Determination of prilocaine HCl in pharmaceutical preparations by GC-MS method

Article abstract of DOI:10.14233/ajchem.2014.17150

The novel analytical method using gas chromatography with mass spectrometry detection (GC-MS) method for determination of prilocaine HCl in pharmaceutical preparations were developed and validated. The using lidocaine HCl as the internal standard (IS) was

Full text of DOI:10.14233/ajchem.2014.17150

Asian Journal of Chemistry; Vol. 26, No. 19 (2014), 6650-6654
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.17150
Determination of Prilocaine HCl in Pharmaceutical Preparations by GC-MS Method
*
ALPTUG ATILA andYUCEL KADIOGLU
Department of Analytical Chemistry, Faculty of Pharmacy, Ataturk University, 25240 Erzurum, Turkey
*
Corresponding author: Fax: +90 442 2360962; Tel: +90 442 2315212; E-mail: yucelkadi@yahoo.com
Received: 31 January 2014;
Accepted: 30 April 2014;
Published online: 16 September 2014;
AJC-15980
The novel analytical method using gas chromatography with mass spectrometry detection (GC-MS) method for determination of prilocaine
HCl in pharmaceutical preparations were developed and validated. The using lidocaine HCl as the internal standard (IS) was carried out
on HP-5MS capillary column. The calibration curves were linear (R = 0.999) in concentration range of 40-1000 ng/mL of prilocaine HCl.
The LOD and LOQ values were 15 and 31 ng/mL, respectively. The within- and between-day RSD and RE % values of proposed method
were found to be ≤ 3.4 and ≤ 3.5 %, respectively. o-Toluidine was formed in stored on auto sampler during 72 h was determined by
proposed method. The developed method can be directly and easily applied for determination of prilocaine HCl without derivatized in
pharmaceutical preparations using internal standard methodology.
Keywords: GC-MS method, Prilocaine HCl, Pharmaceutical preparations, Validation.
To our best of knowledge, no GC-MS method for the
INTRODUCTION
analysis of prilocaine HCl in any pharmaceutical preparations
was available in the literature. The development and validated
Local anesthetic drugs are mainly used to reversibly block
nerve function in various local or regional treatments. Local
anesthetics play an important role clinically in dentistry and
an effective method for analysis of drugs in pharmaceutical
preparations and biological samples for understanding the
therapeutic and toxic effects of drugs is critically important.
The aim of the present work is to develop and validate a new
GC-MS method for determination of alone prilocaine HCl with
a simple sample preparation without derivatization in pharma-
ceutical preparations using internal standard methodology.
1,2
minor surgery for temporary relief of pain . Prilocaine (2-
propilamino-N-o-tolil-propiyonamit hydrochloride) (Fig. 1-
3
A) is one of local anesthetics with the amide type . Prilocaine,
unlike other amide anesthetics, is a secondary amino derivative
of toludine. It produces less vasodilation and toxicity than
lidocaine and is considered relatively free from an allergic
4
EXPERIMENTAL
reaction . Prilocaine is extensively metabolized by the liver.
Prilocaine's primary limiting factor clinically is the production
of methemoglobinemia, a side effect caused by its metabolite
Prilocaine HCl reference substance and lidocaine HCl used
as internal standard (Fig. 1-B, IS) was a kindly supplied by
Novartis Pharmaceutical Industry (Ankara, Turkey) and Doping
Control Center of Hacettepe University (Ankara, Turkey). The
high-purity all other reagents were purchased from Merck
(Germany).All gases were supplied by Havas (Ankara, Turkey)
Pharmaceutical preparations: The following pharma-
ceutical preparations of prilocaine HCl were obtained from
local sources in Erzurum (Turkey):
5
-7
o-toludine .
The literature shows that capillary electrophoresis , HPLC
3
8
-19
with different detection , Liquid chromatography-tandem
20
mass spectrometry , sequential injection chromatography with
21
22
Franz cell and adsorptive square wave method have been
reported for determination of alone prilocaine and the binary
mixture with other local anesthetics of prilocaine in biological
samples (plasma and serum) and pharmaceutical preparations.
Besides the determination of prilocaine and the mixtures with
other local anesthetics of prilocaine in biological materials
®
Emla cream (5 % cream,Astra ZenecaA.S., Turkey) con-
taining 25 mg prilocaine HCl, 25 mg lidocaine HCl, 55 mg
poloxamer188 and 155 mg poloxomer 407.
®
(
plasma and urine) have been done with gas chromatography-
Citanest injection (2 % flacon,Astra ZenecaA.S., Turkey)
2
3,24
mass spectrometry method
method
and gas chromatography
containing prilocaine HCl 400 mg/20 mL, methyl para-
hydroxybenzoate and sodium chloride.
2
5-31
.

Vol. 26, No. 19 (2014)
Determination of Prilocaine HCl in Pharmaceutical Preparations by GC-MS Method 6651
solution (100 µg/mL) of prilocaine HCl in methanol and stored
H
N
at -20 °C when not in use.
HCl
A
N
H
Internal standard (lidocaine HCl, IS) working solution was
prepared at the final concentration of 100 ng/mL.
O
Procedure of pharmaceutical preparations
®
Citanest injection (2 % flacon): The content of a flacon
were mixed into flask and an aliquot of the solution equivalent
to 20 mg prilocaine was quantitatively transferred to 50 mL-
calibrated measuring flask and made up to the mark with
methanol. The solution was filtered through a 0.22 µm milli-
pore filter. The filtrate was adjusted to volume with methanol
to obtain a 150 ng/mL concentration of prilocaine HCl for
flacon.
H
N
HCl
B
N
O
Fig. 1. Chemical structure of prilocaine HCl (A) and IS [lidocaine HCl (B)]
Determination was performed HP 6890 Series II gas
chromatography equipped with a HP 7673 autosampler,
electronic pressure control, Hewlett-Packard automatic injector
®
Emla cream (5 %): An amount of the cream equivalent
to about 25 mg of prilocaine HCl, accurately weighed into a
centrifuge tube, was mixed with methanol for 5 min in ultra-
sonic bath and centrifugated for 5 min 4000 rpm. The super-
natant was filtered through a 0.22 mm millipore filter into
calibrated flask. The filtrate was adjusted to volume with
methanol to obtain a 150 ng/mL concentration of prilocaine
HCl for cream sample.
About 100 ng/mL concentration of IS (i.e., lidocaine) was
added into the solutions prepared from cream and injections.
The solutions were analyzed as described earlier. The content
of prilocaine HCl in pharmaceutical preparations was quan-
tified using regression equation.
(
Model 7673) and an Agilent 5972 series mass selective
detector with electron ionization. The chromatographic sepa-
ration was performed on 5 % methyl phenyl silicone HP-5MS
capillary column (30 m × 0.25 mm i.d., 0.25 µm film thick-
nesses) purchased from Hewlett-Packard (Waldbronn,
Germany). The split mode (10:1) was used with helium carrier
gas and the flow rate of carrier gas was kept constant during
run at 1 mL/min. The injector volume was 1.5 µL. The injector
and detector temperatures were set at 270 °C. The GC tempe-
rature gradient program was as follows: initial temperature
8
1
0 °C, held for 1.5 min; to finally temperature 260 °C at a rate
0 °C 1/min, held for 5 min. The MS detector parameters were:
Method validation: The validation of method was carried
out by establishing specificity, linearity, recovery values,
precision, accuracy and sensitivity according to international
transfer line temperature 240 °C, solvent delay 2.5 min and
electron energy 75 eV. The MS was run in the electron ioniza-
tion (EI) mode with selected ion monitoring (SIM) with a 0.5
s/scan for quantitative analysis (m/z 86 for both prilocaine HCl
and lidocaine HCL; m/z 106 for o-toluidine). The MS spec-
trums were given at Fig. 2 A-B.
32
conference on harmonization guidelines for validation of
analytical procedures.
RESULTS AND DISCUSSION
Specificity: The specificity of method has been demons-
trated by the representative chromatograms for prilocaine HCl
and IS in standard solutions shown in Fig. 3. The retention
time of prilocaine HCl and lidocaine HCl is 5.84 min and 6.58
min, respectively. Different temperature gradient programs
were investigated for exception of matrix interference. At
the end of this investigation, the best temperature program
described earlier was selected for a good resolution.
8
6
1
1
1
4000
2000
0000
(A)
CH
3
CH3
H
H
N
C
O
C N
2 2 2
CH CH CH HCl
H
8
6
4
2
000
000
000
000
0
5
8 65
1
07
220 234
50
60 70 80 90 100 110 120 130 140150160 170 180 190 200 210 220 230 240
m/z
5
4
3
2
1
0
y = 0.004x + 0.0414
2
8
000
7000
000
5000
1
06
R
= 0.9992
(
B)
2
1
1
000
500
000
6
Prilocaine
NH
2
0
500 1000 1500
Concentration (mg/mL)
4
3
2
1
000
000
000
000
0
CH
3
4
4
IS (lidocaine)
500
77
5
30 540 550 560 570 580 590 600 610 620 630 640 650 660
Time (min)
5
6
91
207
18 13314 71 58170191
2
33251263 282
315 336 349 361
1
0
Fig. 3. Chromatograms of standard solutions (40, 100, 250, 500, 750 and
1000 ng/mL) of prilocaine and IS (100 ng/mL)
40
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380
m/z
Fig. 2. MS spectra of prilocaine HCl (A) and o-toluidine (B)
Linearity: Linearity of method was demonstrated over a
linear range of A volume of 40 to 1000 ng/mL at six concen-
trations (40, 100, 250, 500, 750 and 1000 ng/mL) with 100
ng/mL concentration of IS (n = 6). The calibration curve was
plotted by the ratio of the peak areas of prilocaine HCl and IS,
Preparation of standard solution and quality control
solutions: The standard working (SW) solutions (40, 100, 250,
00, 750 and 1000 ng/mL) and quality control (QC) solutions
100, 250 and 500 ng/mL) were daily prepared from stock
5
(

6
652 Atila et al.
Asian J. Chem.
versus the concentrations of prilocaine HCl (Fig. 3). The linear
and inter-day accuracy were found to be ≤ 3.5 %. Precision
and accuracy studies in pharmaceutical preparations showed
acceptable RSD and RE % values as shown in Table-2.
Analytical recovery: To double check the accuracy of
the proposed method, the standard addition technique was
applied. The QC solutions were added to 150 ng/mL concen-
tration of pharmaceutical preparations solutions and assayed
with GC-MS method. The present analytical recovery of the
added standard to the assay samples was calculated from
followed equation:
regression equation was calculated by the least squares method
®
using Microsoft Excel program and summarized in Table-1.
TABLE-1
RESULTS OF REGRESSION ANALYSIS OF PRILOCAINE HCl
Parameters
GC-MS
40-1000
Linearity (ng/mL)
Regression equation
Standard deviation of slope (Sa)
Standard deviation of intercept (Sb)
The standard deviation of the
residuals, (Sy/x)
a
y = 0.004x + 0.0414
-
4
6.6 × 10
6.4 × 10
9.9 × 10
-4
-2
Analytical Recovery % = [(C
where C is total concentration of the analyte determined; C
is the concentration of the pure analyte added to the prepa-
ration; C is the concentration of the present analyte in the
t
-C
u
)/C
a
] × 100
t
u
Correlation coefficient
Standard deviation of correlation
coefficient
0.9992
4.57 × 10
-3
a
preparation. The average per cent recoveries were quanti-
tatively determined as 99.6 % for both preparations of method,
indicating good accuracy of the method. No interference from
the common excipients was observed. The RSD % values of
recovery were found as ranged from 0.8 to 1.9 % (Table-3).
Interferences study: The effects of common excipients
and additives were tested for their possible interferences in
the assay of prilocaine HCl. In addition to the active ingredient,
prilocaine HCl, flacon and cream content contains the follo-
wing inactive ingredients: methyl para-hydroxybenzoate,
sodium chloride, 55 mg poloxamer188 and 155 mg poloxomer
407. It has been determined any interference of these subs-
tances at the levels found in dosage forms.
Limit of detection (LOD, ng/mL)
Limit of quantification (LOQ, ng/mL)
15
31
a
Average of six replicate determinations
Sensitivity: The limit of detection (LOD) is the lowest
amount of analyte in a sample which can be detected but not
necessarily quantitated as an exact value. The limit of quanti-
tation (LOQ) is the lowest amount of analyte which can be
quantitatively determined with suitable precision. The LOD
and LOQ values were determined as 3:1 and 10:1 of the signal/
noise ratio, respectively. LOD and LOQ values were given in
Table-1. Both accuracy and precision of these values were well
within the proposed criteria (RSD % < 20 %).
Precision and accuracy: The repeatability (intra-day)
and intermediate precision (inter-day) were done in precision
studies. The intra-day studies were evaluated by measuring
QC samples, using six replicates at same concentrations and
during the same day. The inter-day studies were done by
comparing the assay the same samples on six different days.
The precision of method were given as the relative standard
deviation (RSD % = 100 × standard deviation/mean) and the
accuracy were reported with percent relative error [RE % =
Stability: Stability studies were performed with standard
solutions of prilocaine HCl (250, 500 and 750 ng/mL) and these
solutions were stored at 4 °C (refrigerator), room temperature
2
4,48
and on auto sampler at
and 72 h time and then changes in
concentration of solutions under conditions of the study were
evaluated using the proposed method. One set of these
solutions were assayed immediately and taken as standard (100
%). Stock solution of prilocaine HCl was found to be stable
for three month. Standard solutions of prilocaine HCl were
found to be stable for 48 h at room temperature and on auto
sampler. After 72 h at room temperature and auto sampler, it
was observed that prilocaine were converted to its metabolite
(
found concentration-known concentration) × 100/known
concentration]. The RSD % values for intra-day and inter-day
precision were found to be ≤ 3.4 %. The RE % for intra-day
TABLE-2
PRECISION AND ACCURACY OF PROPOSED METHOD
Added
Intra-day
Inter-day
(
ng/mL)
Found ± SD
96.5 ± 3.2
247.4 ± 6.3
486.9 ± 8.6
Precision RSD (%) Accuracy RE (%)
Found ± SD
101.5 ± 3.5
249.9 ± 6.6
489.2 ± 6.4
Precision RSD (%) Accuracy RE (%)
100
250
500
3.3
2.5
1.8
-3.5
-1.0
-2.6
3.4
2.6
1.3
1.5
-0.04
-2.1
SD: Standard deviation of six replicate determinations, RSD: Relative standard deviation, RE: Relative error
TABLE-3
ANALYTICAL RECOVERY VALUES BY STANDARD ADDITION METHOD (n = 6)
Pharmaceutical
preparations
Amount taken
(ng/mL)
Amount added
(ng/mL)
Total amount found
(ng/mL) (mean ± SD)
Recovery (%)
RSD (%)
1
250
00
247.9 ± 2.6
398.9 ± 4.7
649.2 ± 5.1
249.9 ± 4.9
400.9 ± 5.2
649.7 ± 7.6
98.6
99.3
99.5
99.9
100.6
99.8
1.1
1.2
0.8
1.9
1.3
1.2
®
Emla cream (5 %)
150
5
1
00
00
®
Citanest injection
150
250
00
(
2 % flacon)
5

Vol. 26, No. 19 (2014)
Determination of Prilocaine HCl in Pharmaceutical Preparations by GC-MS Method 6653
(
o-toluidine) (Fig. 4). Because of its chemical structure, prilo-
Prilocaine
caine is readily hydrolyzed in alcohol medium. The formation
of o-toluidine as a major degradation product in solutions of
prilocaine HCl was checked by spiking of the standard solution
of o-toluidine. o-Toluidine could be formed during degradation
of prilocaine HCl was based on information in literature that
amide anesthetics were degraded to o-toluidine in temperature
3
5000
0000
Citanest injection
Emla cream
3
IS (lidocaine)
25000
20000
15000
1
6,17
change
.
Prilicaine + lidocaine
1
1
20000
00000
Alone prilocaine
Alone lidocaine
Prilocaine
8
6
4
2
0000
0000
0000
0000
0
o-Toluidine
1
0000
5000
4
5
6
7
Time (h)
Fig. 5. Chromatograms of pharmaceutical preparations
2
00 300 400 500 600 700 800 900 10001100 1200 13001400 1500 1600 1700
Time (min)
Fig. 4. Stability of prilocaine HCl on auto sampler for 72 h time
be accepted as a reliable and applicable method. The precision
of method were adequate, because the RSD % values were
less than 3.4 %. Accuracy of method (RE %) was less than
3.5 %. There are several advantages of this method which are
high specificity, good accuracy and precision values, short run
time (6.3 min). In the chromatograms taken, the following
peaks: prilocaine HCl with retention time approx. 5.82 min;
lidocaine HCl used IS with retention time approx. 6.08 min
and the degradation product o-toluidine with retention time
of approx. 1.96 min (Fig. 3, 4). Under the described conditions
(Fig. 3) a linear relationship between the peak-area ratio (y:
prilocaine peak/IS peak) and analyte concentration (x) were
obtained (Table-1). In addition to these, the analytical recovery
percentage of proposed method is high.
Application of the method for analysis of pharma-
ceutical preparations: The proposed method was evaluated
in the assay of commercially available flacon containing
prilocaine HCl 400 mg/20 mL and cream containing 25 mg
prilocaine and 25 mg lidocaine. Evaluation was performed
using the calibration curve method since no significant diffe-
rence between the slopes of the calibration curves for standards
and pharmaceutical preparations solution was observed. The
results obtained are satisfactorily accurate and precise as indi-
cated by the excellent % recovery (Table-4). Experiments
showed that there was no interference from the additions and
excipients (Fig. 5). The determination repeated for six times,
final recovery of method for both preparations was obtained
approximately to be 99.6 and 99.2 %, with an RSD % of 2.85
and 1.29 %.
In the present study, we report a highly selective gas chro-
matography (GC) combined mass spectrometry detection (MS)
that enabled us to quantify the prilocaine HCl without deriva-
tization in pharmaceutical preparations. Prilocaine HCl is one
of local anesthetic substances. An important aspect of the
implementing a new assay in routine quality control analysis
is that it should be thoroughly evaluated before introduction
for routine use. GC method with different detections can be
considered to be a very appropriate method for analysis of
local anesthetic substances that these are very volatile subs-
tances. A rapid and reliable GC-MS method were developed
and validated for the determination of prilocaine HCl in
pharmaceutical preparations in the form of cream and flacon.
The proposed method has supplied all the requirements in
terms of accuracy, linearity, recovery and precision that could
There are many preparations for local anesthesia on the
pharmaceutical market, in which prilocaine HCl and lidocaine
HCl can occur as active substances. Both drugs were served
as internal standard for each other. Prilocaine used in anesthetic
practice is least toxic than lidocaine. o-Toluidine is a metabolite
of prilocaine HCl. Prilocaine metabolizes to o-toluidine during
biotransformation, which may oxidize hemoglobin to methe-
moglobin and also o-toluidine has been shown to be carcino-
genic in laboratory animals in NTP studies. o-Toluidine can
be potential technological impurities of medicinal products
because they are used as substrates in the synthesis of pharma-
ceuticals. In addition, the hydrolysis of the amide linkage of
prilocaine results in the formation of decomposition product
16,17,33
o-toluidine during storage of drugs containing prilocaine
.
After 72 h at room temperature and auto sampler, it was
observed that prilocaine were converted to its metabolite
(o-toluidine).After 72 h storage of solutions of prilocaine HCl,
it was observed that has formed of o-toluidine in study. The
TABLE-4
DETERMINATION OF PRILOCAINE HCl IN PHARMACEUTICAL PREPARATIONS
a
Pharmaceutical preparations
Found ± SD (mg)
24.91 ± 0.71
Recovery (%)
99.6
RSD (%)
2.85
Confidence Interval
98.0-101.0
®
Emla Cream (5 %) (25 mg prilocaine)
®
Citanest Injection (2 % flacon, prilocaine HCl 400 mg/20 mL)
396.8 ± 5.12
99.2
1.29
98.8-100.0
a
SD: Standard deviation, RSD: Relative standard derivation, Average of six replicate determinations

6
654 Atila et al.
Asian J. Chem.
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8
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In this study, new GC-MS method has been developed to
provide a sensitive, simple and quantitative assay of prilocaine
HCl in pure and pharmaceutical preparations. When the solu-
tions of prilocaine HCl were stored at room temperature and
on auto sampler during 72 h, it was observed that o-toluidine
was formed. o-Toluidine was determined by proposed method.
The preparation of samples and their analysis was performed
in a relative short time. Therefore, it was suggested that the
GC-MS method was equally applicable. Besides, the proposed
method can be applicable in routine quality control and clinical
laboratories.
6
5
1
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