Indukaka Ipcowala College of Pharmacy, Beyond GIDC, P.B. No. 53, Vitthal Udyognagar- 388 121, Gujarat, India.
Received: 18/03/2014 Accepted: 16/05/2014 Revised: 09/05/2014
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A sensitive, selective and precise high performance liquid chromatographic method has been developed and validated for the simultaneous determination of Cyclobenzaprine Hydrochloride and Aceclofenac both as a bulk drug and in formulation. The method employed Luna C18 column (250 x 4.6 mm id, 5 μm particle size) as the stationary phase while Methanol and 0.02 M KH2PO4 (75: 25 v/v, pH 3) was used as mobile phase. The Rt of Cyclobenzaprine Hydrochloride and Aceclofenac were observed to be 4.2 and 9 minutes, respectively. Analysis was carried out in absorbance mode at 280 nm. The linear regression analysis data for the calibration plots showed a good linear relationship for Cyclobenzaprine Hydrochloride and Aceclofenac over a concentration range of 0.01-20 μg/ml and 0.1-40 μg/ml respectively with correlation co-efficient of 0.9990 for Cyclobenzaprine Hydrochloride and 0.9990 for Aceclofenac. The LOQ was found to be 0.01 and 0.1 μg/ml respectively for Cyclobenzaprine Hydrochloride and Aceclofenac. The method was validated as per ICH guideline and it was found to be accurate, precise and robust. Marketed formulation was analyzed successfully.
Cyclobenzaprine Hydrochloride (CBP), Aceclofenac (ACF), Liquid Chromatography, Validation
Cyclobenzaprine Hydrochloride is chemically known as 3-(5H-dibenzo [a,d]cyclohepten-5-ylidene)- N,N-dimethyl-1-propanamine [2,3]. CBP is used in treatment of as an adjunct to rest and physical therapy for relief of muscle spasm associated with acute, painful musculoskeletal conditions . Aceclofenac; 2- [2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetyl]oxyacetic acid . It is used for the Used for the relief of pain and inflammation in rheumatoid arthritis, osteoarthritis [5,6]. Literature survey revealed there is no published RP-HPLC method for this combination of drug. The present paper describes a simple, accurate and precise method for simultaneous estimation of CBP and ACF in combined tablet dosage form. The proposed method is optimized and validated as per the International Conference on Harmonization (ICH) guidelines .In the present work, a successful attempt has been made to estimate both these drugs simultaneously by RP-HPLC method.
Instrument Perkin Elmer USA, Series 200, Phenomenex Luna C18 column (250 x 4.6 mm id, 5μm particle size) was used for analytical method development. The chromatographic data were processed by Totalchrom navigator HPLC version 6.3.1 Software.
FLEXABENZE PLUS (15 mg CBP and 200 mg ACF) manufactured by Macleods pharmaceuticals LTD, Mumbai. All chemicals and reagents used were of AR grade.
All the chemicals used were of AR grade.
The sensitivity of HPLC method that uses UV detection depends upon proper selection of detection wavelength. An ideal wavelength is the one that gives good response for the drugs that are to be detected. Overlay UV spectra of both the drugs showed that CBP and ACF absorbed appreciably at 280 nm, so detection was carried at this wavelength (Figure 1).
2.72 gm of KH2PO4 was weighed and dissolved in 1000 ml of HPLC water to prepare 0.02 M KH2PO4 buffer. Mobile phase was prepared by mixing 750 ml of Methanol and 250 ml 0.02 M KH2PO4. The pH was adjusted to 3 using o-phosphoric acid (1%). Solution was filtered through Whatman filter paper (No. 41) and sonicate for 10 min and this solution was used as a mobile phase.
CBP (10 mg) and ACF (10 mg) were accurately weighed and transferred to two separate 10 ml volumetric flask and dissolved in few ml of methanol. Volumes were made up to the mark with methanol to yield a solution containing 1000 μg/ml of CBP and 1000 μg/ml of ACF, respectively. Appropriate aliquots from above solution were taken and diluted with mobile phase to obtain 100 μg/ml of CBP and 100 μg/ml of ACF, respectively.
A Phenomenex® C-18 (250 x 4.6 mm id) chromatographic column equilibrated with mobile phase Methanol: 0.02 M KH2PO4 (75:25, % v/v). pH 3 was adjusted using o-phosphoric acid (1%). Mobile phase flow rate was maintained at 1 ml/min and effluents were monitored at 280 nm. The sample was injected using a 20 μL fixed loop, and the total run time was 12 min.
Appropriate aliquot of stock solution of CBP and ACF was taken in same 10 ml volumetric flasks. The volume was made up to the mark with mobile phase to obtain final concentration of 0.01, 0.1, 1, 5, 10, 20 μg/ml of CBP and 0.1, 0.5, 2, 10, 20, 40μg/ml of ACF, respectively.
The calibration curve for CBP was found to be linear in the range of 0.01-20 μg/ml with a correlation coefficient of 0.9990. The calibration curve for ACF was found to be linear in the range of 0.1-40 μg/ml with a regression coefficient of 0.9990. The regression analysis of calibration curves are reported in table 2 and 3.
The intraday studies were carried out by measuring response for 3 concentrations for 3 times a day. The % RSD values for CBP & ACF were found to be 0.49 – 1.02 and 0.51- 0.93 for intraday respectively. These %RSD value was found to be less than ± 2.0 indicated that the method is precise (Table 4 & 5).
The repeatability studies were carried out by measuring response for a single concentration for 6 times a day. The % RSD values of repeatability for CBP and ACF were found to be 0.54 and 0.80 (Table 6). These %RSD value was found to be less than ±1.0 indicated that the method is precise.
The accuracy of the method was determined by calculating recoveries of CBP & ACF by method of standard addition. The recoveries found to be 99.57 – 101.10 % and 99.78 –101.81 % for CBP & ACF respectively (Table 7).
LOD is the lowest amount of the analyte that can be detected but not quantified. From the visual observation of chromatogram, the detection limits for CBP & ACF were 0.003 μg/ml and 0.03 μg/ml, respectively. LOQ is the lowest amount of the analyte that can be detected and quantified. The quantitation limits were 0.01 and 0.1 μg/ml respectively. The above data shows that a microgram quantity of both the drugs can be accurately and precisely determined.
Robustness study was carried out by changing the mobile phase composition, pH and flow rate (Table 8). There is no significant change in the results so the proposed method is robust.
The specificity study was carried out to check the interference from the excipients used like Micro crystalline cellulose, Lactose and magnesium stearate in the formulations by preparing synthetic mixture containing both the drugs and excipients. The specificity study was carried out to check the interference from the excipients. Recoveries of both the drugs were above 98%. The results of the specificity study are shown in the Table 9.
Stability of standard and sample solution of CBP and ACF were evaluated at room temperature for 24 hr. Both the drugs were found to be stable with a recovery of more than 98% (Table 10).
System suitability test was carried out and the results are summarized in Table 11.
The validation parameters are summarized in Table 12.
Marketed formulation was analyzed using proposed method which gave percentage recovery for CBP & ACF were 99.49% and 110.01% respectively. Rt of 4.2 min and 9.0 min were observed in the chromatogram for CBP and ACF, and no interference from the excipients present in the marketed tablet formulation was observed (Table 13).
The HPLC method for the estimation of CBP and ACF has been developed. The proposed method was validated as per ICH Q2 (R1) guideline for accuracy, precision, linearity, specificity and robustness. The developed method was successfully applied to marketed formulation.