at >200 assay allowed a measurement of free LNG levels with only
pg/ml. However, the researcher was interesed in a modest increase in sample volume. Differences in
developing a measurement for “free LNG”. Most steroids, bound vs. free levels may be able to explain differences in
including LNG, circulate in the blood bound to SHBG or efficacy and/or off-target effects seen between individuals
serum albumin. Only the unbound fraction (usually ~1%) on this drug.
is available for biological function. Having a very sensitive
313.10>109.20(+)
3000 313.10>245.25(+)
2750
2750 Free LNG
2500 13 pg/mL 2500
2250
2250
2000 2000
1750 1750
1500
1500
1250
1250
1000
1000
750
500 750
250 500
0
250
1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 min 4.25 4.50 4.75
Measurement of (free) LNG in Human Serum Mass chromatogram for

“free” LNG in human
serum. The LOD was
13 pg/mL.
5
Results - Sheep And Monkey Assay

E2 is very important to have sensitive assays because it sensitive assays.
typically circulates at 50-200 pg/ml in normal adult Results here show 5 pg/mL of E2 and P4 on column in
women; but is frequently <15 pg/ml in men, children & both sheep and monkey assays. E2 was analyzed in
post-menopausal women. Studies looking at negative mode and P4 was analyzed in positive mode in
development, puberty and aging really need to have the same chromatographic run.
E2 700
271.00>183.20(-)
271.00>144.90(-)
P4 1100
315.30>97.25(+)
315.30>109.00(+)
5 pg/col 5 pg/col
1000
600
900
500 800
700
400
600
300 500
400
200
300
200
100
100
1.50 1.75 2.00 5.25 5.50 5.75
Chromatogram for Sheep Sample
E2 22500 271.00>183.20(-)
271.00>144.90(-)
P4 3000 315.30>97.25(+)
315.30>109.00(+)
5 pg/col 20000
5 pg/col 2750
2500
17500
2250
15000
2000
12500 1750
1500
10000
1250
7500
1000
5000 750
500
2500
250
0
0
1.50 1.75 2.00 5.25 5.50 5.75
Chromatogram for Monkey Sample
6
Results - Calibration Curves

Area Ratio Area Ratio Area Ratio Area Ratio
[*10^1] [*10^2] [*10^1] [*10^1]
9.0 6.0 2.0
1.0
P4 MPA E2 EE2
1.8
8.0
r2 = 0.997 r2 = 0.995 r2 = 0.998 r2 = 0.999
5.0
0.8 1.6
7.0
1.4
6.0 4.0
0.6 1.2
5.0
3.0 1.0
4.0
0.4 0.8
3.0 2.0
0.6
2.0
0.2 0.4
1.0
1.0 0.2
0.0 0.0 0.0 0.0

0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2
Conc.(Ratio) [*10^1] Conc.(Ratio) [*10^1] Conc.(Ratio) [*10^1] Conc.(Ratio) [*10^1]
Area Ratio Area Ratio Area Ratio

[*10^1] [*10^0] [*10^1]
9.0 4.0 8.0
Net ENG LNG
8.0
r2 = 0.998 3.5 r2 = 0.996 7.0 r2 = 0.999
7.0
3.0 6.0
6.0
2.5 5.0
5.0
2.0 4.0
4.0
1.5 3.0
3.0
1.0 2.0
2.0
1.0 0.5 1.0
0.0 0.0 0.0

0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2
Conc.(Ratio) [*10^1] Conc.(Ratio) [*10^1] Conc.(Ratio) [*10^1]
Calibration Levels for all Analytes
Limit of Quantitation
Analyte LOQs pg/col LOQ pg/ml

MPA 0.49 9.8
P4 0.49 9.8
E2 0.49 9.8
EE2 0.49 9.8
LNG 0.49 9.8
ENG 1.95 39
NET 0.49 9.8
7
Conclusions
A robust and rapid sub eight minute method for trillion (pg/mL) range. Low levels of P4 and E2 were
evaluating multiple synthetic contraceptive hormones was measured in both sheep and monkey serum samples. In
developed using ultra-fast liquid chromatography mass addition, very low levels of free LNG levels in the blood
spectrometry. All analytes were detected in the parts per were measured in this research assay.
Acknowledgements
I would like to thank Steven Blue and David Erikson from Oregon National Primate Research Center/Oregon Health and
Science University for providing the excellent data.
For research purposes only. Not for use in diagnostic procedures.

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Method development of high

ASMS 2016 ThP 002

Motoshi Sakakura1, Teruhisa Shiota1, Jun Watanabe2

Methods and Materials

Figure 1 DART-MS System; LCMS-2020 equipped with DART-OS

Positive Mass Spectum

6.0 DAG TAG

2.0 TAG • DAG

Figure 2 Positive mass spectra of samples analyzed by DART-MS

2.0 TAG • DAG DAG from TAG from

Figure 3 Positive mass spectum of margarine added butter analyzed by DART-MS

Negative Mass Spectrum

Figure 4 Positive mass spectra of samples analyzed by DART-MS

Soybean milk Milk Margarine Butter

Figure 5 TIC chromatogram and XIC chromatogram (m/z 215)

From the results of food samples;

First Edition: June, 2016

For Research Use Only. Not for use in diagnostic procedure.

Development of a flavor release

ASMS 2016 ThP 005

Takehiko Sagawa1, Keiko Matsumoto2, Jun Watanabe2,

Methods and Materials

Figure 1 Structure of volatile compounds in this study

Ultra Fast Polarity Switching

Figure 2 LCMS-8030 triple quadrupole mass spectrometer

Figure 3 Standard sample (vapor)

Sample vial opened its cap was placed

Figure 4 Positive mass spectra of limonene and a-terpineol

Figure 5 Shonan Gold

Shonan Gold Orange

1.0 MRM 170/153

1.0 MRM 135/107

Set fruit in Squeezing Set fruit in Squeezing

Figure 5 MRM chromatograms of Shonan Gold and Orange

Figure 6 DART & Volatimeship System

First Edition: June, 2016

For Research Use Only. Not for use in diagnostic procedure.

A Combined MRM and SIM Method

ASMS 2016 TP740

Column : Intrada Amino Acid (100 x3 mm, 3µm) Interface : ESI

Results and Discussion

(x1,000,000) Area (x1,000,000) Area (x1,000,000) Area (x1,000,000)

MRM mode Leucine Methionine 2.0 Glutamic

SIM mode Proline Threonine Glycine

1.5 (MRM) (MRM) (SIM)

100.00 100.00 100.00 100.00 100.00 106.47

0.00 0.00 0.00 0.00 0.00 30.59

100.00 102.79 101.89 102.86 102.21 101.12

0.00 8.94 26.42 12.38 36.76 40.78

RT MRM Method (nmo//mL) SIM Method (nmol/mL)

Analysis of amino acid in biological and beverage samples

(c) Red wine 2.0

Biological Sample Conc. (nmol/mL) Beverage Sample Conc. (nmol/mL)

First Edition: June, 2016

For Research Use Only. Not for use in diagnostic procedure.

Development of LC/MS/MS Method

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Column : Kinetex C18 100A (100 x 2.1mm, 1.7µm)