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Original Article
Introduction
Local anesthesia is described as any technique
that induces the absence of sensation in part of
the body, generally for the aim of inducing local
analgesia, that is, local insensitivity to pain,
although other local senses may be affected as
well (1, 2).
In 1943, lidocaine was first synthesized under
the name Xylocaine by Swedish chemist Nils
Lfgren as an alternative class of anesthetics.
Lidocaine HCl, 2-diethylamino-N-(2,6-dimethyl
* Corresponding author:
E-mail: yucel@atauni.edu.tr
Figure 1. Chemical structure of lidocaine HCl (A) and IS [prilocaine HCl (B)].
method (6, 7), spectrophotometric methods (813), HPLC methods with different detection (10,
13-19), capillary electrophoretic method (20) and
membrane electrode method (21, 22) have been
reported for determination of lidocaine HCl in
pharmaceutical formulations. The determination
of mixtures with other local anesthetics of
lidocaine HCl in biological samples (23-28) and
pharmaceutical formulation (29) have been done
with the GC-MS and GC methods. There are
only a few studies for determination of lidocaine
HCl with the GC method in whole blood (30),
serum (31), and urine (32).
According to the current good manufacturing
practices, the stability-indicating assay method
before release must be assessed for all drugs. A
forced degradation study of the drug substance
can help to identify the possible degradation
products, which can help to establish the
degradation pathways and the intrinsic stability
of the molecule and validate the stabilityindicating power of the analytical procedures
used (33). The nature of the forced degradation
will depend on the individual drug substance and
the type of the drug product involved (33).
So far, according to our present knowledge,
neither the GC-FID method for the analysis
of lidocaine HCl alone in any pharmaceutical
formulation nor the study of GC-FID and NMR
spectroscopy for the degradation behavior of
lidocaine HCl under various ICH-prescribed
degradation conditions is available in the
literature. There are a few studies about the
stability of lidocaine HCl. These studies are
described as follows: the relationship between
pH and concentrations of antioxidants and
660
Experimental
Lidocaine HCl that was used as reference
substance and prilocaine HCl that was used as
internal standard (IS; Figure 1 B) were purchased
from Novagenix Bio Analytical R&D Centre
(Ankara, Turkey). All other chemicals were
purchased from Merck (Germany). All gases
were supplied by Havas (Ankara, Turkey).
The following pharmaceutical formulations of
lidocaine HCl were obtained from local sources
in Erzurum (Turkey): Emla Cream (5% cream,
Astra Zeneca A.S., Turkey) containing 25 mg of
lidocaine HCl, 25 mg of prilocaine HCl, 55 mg
of poloxamer188 and 155 mg of poloxamer407.
Jetokain Ampoule (2 mL ampoule, Adeka A.S.,
Turkey) containing about 20 mg of lidocaine HCl,
0.125 mg of epinephrine, 4.5 mg of sodium chloride
and 1.0 mg of sodium metabisulfite per mL.
Jetmonal Ampoule (2% 5 mL ampoule, Adeka
A.S., Turkey) containing about 20 mg of lidocaine
HCl and 4.6 mg of sodium chloride per mL.
Chromatographic Conditions
Separation was achieved using a HP-5 capillary
column (5% - phenyl -methylpolysilocone 30 m
x 0.320 mm i.d., 25-m film thickness, USA)
with 2.0L of injector volume. The split mode
(10:1) was used with nitrogen carrier gas and
the flow rate of carrier gas was kept constant
during analysis at 1.6mL/min. Hydrogen and
synthetic air were used as auxiliary gases for
the detector (FID). The injector and detector
temperatures were 260C. The oven temperature
programs were as follows: initial temperature
of 80C, held for 1.5 min at 210oC, ramp rate
of 10C/min and final temperature of 230C,
where the temperature was held for 0.5 min. The
chromatograms under these circumstances were
shown in Figure 2.
Equipment
The equipment used was: an Agilent 6890N
Network gas chromatography system provided
with an Agilent 7683 series auto sampler, a split/
splitless injector, flame ionization detector and
Agilent chemstation software.
1
H and 13C NMR spectra were recorded
on a Varian 400 spectrometer in deuterated
661
Peak areaa
7.53
22.58
SD
0.09
0.66
RSD %
1.23
2.92
based on six analysis, SD: standard deviation, RSD: relative standard deviation
Results
System suitability
The system suitability tests were assessed by
six replicate analyses of 5.0 g/mL concentration
of lidocaine HCl. In this study, the acceptance
criterion was considered as 2% deviation from
the percent relative standard deviation (RSD %)
of peak area and retention time of lidocaine HCl
(Table 1). The number of theoretical plates (N)
and the height of theoretical plate [(mm), mean
(RSD %)] that was expressed as the efficiency of
the column were found to be 186712.6 (2.56%)
and 0.161 (1.56%), respectively, for injections of
six replicate.
Linearity
Linearity of method was demonstrated
over a linear range of 0.1- 5.0 g/mL at six
concentrations with 2.0 g/mL concentration of
IS (n = 6). The calibration curve was plotted by
the ratio of the peak areas of lidocaine HCl and
IS, versus the concentration of lidocaine HCl.
The linear regression equation and statistical
parameters was calculated by the least squares
method using Microsoft Excel program and
summarized in Table 2.
Cream
An amount of the cream containing
approximately 25 mg of lidocaine HCl was
transferred with methanol to a centrifuge
tube, and vortexed for 5 min. This solution
was centrifuged for 5 min at 4000 rpm. The
supernatant was filtered through a 0.22-m
Millipore filter into a volumetric flask. The
filtrate was adjusted to volume with methanol to
obtain a 0.5 g/mL concentration of lidocaine
HCl for the cream sample.
About 2.0 g/mL concentration of IS was
added into the solutions prepared from cream
and injections. The solutions were analyzed
as described in section Chromatographic
Conditions.
Sensitivity
The limit of detection (LOD) is the lowest
amount of analyte in a sample that can be detected
but not necessarily quantitated as an exact value.
The limit of quantitation (LOQ) is the lowest
amount of analyte that can be quantitatively
determined with suitable precision (42). The
LOD and LOQ values were determined as 3:1
and 10:1 of the signal/noise ratio, respectively,
and given in Table 2.
Selectivity
The selectivity of an analytical method may be
662
0.1-50
a
Regression Equation
y = 1.726x + 0.0004
7.52 x 10-7
2.96 x10-2
Correlation Coefficient
0.9992
4.62x10-3
5.88x10-2
0.03
0.09
Average of six replicate determinations. b y: Peak-area ratios (lidocaine HCl/IS), x: lidocaine HCl concentration.
Between-day
Added
( g/mL )
FoundSD
( g/mL )
Precision
b
RSD%
Accuracy
c
RE%
FoundSD
( g/mL )
0.20
0.190.002
1.05
-5.00
1.25
1.260.02
1.59
4.00
3.950.03
0.76
Precision
b
RSD%
Accuracy
c
RE%
0.210.004
1.91
5.00
0.80
1.270.01
0.79
1.60
1.12
4.040.04
0.99
1.00
SD: standard deviation RSD: relative standard deviation, RE: relative error, (n = 6).
663
Jetokain Ampoule
Jetmonal Ampoule
Emla Cream
Taken Amount
(g/mL)
Added Amount
(g/mL)
Recovery
(%)
RSD
(%)
0.20
0.6980.01
99.6
1.43
0.50
1.25
1.7410.08
98.2
4.60
0.50
0.50
4.00
4.4960.06
99.2
1.33
0.20
0.6960.02
99.2
2.87
1.25
1.7390.05
97.8
2.88
4.00
4.5010.08
100.2
1.78
0.20
0.6840.01
96.8
1.46
1.25
1.7300.02
96.0
1.16
4.00
4.4920.04
98.4
0.89
2,5-dimethyl aniline
1
H-NMR (CDCl3, 400 MHz, ppm) =2.10 (s,
6H), 7.25-6.92 (m, 3H),
13
C-NMR (CDCl3, 100 MHz, ppm) = 135.2,
133.2, 128.3, 127.8, 18.3.
2-(diethylamino)acetic acid
1
H-NMR (CDCl3, 400 MHz, ppm) =1.25 (t,
J=6.8 Hz, 6H), 3.23 (brq, J= 6.8 Hz, 4H), 4.17
(brs, 2H) 10.01 (brs, 1H). 13C-NMR (CDCl3, 100
MHz, ppm) =163.5, 52.0, 48.9, 9.3
Base-induced degradation studies
In the study in alkaline condition, the solution
of lidocaine HCl (0.43 mmol) was heated to
reflux temperature and 2.5 mL of 1.0 M NaOH
solution was added slowly to this solution. The
reaction mixture was stirred magnetically for
24 h, 48 h and 72 h at the same temperature.
After the stirring process, the mixture was
neutralized by 1.0 M HCl solution and then
diluted. The reaction mixture was extracted with
ethyl acetate (3 x 25 mL), and the organic layer
was evaporated. The residue was monitored by
proton NMR spectroscopy. The degradation
products were not observed.
Label Claim
Jetokain ampoule
Jetmonal ampoule
Emla cream
MeanSD
Recovery (%)
RSD (%)
20 mg per mL
19.981.21
99.9
6.06
20 mg per mL
19.961.12
99.8
5.61
25 mg
20.010.99
100.1
4.95
665
Discussion
In the pharmaceutical industry, the
disciplines primarily involved with stability
are pharmaceutical analysis and product
development. The requirement of the
establishment of a stability-indicating assay
has become more clearly mandated with the
International Conference on Harmonization
(ICH) guidelines (33). The guidelines explicitly
require conduct of forced degradation studies
under a variety of conditions, like pH (acidbase), photolytic, oxidation and dry heat (33, 43).
Understanding drug degradation in formulated
product is important in pharmaceutical
development as drug stability and degradation
products could have significant impacts on
formulation development, analytical method
development, package development, storage
conditions, shelf-life determination, and safety
and toxicology concerns (43). Consequently,
we aimed to designate the GC-FID method for
666
Scheme 1
Conclusions
A rapid and sensitive gas chromatography
method with flame ionization detection (GCFID) was developed and validated for the
determination of lidocaine HCl in pharmaceutical
formulations in the form of cream and injections.
The proposed method has several advantages,
which are high specificity, good accuracy and
precision values, short chromatographic run time
(8.5 min) and high analytical recovery value.
In forced degradation studies, many forms of
degradation and fragmentation were not observed
in destruction reactions. It was found that the
structure of the lidocaine HCl molecule was very
stable in alkaline, acidic, oxidation, dry heat and
photolytic media. Furthermore, trifluoroacetic
acid (TFA) was used as the acidic media. In this
media, the decomposition products occurred. The
developed GC-FID method, together with NMR
spectroscopy, for determination of lidocaine HCl
in pharmaceutical formulations can be used for
both routine analysis in quality control studies
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