International Journal of Current Pharmaceutical Research

ISSN- 0975-7066 Vol 9, Issue 6, 2017

Original Article
STABILITY INDICATING RP-LC ASSAY METHOD FOR CARISOPRODOL

M. SANGEETHAa*, TIRUMALAa, NAGAMALLIKAb

aVijay College of Pharmacy, Das Nagar, Nizambad, Telangana, bQIS College of Pharmacy, Ongole, Andhrapradesh
Email: [email protected]
Received: 21 Aug 2017, Revised and Accepted: 13 Oct 2017
ABSTRACT
Objective: A reverse phase stability-indicating HPLC method was developed for the determination of Carisoprodol in pharmaceutical dosage forms.
The chromatographic elution was achieved on C18, 250 mm × 4.6 mm, 5-μm particle size column.
Methods: The mobile phase contains a mixture of water and acetonitrile in ratio of 60:40 v/v. The flow rate was 1.0 ml min-1 and was detected by
Refractive index detector.
Results: The method was proven to be linear over a range of 1 to 4 mg/ml with a mean correlation coefficient of 0.99998. The %mean recovery is in
the range of 100.55% to 101.11% and %RSD was less than 1.0% between preparations. The % RSD for Assay results of initial sample preparation in
different intervals of 0hr, 24 h, 30 h and 48 h was less than 1.0%. To establish stability-indicating capability of the method, drug product was
subjected to the stress conditions of acid, base, oxidative, hydrolytic, thermal and photolytic degradation. The degradation products were well
resolved from Carisoprodol.
Conclusion: The developed method was validated as per international ICH guidelines with respect to specificity, linearity, accuracy, precision and
robustness.
Keywords: Carisoprodol, Stability-Indicating HPLC Method, Stress Conditions
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijcpr.2017v9i6.23434

INTRODUCTION addition, the gradient mode of elution increases the use of solvents
and the method is more concentrated on the characterization of
Carisoprodol is chemically (1-Methylethyl) carbamic acid 2-(((amino- impurities rather than the assay of carisoprodol. The main objective
carbonyl)oxy)methyl)-2-methyl pentyl ester marketed under the of the present investigation is to develop and validate a simple,
brand name soma since 1959 and is used as skeletal muscle relaxant sensitive, cost effective, selective and reproducible stability
[1-2]. It belongs to the class of carbamates and produces effecs indicating HPLC method with UV detector for quantitative
associated with barbitutates [3-4]. Mechanism of the activity of determination of carisoprodol and also to study the stability of
carisoprodol of relieving discomfort associated with acute painful carisoprodol. The present study is aimed to develop a reverse phase
musculoskeletal condition has not been clearly known. In animal HPLC assay which is specific, precise, linear and accurate that can be
studies, muscle relaxation induced by carisoprodol was found to be used for routine analysis and stability study.
associated with altered inter-neuronal activity in the spinal cord and
in the descending reticular formation of the brain, resulting in blocking MATERIALS AND METHODS
pain sensations between the nerves and the brain [4].
Materials
Literature review reveals few reports for carisoprodol assay. They
include liquid chromatography-tandem mass spectrometry [5-6], HPLC grade acetonitrile was purchased from Merck India Limited,
gas chromatography [7-8], high-performance thin-layer Mumbai, India. Analytical grade potassium dihydrogen phosphate,
chromatography [9] and homogeneous immunoassay [10]. All these hydrochloric acid, sodium hydroxide and hydrogen peroxide were
analytical techniques have been employed for carisoprodol from Sdfine-Chem limited, Mumbai, India. Milli-Q-water was used
determination in biological samples such as urine and serum of throughout the process. The 2-Methyl-2-propyl-1,3-prpanediol;
equine and urine and plasma of human. Furthermore the reported Carisoprodol Impurity A CRS Lot no: 1; Carisoprodol Sample and
methods are cumbersome and require sophisticated equipment. USP Carisoprodol RS were donated by Lee pharma as gift sample.
Drugs in bulk and pharmaceutical dosage forms can be analysed in
Instrumentation
quality control laboratories and cost effective methods like
uv/visible spectroscopic method or HPLC with UV/Visible detector. Separation and quantization of carisoprodol was performed on a
Three extractive spectrophotometric methods for the quantification High Pressure Liquid Chromatography (Waters, HPLC) equipped
of carisoprodol in pure and pharmaceutical formulations were with 2345 Quaternary gradient pump and 2414 RI detector. The
determined by Ravi et al. [11], which were based on formation of HPLC data were processed using Empower soft ware.
colored chloroform extractable ion-pair complexes with dyes like
bromocresol green, bromothymol blue and bromophenol blue in Chromatographic conditions
acidic medium. However, these methods suffer from one or the other The present study was aimed to carry out by reversed-phase
disadvantage such as extraction of ionpair complex, poor sensitivity,
chromatography to determine Carisoprodol; the column used was
unstable color and rigid experimental conditions.
Sunfire-C 18 column (250 mm x 4.6 mm, 5 µm) at 30°C, mobile phase
Assay of carisoprodol and its impurities with 2-methyl-2- was prepared by mixing water with acetonitrile in the ratio
propylpropane-1, 3-diyl dicarbamate and N-isopropyl-2-methyl-2- 60:40v/v and refractive index detector at 30°C. The mobile phase
propyl-3-hydroxy propyl carbamate using UV-HPLC was presented was delivered at a flow rate of 1.0 ml per min. The mobile phase was
by Rohith et al. [12], but has drawbacks in terms of precision, filtered through a Millipore membrane filter paper and sonicated for
accuracy, retention time (16.855 min) and run time (50 min). In 15 min for degassing prior to use.
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Preparation of standard solutions Thermal degradation

Water and acetonitrile in the ratio of 60:40 (v/v) is used as diluent Thermal degradation studies were performed at 60 °C for 5 h. For this
for the preparation of standard solutions. A standard stock solution study, carisoprodol powder was taken in glass petric dish and placed
of carisoprodol (5 mg/ml) was prepared by dissolving 50.0 mg under sunlight at 60 °C for 5 h. After specified time, the sample was
portion of USP Carisoprodol RS in 10-mL of mobile phase. Working cooled, transferred to a 100 ml volumetric flask and dissolved in 30 ml of
standard solutions were prepared after the dilution of the stock diluent and made up to mark with the same solvent.
solution with the same solvent. Five series of carisoprodol
calibration solutions at the concentration values of 1, 2, 3, 4, 5 After degradation, all stress degraded samples were diluted to give a
mg/ml were prepared from the stock standard solution by final concentration of 2 mg/ml and filtered through a millipore
appropriate dilution with the diluent. membrane filter paper before injection in the chromatographic system.

Preparation of stress degradation samples Assay procedure

Stress degradation samples were prepared using different ICH The Assay preparation was dissolving 20.0 mg portion of sample in
recommended stress conditions such as acidic, alkali, oxidative and 10-mL of mobile phase.
thermal. RESULTS AND DISCUSSION
Acid degradation Method development
For acid degradation, carisoprodol was dissolved in 5 ml of 1N Initial trial, as suggested in the test plan was carried out by reversed-
Methanolic HCl in a 100 ml volumetric flask. The resulting solution phase chromatography to determine Carisoprodol; the operating
was refluxed for 8 h at 60 °C on a heating mantle. After completion of conditions were Sunfire-C18 column (250 mm x 4.6 mm, 5 µm) at 30 °C,
the stress the solution was cooled and diluted to the volume with the mobile phase was prepared by mixing water with acetonitrile (60/40,
diluents. v/v) and refractive index detector at 30 °C. The mobile phase was
delivered at a flow rate of 1.0Ml per min. The diluent was prepared by
Alkali degradation
mixing 0.01N Sulfuric acid and methanol in ratio of 40:60 (v/v). The
For alkali degradation, carisoprodol was dissolved in 5 ml of 0.1N sample concentration was 3.5 mg per ml of Carisoprodol in diluent. The
Methanolic KOH for 1 h refluxing at 60 °C in a 100 ml volumetric peak response was good neither with the diluents nor with the mobile
flask. The resulting solution was refluxed for 1 h at 60 °C on a phase. Therefore, to increase the peak response, the column and
heating mantle. After completion of the stress the solution was detector temperature was increased to 45 °C (fig. 2).
cooled and diluted to the volume with the diluent.
Different concentrations were tried to get good peak shape and
Oxidative degradation response. This was achieved with 5.0 mg/ml concentration of
Carisoprodol and 20 µl injection load. Peak tailing of 1.48 and the LOD
Oxidative degradation was carried out at 60 °C using 3% Ethanolic was found at a concentration of 0.015 mg per ml. Based on the
H 2 O 2 . To perform this, carisoprodol was dissolved in 5 ml of 3% equivalent concentration of 5.0 mg/ml concentration of Carisoprodol
Ethanolic H 2 O 2 in a 100 ml volumetric flask. The resulting solution and 20 µl injection load, we checked with the concentration of 2.0 mg per
was refluxed for 24 h at 60 °C. After completion of the stress the ml and 50 µl injection load. The observed peak tailing of 1.32 and the
solution was cooled and diluted to the volume with the diluent. LOD was found at a concentration of 0.01 mg per ml.

Fig. 1: A representative chromatogram for carisoprodol concentration optimized to 5 mg/ml

Validation of the method An accurately weighed amount of 24.0 mg of Carisoprodol Impurity

C (2-Methyl-2-propyl-1,3-prpanediol), 35.0 mg of USP Carisoprodol
The developed method was validated as per ICH guidelines by
RS and 4 ml of Carisoprodol impurity A stock solution, are dissolved
evaluating parameters like system suitability, linearity, limit of
detection, limit of quantitation, precision, accuracy, specificity, in 10 ml of Mobile phase.
robustness and ruggedness. Resolution between impurity C and impurity A of Carisoprodol to
System suitability that of Carisoprodol is 18.0 and 2.34 respectively. The relative
retention time for Carisoprodol impurity C was 0.4 and that of
Carisoprodol impurity A stock solution Carisoprodol Impurity A was 1.1 whereas for Carisoprodol it was
An accurately weighed amount of 10.0 mg of carisoprodol Impurity 1.0. USP tailing for Carisoprodol peak for standard solution was 1.23
A CRS Lot no: 1a into volumetric flask and dissolved it in mobile and % RSD for 5 replicate injections of standard solution was 0.42%
phase and diluted up to the mark with mobile phase. (as shown in table 2).

Carisoprodol impurity C stock solution Assay precision

An accurately weighed amount of 24.0 mg of 2-Methyl-2-propyl-1,3- The % relative standard deviation of carisoprodol from 6 sample
prpanediol is dissolve in 10 ml of mobile phase. preparations was 0.35% (table 2).

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Table 1: Performance parameters from the system suitability solution and standard solution
Parameter Impurity C Carisoprodol Impurity A
t R , min. 2.2 5.3 5.8
Relative t R 0.4 1.0 1.1
Resolution 18.0 2.3
Tailing factor for Carisoprodol peak from 1.2
standard solution
Repeatability from Standard solution Mean peak area for replicate injections 325768
% RSD for replicate injections 0.42

Fig. 2: A representative chromatogram of a system suitability solution meets the requirements from precision

Table 2: The validation parameter, assay precision results

Preparations % w/w of assay
Preparation-1 99.79
Preparation-2 99.87
Preparation-3 99.87
Preparation-4 99.76
Preparation-5 100.13
Preparation-6 100.67
mean±SEM (n=6) 100.01±0.14
% RSD 0.35

Linearity Linearity solution 100%: Pipette 25 ml of the linearity stock solution

into 50 ml volumetric flasks, dilute to volume with mobile phase.
Linearity stock solution: A 200-mg portion of Carisoprodol is
dissolve in 50 ml volumetric flask, dissolves and diluted to volume Linearity solution 75%: Pipette 15 ml of the Linearity solution 100%,
with mobile phase. into 20 ml volumetric flasks, dilute to volume with mobile phase.
Linearity solution 150%: Pipette 15 ml of the linearity stock solution Linearity solution 50%: Pipette 5 ml of the Linearity solution 100%,
into 20 ml volumetric flasks, dilute to volume with mobile phase. into 10 ml volumetric flasks, dilute to volume with mobile phase.

Table 3: The validation parameter, linearity results

Concentration (in mg/ml) Mean area
1.002 162831
1.503 246392
2.004 331649
3.006 496392
4.007 662755
Correlation coefficient (R2) 0.99998

Accuracy and 120% levels against USP Carisoprodol RS. At each level, three
sample preparations were used. The average % recovery of these
Assay accuracy preparations at 80%, 100% and 120% level for
three preparations at their respective concentration level was
Carisoprodol: Weigh 40.0 mg, 50.0 mg and 60.0 mg of Carisoprodol,
100.55%, 100.67% and 101.11% respectively. The results were found
each in triplicate, into a 25 ml of volumetric flask, dissolve it and
diluted to volume with mobile phase. to be within the desired acceptance criteria according to which, the %
Recovery should not be less than 98.0% and not be more than 102.0
The accuracy of the method is determined by recovery experiments. %. Typical chromatograms of both Standard and assay preparations
The recovery was performed by Carisoprodol sample at 80%, 100% are shown in fig. 6 to fig. 8. The assay data are listed in table 4.

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Fig. 3: Calibration curve for carisoprodo l from linearity study

Table 4: The validation parameter, accuracy results

Level of accuracy S. No. Drug added Drug recovered (in mg) % assay mean±SEM % RSD of assay, (n=3)
(in mg) (n=3)
at 80% 1 40.49 40.79 100.55±0.27 0.97
2 40.20 40.23
3 40.18 40.03
at 100% 1 50.30 50.19 100.67±0.39 0.67
2 50.05 50.09
3 50.52 50.72
at 120% 1 60.63 60.39 101.11±0.23 0.39
2 60.50 60.79
3 60.44 60.81

Fig. 4: A representative chromatogram of an accuracy 80% solution exhibit carisoprodol peak

Fig. 5: A representative chromatogram of an accuracy 100% solution exhibit carisoprodol peak

Stability of mobile phase Degradation studies

The % RSD of retention times for different intervals of 0h, 24h, 30h From the forced degradation studies, it was observed that
and 48h was found to be 0.13% and similarly, the assay results for carisoprodol was quickly degraded to impurity A in alcoholic
fresh sample preparations in different intervals was found to be alkaline media which was confirmed by injecting the carisoprodol
0.58% (table 5). impurity A CRS Lot no: 1a solution into chromatographic system,
and resolution between carisoprodol peak and Impurity A obtained
Sample solution stability was 2.3. The RRT of impurity A was 1.1.
The % RSD for Assay results of initial sample preparation in From table no: 8 of degradation studies, in 0.1N methanolic KOH,
different intervals of 0hr, 24 h, 30 h and 48 h was found to be 0.46% the controlled degradation of carisoprodol in 0.1N methanolic
(table 5). KOH for 1 h refluxing at 60 °C, showed 10.6% degradation as

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Int J Curr Pharm Res, Vol 9, Issue 6, 79-85

shown in fig. 7. In 1N Methanolic HCl, refluxing for 8 h at 60 °C, reflux at 60 °C, showed 9.1% degradation which is shown in fig.
the degradation of Carisoprodol in 0.1N methanolic HCl showed 9. Upon Thermal exposure, no degradation was observed from
5.1% degradation as shoen in fig. 8 and in 3% ethanolic H 2 O 2 , solution form as well as powder form of carisoprodol at 60 °C for
the degradation of carisoprodol in 3% ethanolic H 2 O 2 for 24 h 24 h (fig. 10-13).

Fig. 6: A representative chromatogram of an accuracy 120% solution exhibit carisoprodol peak

Table 7: The validation parameter, stability of mobile phase and analyte solution results
Time interval Retention time (in min) Mobile phase stability Analyte solution stability
0 H (Initial) 5.23 99.69 99.69
24 H 5.25 99.92 99.51
30 H 5.25 99.17 100.45
48 H 5.24 100.58 99.47
mean±SEM (n= 4) 5.24±0.00 99.84±0.34 99.78±0.22
%RSD 0.13 0.58 0.46

Table 8: Representing controlled degradation for Forced degradation study

Stress condition % degradation Optimized time
1 N Methanolic Hcl, at 60 °C 5.1 8h
0.1N Methanolic KOH, at 60 °C 10.6 1hr
3% H 2 0 2, at 60 °C 9.1 24 h
Heat at 60 °C 0 24 h
UV-short wave length 0 24 h
Sunlight 0 5h

Fig. 7: A representative chromatogram of about 5% degradation in stress study for carisoprodol in acid media (1N Hcl, Methanolic-8h
reflux, at 60 °C)

Fig. 8: A representative chromatogram of about 10% degradation in stress study for carisoprodol in acid media (1N KOH, methanolic-1h
reflux, at 60 °C)

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Fig. 9: A representative chromatogram of about 10% degradation in stress study for carisoprodol in acid media (3%H 2 O 2 , alcoholic-24h
reflux, at 60 °C)

Fig. 10: A chromatogram representing no degradation in carisoprodol test solution heated at 60 °C for 24 h during stress study

Fig. 11: A chromatogram representing no degradation in carisoprodol powder, heated at 60 °C for 24 h during stress study

.
Fig. 12: A chromatogram representing no degradation in carisoprodol test solution under sunlight for 5 h during stress study

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Fig. 13: A chromatogram representing no degradation in carisoprodol powder under sunlight for 5 h during stress study

CONCLUSION 5. Mohrhaus AS, Gratz SR. Identification and determination of

carisoprodol in tablets by liquid chromatography/mass
The Assay method adopted for Carisoprodol is specific, precise, spectrometry. Microgram 2004;2:36.
linear and accurate. The Analyte solution was found to be stable 6. Skinner W, McKemie D, Stanley S. Quantitative determination
up to 48 h under ambient conditions and mobile phase was found of carisoprodol and its metabolites in equine urine and serum
to be stable up to 48 h. Hence this method can be used to replace by liquid chromatography-tandem mass spectrometry.
the existing titration method for routine analysis and stability Chromatographia 2004;59:S61-7.
study of carisoprodol. 7. Kucharczyk N, Segelman FH, Kelton E, Summers J, Sofia RD,
CONFLICT OF INTERESTS Mahrous H, et al. Gas chromatographic determination of
carisoprodol in human plasma. J Chromatogr B: Biomed Sci
Declared none Appl 1986;377:384-90.
8. Kintz P, Mangin P, Lugnier AA, Chaumont AJ. A rapid and
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