Determination of prilocaine HCL in bulk drug and pharmaceutical formulation by GC-NPD method

Abstract

A novel analytical method was developed and validated for determination of prilocaine HCl in bulk drug and pharmaceutical formulation by gas chromatography-nitrogen phosphorus detection (GC-NPD). The chromatographic separation was performed using a HP-5MS column. The calibration curve was linear over the concentration range of 40-1000 ng ml(-1) with a correlation coefficient of 0.9998. The limits of detection (LOD) and quantification (LOQ) of the method were 10 and 35 ng m(-1), respectively. The within-day and between-day precision, expressed as the percent relative standard deviation (RSD%) were less than 5.0%, and the accuracy (percent relative error) was better than 4.0%. The developed method can be directly and easily applied for determination of prilocaine HCl in bulk drug and pharmaceutical formulation using internal standard methodology.

Full text

1
SCIENTIFIC PAPER
DETERMINATION OF PRILOCAINE HCl IN BULK DRUG AND
PHARMACEUTICAL FORMULATION BY GC-NPD METHOD
ALPTUG ATILA1, YUCEL KADIOGLU1*
1Department of Analytical Chemistry, Faculty of Pharmacy, Ataturk University, 25240,
Erzurum, Turkey
Received 24.01.2012.
Revised 17.6.2012.
Accepted 18.6.2012.
* Corresponding author: Y. Kadioglu; Department of Analytical Chemistry, Faculty of Pharmacy, Ataturk
University, 25240, Erzurum, Turkey; Tel.: +90-442-2315212; Fax.;+90-442-2360962; E-mails:
yucel@atauni.edu.tr ; yucelkadi@yahoo.com

2
ABSTRACT
The novel analytical method was developed and validated for determination of
prilocaine HCl in bulk drug and pharmaceutical formulation by gas chromatography-
nitrogen phosphorus detection (GC-NPD). The chromatographic separation was
performed using a HP-5MS column. The calibration curve was linear over the
concentration range of 40-1000 ng ml-1 with a correlation coefficient of 0.9998. The
limits of detection (LOD) and quantification (LOQ) of method were 10 ng ml-1 and 35
ng ml-1, respectively. The within-day and between-day precision, expressed as the
percent relative standard deviation (RSD%) was less than 5.0%, and accuracy (percent
relative error) was better than 4.0%. The developed method can be directly and easily
applied for determination of prilocaine HCl in bulk drug and pharmaceutical
formulation using internal standard methodology.
Key words: GC-NPD, prilocaine HCl, pharmaceutical formulation, validation

3
INTRODUCTION
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 minor surgery for temporary relief of pain [1,2]. Prilocaine, 2-
propilamino-N-o-tolil-propiyonamit hydrochloride (Fig. 1 A), is a local anesthetic of the
amide type [3]. 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 reaction [4]. 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 o-toludine [5,6].
< Incorporate Fig. 1>
Several methods have been reported for determination of prilocaine HCl in
biological samples and pharmaceutical formulations including the capillary
electrophoresis method [3], HPLC with different detection [5,6-16], the liquid
chromatography-tandem mass spectrometry [17], the sequential injection
chromatography with Franz cell [18], adsorptive square wave method [19] and
spectrophotometry method [20]. In addition, the determination of mixtures with other
local anesthetics of prilocaine HCl in biological samples have been done with the GC-
MS [21,22] and GC methods [23-29].
So far, according to our present knowledge, no GC-NPD method for the analysis
of prilocaine HCl alone in any pharmaceutical formulations is available in the literature.
The development a selective and effective method for drug analysis is important for
keeping abreast of therapeutic and toxic effects in biological samples and quality
controlling studies in pharmaceutical formulations. The aim of the present work is to
develop and validate a new GC-NPD method for determination of prilocaine HCl in
pharmaceutical formulation with a simple sample preparation using internal standard
methodology. The proposed method was validated with validation parameters, which
are sensitivity, specificity, linearity, precision, accuracy, stability and analytical
recovery in accordance with International Conference on Harmonization (ICH)
guidelines [30].

4
EXPERIMENTAL
Materials and Reagents
Prilocaine HCl that used as reference substance (99.9% purity) was supplied by
Novartis Pharmaceutical Industry (Ankara, Turkey) and lidocaine HCl (99.8% purity)
that was used as internal standard (IS; Fig. 1 B) was supplied by Doping Control Center
of Hacettepe University (Ankara, Turkey). The high-purity grade methanol and all other
reagents were purchased from Merck (Germany). All gases were supplied by Havas
(Ankara, Turkey)
The following pharmaceutical formulation of prilocaine HCl was obtained from
local sources in Erzurum (Turkey):
- Citanest® Injection (2% flacon, Astra Zeneca A.S., Turkey) containing prilocaine HCl,
para-hydroxybenzoate and sodium chloride with the concentration of 20 mg ml-1, 1 mg
ml-1 and 0.46 mg ml-1, respectively.
Equipment
The chromatographic analysis was performed by a HP 6890 Series II gas
chromatography system equipped with a HP 7673 auto sampler, Hewlett-Packard
automatic injector (Model 7673), HP 5890 nitrogen-phosphorus detector (NPD) and HP
software.
Chromatographic Conditions
The chromatographic separation was achieved using a HP-5MS column (25
m×0.2 mm i.d. × 0.33 μm film thickness, cross-linked [5% phenyl]-methylpolysiloxano,
Germany). The split mode (10:1) was used with helium carrier gas and the flow rate of
carrier gas was kept constant during analysis at 0.7 ml min-1. Hydrogen (4 ml min-1) and
dried air (60 ml min-1) were used as auxiliary gases for the detector (NPD). The injector
volume was 3.0 μl. The injector and detector temperatures were 250 °C. The oven
temperature programs were as follows: initial temperature of 80°C, then ramp rate of
15°C min-1 and final temperature of 300°C, where the temperature was held for 4 min.
Chromatograms, which are obtained in these operating conditions for standard
solutions, are shown in Fig. 2.

5
< Incorporate Fig. 2>
Preparation of Standard Solutions
The standard working (SW) solutions (40, 100, 250, 500, 750 and 1000 ng ml-1)
and quality control (QC) samples (100, 250 and 500 ng ml−1) were prepared in methanol
from stock solution (100 µg ml-1). All solutions were prepared daily and stored at -20 C
when not in use.
SW solution of lidocaine HCl (Internal Standard, IS) was prepared at 100 ng ml-
1 concentration with methanol from the stock solution (50 µg ml-1).
Preparations of Pharmaceutical Formulation
Prilocaine (Citanest® flacon) is a drug that is injected during various surgical or
dental procedures. The content of a flacon was mixed into a volumetric flask and an
aliquot of the solution equivalent to 20 mg prilocaine HCl 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 Millipore filter. The filtrate was diluted with
methanol to obtain a 150 ng ml-1 concentration of prilocaine HCl for pharmaceutical
formulation.
About 100 ng ml-1 concentration of IS was added into the solution prepared from
flacon. The solution was analyzed as described in section Chromatographic Conditions.
Data Analysis
All statistical calculations were performed with the Statistical Product and
Service Solutions (IBM SPSS) for Windows, version 20.0. Correlations were
considered statistically significant if calculated P values were 0.05 or less.

6
RESULTS
Specificity
Specificity should confirm the ability of the method to unequivocally assess the
analyte in the presence of other components that may be present (for example:
impurities, degradation products and matrix components). The specificity of method
was demonstrated by the representative chromatograms for prilocaine HCl and lidocaine
HCl (IS) in standard solutions shown in Fig. 2. The retention time of prilocaine HCl and
lidocaine HCl is 6.78 min and 7.22 min. Different temperature programs were
investigated for exception of matrix interference. At the end of this investigation, the
best temperature program was selected for a good resolution and thus the all
experiments was used the oven temperature program described at section
Chromatographic Conditions. When the ramp rate was more or less than 15 ºC min-1,
the good resolution of the peaks (analyte peak and matrix interference peaks) was not
obtained.
Linearity
Linearity was established over a linear range of 40 - 1000 ng ml-1 at six
concentrations with a constant concentration of IS (100 ng ml-1). The calibration curve
was constructed by plotting the ratio of the peak areas of prilocaine HCl and IS, versus
the concentrations of prilocaine HCl (Fig. 3). The linear regression equation and
statistical parameters was calculated by the least squares method using Microsoft
Excel® program and summarized in Table 1. Relative rezidual standard deviation
(S∆y/y,n-2) is also included in the table [31].
< Incorporate Fig. 3>
< Incorporate Table 1>
Limit of Detection and Quantification
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
quantification (LOQ) is the lowest amount of analyte which can be quantitatively
determined with suitable precision [30]. The LOD and LOQ values of the developed

7
method were determined as 3:1 and 10:1 of the signal/noise ratio, respectively, by
injecting progressively low concentration of the standard solution under the
chromatographic conditions. These values are also listed in Table 1.
Precision and Accuracy
Assay precision was determined by repeatability (within-day) and intermediate
precision (between-day). The within-day was evaluated by assaying samples, at same
concentration and during the same day. The between-day was studied by comparing the
assay on different six days. The accuracy of this analytic method was evaluated by
checking at three different concentrations of prilocaine HCl with IS. The precision of
method were given as the relative standard deviation [RSD %= (100 x standard
deviation)/mean] and the accuracy of method were given with percent relative error
[RE% = (found concentration known concentration) x 100 known concentration].
The RSD % values for within-day and between-day precision of proposed method were
found to be 4.9%. The RE% for within-day and between-day accuracy for method
were found to be 3.8%. Precision and accuracy studies in pharmaceutical formulation
showed acceptable RSD % and RE % values. The results were shown in Table 2.
< Incorporate Table 2>
Analytical Recovery
To double check the accuracy of the proposed method, the recovery study was
performed with two different ways. In first method, the standard addition technique was
used. The three different concentrations (100, 250 and 500 ng ml-1) of standard sample
were added to 150 ng ml-1 concentration of solution of pharmaceutical formulation and
assayed with GC-NPD method. The analytical recovery values of proposed method
were calculated from followed equation:
Analytical Recovery % = [(Ct-Cu) / Ca] x 100
where Ct is total concentration of the analyte determined; Cu is the concentration of the
pure analyte added to the formulation; Ca is the concentration of the analyte present in
the formulation. The average percent recoveries were determined between 98.6% and
99.6% for proposed method, indicating good accuracy of the method. No interference

8
from the common excipients was observed. The RSD % values of recovery were found
as ranged from 0.9% to 1.4% (Table 3).
In second method, the technique of proportioning was used. The solutions in
three different concentrations (100, 250 and 500 ng ml-1) from pharmaceutical
formulation were prepared and assessed with same procedures. The percent recovery
values were calculated from followed equation for each case: Recovery % = (Cfound /
Cformulation) x 100. The average recovery values for second method were determined
between from 100.1 and 98.8 %. The RSD % values of recovery were found as ranged
from 1.9% to 1.2% (Table 3).
The recovery values of both methods were compared statistically by One-
Sample t-test at 95 % confidence level with five degrees of freedom. The t-values were
obtained as t=529.2 for first method and t=415.5 for second method. There was no
significant difference between both recovery methods (p 0.05).
< Incorporate 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 content contains the following inactive ingredients: methyl para-
hydroxybenzoate and sodium chloride. It has been determined any interference of these
substances at the levels found in dosage forms.
Stability
Stability studies were performed on pharmaceutical formulation and standard
solutions of prilocaine HCl (250, 500 and 750 ng ml-1) and these solutions were stored
at 4oC (refrigerator), room temperature and auto sampler at 24, 48 and 72 h time and
then changes in concentration of standard solutions and pharmaceutical formulation
under conditions of the study were evaluated using the GC-NPD 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 and pharmaceutical solution 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

9
was observed that prilocaine were converted to its metabolite (o-toluidine) (Fig. 4).
Because of its chemical structure, prilocaine 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 change [5,14].
< Incorporate Fig. 4>
Application of Method for Analysis of Pharmaceutical Formulation
The proposed method was evaluated in the assay of commercially available
flacon containing prilocaine HCl 400 mg/20 ml (Citanest® flacon). Evaluation was
performed using the calibration curve method since no significant difference between
the slopes of the calibration curves for pharmaceutical formulation and standard
solutions was observed. The amount of flacon containing 400 mg prilocaine HCl per 20
mL was determined of six replicates. The results obtained are satisfactorily accurate and
precise as indicated by the excellent % recovery and SD<4.25. Experiments showed that
there was no interference from the additions and excipients. The determination repeated
for six times, final recovery of formulation was obtained approximately 98.9%, with an
RSD % of 1.07.

10
DISCUSSION
In the present study, a highly selective gas chromatography (GC) with nitrogen-
phosphorous detection (NPD) that enabled us to quantify the prilocaine HCl without
derivatization in pharmaceutical formulation was developed and validated. Prilocaine
HCl is one of local anesthetic substances. Chromatographic analyses of pharmaceutical
compounds have evolved as drug industry matured. Gas-liquid chromatography (GLC)
developed from early 1950s to the present with many concurrent innovations in
chromatography columns and detection systems [32]. Gas chromatography has found its
niche in the monitoring of certain impurities, measuring and characterizing excipients,
preservatives, and active drugs. In assays where sensitivity is required, gas
chromatographic methods are still unsurpassed [32]. 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 substances. The proposed method has
supplied all the requirements in terms of accuracy, linearity, recovery and precision that
could be accepted as a reliable and applicable method. The precision of method was
adequate, because the RSD % values were less than 5.0%. Accuracy of method (RE %)
was less than 4.0%. There are several advantages of this method which are high
specificity, good accuracy and precision values, short chromatographic run time (7.5
min). In the chromatograms taken with proposed method, the following peaks:
prilocaine HCl with retention time approximately 6.78 min; lidocaine HCl (IS) with
retention time approximately 7.22 min and the degradation product o-toluidine with
retention time of approximately 2.15 min (Fig. 2 and Fig. 4), were identified. Under the
described chromatographic conditions (Fig. 3), a linear relationship between the peak-
area ratio (y = prilocaine peak area/IS peak area) and analyte (prilocaine) concentration
(x) were obtained (Table 1). In addition to these, the analytical recovery percentage of
proposed method is high.
There are many preparations for local anesthesia on the pharmaceutical market,
in which prilocaine and lidocaine can occur as active substances. Both drugs were

11
served as internal standard for each other. Prilocaine used in anesthetic practice is least
toxic than lidocaine. o
‑
Toluidine is a metabolite of prilocaine. Prilocaine metabolizes to
o-toluidine during biotransformation, which many oxidize hemoglobin to
methemoglobin and also o-toluidine has been shown to be carcinogenic in laboratory
animals in the National Toxicology Program (NTP) studies [33]. o-Toluidine can be
potential technological impurities of medicinal products because they are used as
substrates in the synthesis of pharmaceuticals. In addition, the hydrolysis of the amide
linkage of prilocaine results in the formation of decomposition product o-toluidine
during storage of drugs containing prilocaine [5, 14, 33]. After 72 hours storage of
standard solutions of prilocaine and pharmaceutical formulation, it was observed that
prilocaine were converted to its metabolite (o-toluidine) in study. The content of o-
toluidine in standard solutions and pharmaceutical formulation chosen for this study
was determined by the standard addition method, while the content of local anesthetics
in pharmaceutical formulation studied was assayed by GC-NPD method.
Conclusion
In this study, new GC-NPD method was developed to provide a very sensitive
and quantitative assay of prilocaine HCl in bulk drug and pharmaceutical formulation.
In addition, o-toluidine was determined in stability studies. The analysis and preparation
of samples was performed in a relative short time. The method can be applied in routine
quality control analysis of pharmaceutical formulations and clinical laboratories.
Acknowledgements
The authors are grateful to Ataturk University for financial support of this works and
Doping Control Centre of Hacettepe University for GC-NPD equipment used.

12
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Electrochem. 46 (2010) 999-1006.

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[20] A. Atila, Y. Kadioglu. IJPSR. 3 (2012) 1018-1021.
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14
Captions of Table
Table 1. Results of regression analysis of prilocaine HCl (n=6)
Table 2. Precision and accuracy of GC-NPD method
Table 3. Analytical recovery values with two methods of proposed method
Table 4. Determination of prilocaine HCl in flacon (400 mg prilocaine HCl/20 ml)

15
Table 1. Results of regression analysis of prilocaine HCl
Parameters
GC-NPD
Linearity (ng ml-1)
40-1000
Regression equationa
by=0.0073x-0.0484
Standard deviation of slope (Sa)
5.4x10-4
Standard deviation of intercept (Sb)
2.8x10-2
Correlation coefficient
0.9998
Standard deviation of correlation
coefficient
0.0045
Relative residual standard deviation
(S∆y/y, n-2)
0.059
Limit of detection (LOD, ng ml-1)
10
Limit of quantification (LOQ, ng ml-1)
35
aAverage of six replicate determinations
b y: Peak-area ratios (prilocaine/IS), x: prilocaine concentration

16
Table 2. Precision and accuracy of GC-NPD method
Within-day
Between-day
Added
(ng ml-1)
Found SDa
(ng ml-1)
Precision
bRSD %
Accuracy
cRE %
Found SDa
(ng ml-1)
Precision
bRSD %
Accuracy
cRE %
100
103.3±2.7
2.6
3.3
101.1 5.0
4.9
1.1
250
251.9 2.6
1.0
0.8
250.8 8.6
3.4
0.3
500
480.9 9.5
2.0
-3.8
495.7±8.9
1.8
-0.9
aSD: standard deviation of six replicate determinations, bRSD: relative standard deviation, cRE: relative error

17
Table 3. Analytical recovery values with two methods of proposed method
Amount
added
(ng ml-1)
aAmount
taken
(ng ml-1)
b Total amount found
(ng ml-1)
(mean ±SD)
Recovery
(%)
RSD
(%)
The standard
addition technique
100
150
248.9±2.8
99.3
1.1
250
397.9±5.7
98.6
1.4
500
649.4±6.1
99.6
0.9
The technique of
proportioning
-
100
98.8±1.9
98.8
1.9
250
250.2±4.4
100.1
1.8
500
497.8±5.8
99.6
1.2
a Solutions of pharmaceutical preparation, b Average of six replicate determinations

18
Table 4. Determination of prilocaine HCl in flacon (400 mg prilocaine HCl/20 ml)
Pharmaceutical
formulation
aFound SD
(mg)
Recovery
(%)
RSD
(%)
Confidence
Interval
Citanest® Injection
(2% flacon)
395.6 4.25
98.9
1.07
98.0-99.9
a Average of six replicate determinations

19
Captions of Figures
Fig. 1. Chemical structure of prilocaine HCl (A) and IS [lidocaine HCl; (B)]
Fig. 2. GC chromatogram of standard solutions (40, 100, 250, 500, 750 and 1000 ng ml-
1) of prilocaine and IS (100 ng ml-1)
Fig. 3. Calibration curve for determination of prilocaine HCl with proposed method
Fig. 4. GC chromatogram of prilocaine and o-toluidine formed during storage at auto
sampler during 72 h period

20
Fig. 1. Chemical structure of prilocaine HCl (A) and IS [lidocaine HCl; (B)]

21
Fig. 2. GC-NPD chromatogram of standard solutions (40, 100, 250, 500, 750 and 1000
ng ml-1) of prilocaine and IS (100 ng ml-1)

22
Fig. 3. Calibration curve for determination of prilocaine HCl with proposed method

23
Fig. 4. GC chromatogram of prilocaine and o-toluidine formed during storage at auto
sampler during 72 h period