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A High Sensitive LC-MS/MS Method for Quantitation of Formoterol in Human Plasma

Dawei Zhou, Wenzhong Liang, Xinping Fang, and Jinn Wu


XenoBiotic Laboratories, Inc., 107 Morgan Lane, Plainsboro, NJ 08536

Experimental

N
H
H
N

CD2
CD
CD3

N
O

The eluted samples were evaporated to dryness for


approximately 30 min in a 96-Well Nitrogen Evaporator
(EvapArray) at approx. 35 C. The residue was
dissolved in 125 L of 100 mM ammonium formate in
water for analysis.

Formoterol-D6

Intensity, cps

0.4 to 100 pg/mL

Calibration Range
Accuracy & Precision

400

Accuracy
Precision
QC Conc. (pg/mL)
RE%
CV%
LLOQ
0.4
2.00
13.57
Low
1
5.00
8.94
Medium
40
9.00
3.81
High
90
8.78
3.49
Compared with Nominal Value (%)
> 71.95
Accuracy

320

Inter-Batch (n=18)

240
160
80
20
800
720

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

0.4 pg/mL of formoterol


extracted from human plasma

(B)

Method Recovery

640
560
480
400

240
160

0.5

1.0

1.5

2.0

2.5
3.0
Time, min

3.5

4.0

4.5

5.0

5.5

149.1

1.8e6
1.2e6

(A)

100 pg/mL of
Formoterol in Plasma

60

4.0e6

351.3

155.1

Intensity, cps

120

180
155.3

240

300

1.9e4

Formoterol-D6

In previous methods larger plasma sample volumes (12 mL) were needed in order to achieve a lower detection
limit. These sample volumes would make preparation
much longer requiring large individual SPE cartridges and
introduce more error in the preparation procedure. The
current method employed special SPE elution solution by
which the extraction method recovery was dramatically
increased. Therefore, only 0.5 mL of human plasma was
required to reach the requisite sensitivity .

A 0.5 mL sample volume afforded the use of a 96-well


SPE plate format, instead of individual cartridges. In
addition, the TOMTEC liquid handling system made
sample preparation quick and consistent.

6.0e5

4.17

Mass Spectrometry
MS System:
AB Sciex API-4000
Condition:
LC/(+)ESI-MS/MS (MRM)
MRM Transition:
Formoterol:
345.2
149.1

RE%
<9.89
< 11.1
< 9.75
< 5.30

327.5
345.3

Figure 1. Ion chromatograms of blank plasma (A), and 0.4 pg/mL


formoterol extracted from plasma (B)

2.3e4

Liquid Chromatography:
HPLC System
Shimadzu LC-10AD
Analytical Column: C8 column, 2.1 x 50 mm, 3 m
Mobile Phase A)
10 mM ammonium Acetate in water
(pH4)
Mobile Phase B)
Methanol
Gradient
Flow rate:
0.5 mL/min
Injection Volume:
25 L

Condition
3 Cycles, < -70 C
4 hrs, Room Temperature
4 Days, Room Temperature
117 Days, < -70 C

Freeze/Thaw
Bench-Top
Autosampler Extract Stability
Long-Term Storage Stability

320

360
333.4
351.6

420

2.5e6

1.5e4
1.1e4

1.0e6
7000.0

60

120

180

0.5
4.8e4

(B)

1.5

2.5

3.5

4.12

4.5

5.5

Formoterol-D6 in Plasma

240

m/z, amu

3000.0

300

360

420

Figure 3. LC/(+)ESI-MS/MS product ion spectra of Formoterol (A) and


Formoterol-D6 (B)

Excellent linearity was obtained with a correlation


coefficient greater than 0.9936. The inter-day precision
(CV%) and accuracy (RE%) for all QC plasma samples,
including LLOQ were 13.6% and 9%, respectively (Table
I). Three freeze/thaw cycles, ambient temperature storage for
up to 4 h prior to analysis, and three month long-term storage
at -20oC appeared to have little effect on the quantitation.

1.14

4.0e4

0.95

3.2e4
2.4e4
1.6e4
8000.0

Formoterol

OH

Formoterol and the internal standard (formoterol-D6) were


transferred onto the preconditioned Strata X-C Polymer
Strong Cation 96-well plate (Phenomenex). The plates
were preconditioned with 1 mL of methanol and 1 mL of
0.1% formic acid aqueous solution. The SPE plates were
then washed with 2 mL of 0.1% formic acid solution, 0.8
mL of acetonitrile, and 0.8 mL of a mixed solution of
dichloromethane and isopropyl alcohol. Both analyte and
internal standard were then eluted using a mixed solution
of dichloromethane and isopropyl alcohol containing 2%
of ammonia.

480

HO
H

Control plasma

560

0.5

1.5

2.5

3.5
Time, min

4.5

5.5

Analyte Area / IS Area

Structures

Table I. Validation Data Summary

640

Intensity, cps

In a recent study for obstructive airway disease, including


asthma and COPD, formoterol exhibited a rapid onset of
action comparable to the short-acting bronchodilator,
VENTOLIN, as well as a duration of action of up to 24
hours. Previous bioanalytical methods have been developed
for 1-2 mL sample volumes, which would necessitate large
individual SPE cartridges making the sample preparation time
longer, and also require the collection of higher total volumes
of plasma. The current method was developed to reduce the
sample preparation time, lower the detection limit and lower
the volume of plasma required. We report a rapid, specific,
and highly sensitive LC-MS/MS method capable of
quantifying formoterol at levels as low as 0.4 pg/mL from 0.5
mL of human plasma.

OH

(A)

80
20

Introduction

HO

Sample Preparation

720

Intensity, cps

In this method, the drug was extracted from a 0.5 mL sample


of plasma using a strong cation exchange solid phase
extraction method. Separation was performed on a reverse
phase C8 column. Detection was achieved using a AB/SCIEX
API-4000 tandem mass spectrometer employing turbo-ion
spray ionization in the positive ion mode along with multiple
reaction monitoring (MRM). The lower limit of quantitation
was 0.4 pg/mL.

Results and Discussion


800

Intensity, cps

Overview
A highly sensitive and specific liquid chromatography- mass
spectrometry (LC-MS/MS) method capable of quantifying
formoterol in human plasma is described.

Linear" Regression ("1 / (x * x)" weighting):


y = 0.00988 x + 0.00417 (r = 0.9986)

Conclusions

0.75
0.60
0.40
0.20

Figure 2. Ion chromatograms of 100 pg/mL formoterol extracted from


plasma (A), and the internal standard extracted from plasma (B)

0.05
0

15

30

45
6
75
Analyte Conc.0(pg/mL).

90

100

Figure 4. Representative calibration curve for the determination of formoterol


in the range from 0.4 pg/mL to 100 pg/mL in human plasma

A rapid and specific LC-MS/MS method was developed and


validated for quantifying formoterol with a lower limit of
quantitation of 0.4 pg/mL from a 0.5 mL of human plasma
sample.