Development and validation of a New HPLC Method for Determination of Clorazepate Dipotassium in Capsule Formulations
Safwan Ashourα & Nuha Kattanσ
A simple, accurate and rapid high performance liquid chromatographic method has been developed and applied for the determination of clorazepate dipotassium in capsules. Rosuvastatin calcium was used as internal standard. The determination was performed on Supelcosil C8 DB column (250×4.6mm i.d., 5μm particle size) at 25 °C using a mobile phase composed of MeOH and 0.1 M HCOOH pH 2.16 (67:33, v/v), pumped at a flow rate 1.0 mL min−1. The photodiode array detector was operated at 245 nm. The retention times for rosuvastatin and clorazepate were about 5.05 and 6.85 min, respectively. Results of the assay were statistically evaluated for its accuracy and precision. Linearity range was 2.15–400.0 µg mL-1. The limits of quantification and detection were 1.31 and 0.39 µg mL-1, respectively. The intra-day precision results, expressed by relative standard deviation values, were lower than 2.35%. The validated method has been successfully applied to the analysis of clorazepate dipotassium in capsules with no interference from the excipients.
Keywords: clorazepate dipotassium; high- performance liquid chromatography; capsules.
Author α σ: Department of Chemistry, Faculty of Science, University of Aleppo, Aleppo, Syria.
Clorazepate, 7-chloro-2,3-dihydro-2,2-dihydroxy -5-phenyl-1H-1,4-benzodiazepin-3-carboxylic acid is used in the form of a dipotassium salt. Clorazepate dipotassium, is a benzodiazepine . Benzodiazepines are among the most widely prescribed drugs and are used in the treatment of stress, anxiety-induced depression, panic, sleep disorders, muscle spasms, alcohol withdrawal and seizures . Unfortunately, they are often subject to overdose in suicide attempts and are frequently encountered in emergency intoxication episodes and drugs-of-abuse testing. Clorazepate dipotassium is official in BP and USP. The BP  and USP  describe potentiometric titration method for the estimation of clorazepate dipotassium. Spectrophotometric methods [4-9] and micellar electrokinetic chromatography  are reported for determination of clorazepate dipotassium in pharmaceutical formulations. Clorazepate dipotassium with other drugs are determined by TLC , HPLC [12-14] and gas-liquid chromatography  in pharmaceuticals and biological samples.
The objective of this work was to develop an analytical HPLC procedure, which would serve as a reliable and rapid method for the determination of clorazepate dipotassium in pharmaceutical preparations. This manuscript describes the development and subsequent validation of isocratic reversed-phase HPLC method using the C8 column as a stationary phase for the above determination. In the proposed LC method, clorazepate dipotassium and rosuvastatin calcium (internal standard) were well separated and eluted before 8 min run time. The precision of the described method for the assay of clorazepate dipotassium has been checked regarding F-test using a reported method as reference. The method serves as an alternative to the methods described in pharmacopoeias.
A liquid chromatography (Hitachi Model L–2000, Japan) system equipped with a binary pump (model L-2130, flow rate range of 0.000-9.999 mL min-1), degasser, column oven (model L-2350, temperature range of 1-85 oC), autosampler and photodiode array (PDA) detector (190-900 nm) containing a quartz flow cell (10 mm path and 13 μL volume). Chromatograms were analyzed and integrated automatically using the Ezchrom Elite Hitachi Software. Other instruments like Metrohm compact titrator, Sartorius analytical balance, WTW pH meter and Dihan sonicator were used in sample and standard preparations.
3.2. Chromatographic conditions
Supelcosil C8 DB column (250×4.6 mm, 5 μm particle size, Macherey-Nagel Germany) was used to achieve the separation. The mobile phase was a mixture of methanol and 0.1 M formic acid (67:33, v/v), filtered through a nylon 0.45 μm membrane filter and degassed by ultrasonic agitation before use. The mobile phase was prepared weekly and has a flow rate of 1.0 mL/min. The injection volume was 10 μL with ambient column oven temperature. Isocratic elution of all analytes was monitored at 245 nm.
3.3. Chemicals and Materials
HPLC grade methanol and water (Labscan, Ireland), formic acid (AR grade, Surechem Products LTD, England) and water (HPLC grade, Merck). Clorazepate dipotassium (CLZ) pure drug substance, its purity was found to be 99.85%, was supplied by Cambrex Profarmaco Milano (Italy) and rosuvastatin calcium (RSV) was obtained from MSN Laboratories Limited (India), respectively. The structures of these compounds are shown in Figure 1. Capsules were purchased from Syrian market, containing clorazepate dipotassium 5 mg and 10 mg per capsule.
Figure 1: Chemical structure of (a) clorazepate dipotassium and (b) rosuvastatin calcium.
3.4. Standard Solutions
Standard stock solutions of CLZ (1.0 mg mL-1) and RSV (0.5 mg mL-1) were prepared by direct weighing of standard substance with subsequent dissolution in methanol. From these stock solutions the working standard solutions were prepared by further diluting of stock solution using methanol. These solutions were stored in the dark at 2–8 °C and found to be stable during the analysis time.
3.5. Assay Procedure for Dosage Forms
The entire content of twenty capsules containing CLZ was weighed and mixed well. Five accurately weighed quantities of the powder equivalent to 50 mg of CLZ were transferred into 100 mL separated volumetric flasks. 70 mL of methanol was then added to each flask and the mixture was sonicated for 5 min. Then, the volume was adjusted to 100mL with methanol. The sample solutions were centrifuged for 10 min and filtered through a millipore filter (0.45 µm). An aliquot (500 µL) from the supernatants was transferred into 10 mL calibrated flasks, 1 mL RSV solution was added and the solutions were diluted with methanol to give a final concentration of about 25 µg mL-1 of CLZ and 50 µg mL-1. Finally, 10 μL of each diluted sample was injected into the column and chromatogram was recorded for the same. Peak area ratios of CLZ to that of RSV were then measured for the determination.CLZ concentration in the samples was then calculated using peak data and standard curves.
3.6. Method validation
A series working standard solutions of CLZ (2.15–400 µg mL-1) were prepared by diluting the stock standard solution with the methanol. In each sample 1 mL of RSV was added (50 µg mL-1 in the final volume). To construct the calibration curve five replicates (10 µL) of each standard solution were injected immediately after preparation into the column and the peak area of the chromatograms was measured. Then, the mean peak area ratio of CLZ to that of RSV (IS) was plotted against the corresponding concentration of CLZ to obtain the calibration graph.
The sensitivity of the method was determined on LOD and LOQ. The limit of detection (LOD) and limit of quantification (LOQ) of the CLZ assay were determined experimentally by calibration curve method. LOD was expressed as the concentration of drug that generated a response to three times of the signal-to-noise (S/N) ratio, and LOQ was 10 times of the S/N ratio, thus, were calculation by using the following equations :
The intraday precision was determined by measuring CLZ samples of 2.15–400 μg mL-1 injected five times on the same day. The relative standard deviation (RSD) of the assay values was calculated.
Recovery studies were performed in view to justify the accuracy of the proposed method. Precision was calculated from percentage relative standard deviation (RSD %) for repeated measurements, whereas accuracy was expressed as % of recovery.
The specificity of the method was established through the study of resolution factor of drug peaks from nearest resolving peak and also among all other peaks. Peak purity of CLZ was assessed to evaluate the specificity of the method.
Robustness of HPLC method was determined by deliberately varying certain parameters like flow rate, the percentage of organic solvent in the mobile phase, pH of mobile phase and the detection wavelength. When the effect altering one set of conditions was tested, the other conditions were held constant at optimum values. The robustness of the method was studied by using five replicates at a CLZ concentration level of 100 μg mL-1.
The system suitability test was performed to confirm that the LC system to be used was suitable for intended application. A standard solution containing 25 μg mL-1 of CLZ in the presence of 500 μg mL-1 of internal standard were injected seven times. The parameters peak area, retention time, resolution, capacity factor, theoretical plates, tailing factor (peak symmetry) and % RSD were determined.
RESULTS AND DISCUSSION
4.1. Optimization of chromatographic conditions
RSV was used as internal standard, to improve the analytical performance and thus control undetermined changes in active pharmaceutical ingredient concentration and instrument response fluctuations, and also to reduce the problem of the many-fold dilution required in the classical batch procedures.
The effect of the composition of the mobile phase on the retention time of CLZ and the internal standard, RSV, was investigated. Drug peak was eluted fast with the solvent front when many solvents and water was used. When methanol and water were used as mobile phase drug eluted late and had broadened. Results of the effect of methanol percentage in the mobile phase are presented in Figure 2a. An increase in the percentage of methanol decreases the retention of CLZ and RSV. Increasing methanol percentage to more than 75% RSV peak is eluted with the solvent front, while at methanol percentage lower than 60% the elution of CLZ peak is seriously delayed. The optimum methanol percentage was found to be 67%.
The effect of pH in the chromatographic elution of the compounds was also investigated by change the concentration values of the aqueous component of the mobile phase from 0.03 to 0.15 M. A satisfactory separation and peak asymmetry for the drug was obtained with mobile phase consisting of methanol: 0.1M formic acid pH 2.16 (67:33, v/v), pumped at a flow rate 1.0 mL min-1 (Figure 2b) at 25 oC.
Supelcosil C8 DB column (250×4.6mm, 5 µm particle size) gave the most suitable resolution between RSV and CLZ peaks (>4) according to the pharmacopeial requirement while the other columns (Nucleodur C8, Hichrom 5 C8, Nucleodur C18, ODS Hypersil C18) cause the peaks of the RSV and CLZ either to be overlapped or to have unsuitable resolution (<4).
Figure 2: Variation of the retention time of CLZ and RSV as a function of (a) methanol percentage in the mobile phase and (b) flow rate of the mobile phase.
The use of isocratic elution was proven to be short retention time for the CLZ peak and helped in the separation of RSV and CLZ. Figure 3 shows a typical chromatogram obtained by the proposed RP-HPLC method, demonstrating the resolution of the symmetrical peaks corresponding to RSV and CLZ with a flow rate of 1.0 mL/min. The retention time of RSV and CLZ was about 5.023 and 6.853 min, respectively. The retention time observed allows a fast determination of the drug, which is suitable for QC laboratories. Quantitation was achieved with UV detection at 245 nm based on peak area.
Figure 3: A typical chromatogram of a mixture of RSV (50µg mL-1) and CLZ (25 µg mL-1) at retention times 5.020 and 6.740 min, respectively, under optimal conditions.
4.2. Method validation
The method was validated in accordance with the International Conference on Harmonization (ICH) guidelines . The following validation characteristics were addressed:
The system suitability was determined by making six replicate injections and analyzing each solute for their peak area, resolution and peak tailing factor. The system suitability requirements for 25 μg mL-1 of CLZ in the presence of 50 μg mL-1 of internal standard was a %RSD for peak area less than 0.38, a peak tailing factor less than 1.1 and Rs greater than 6.0 between adjacent peaks for all analytes. This method met these requirements. The results are shown in Table 1.
Table 1: System suitability parameters
Capacity factor (k')
Number of theoretical plates (N)
Tailing factor (T)
% RSD for six injections
Under the optimal conditions for HPLC, the calibration curve obtained between the peak area ratio of CLZ to that of the internal standard and the corresponding concentration of CLZ was linear over the concentration range of 2.15-400.0 µg mL-1, as shown by the equation presented in Table 2. The calibration curve could be represented by the linear regression equation . Correlation coefficients (r) of the regression equations were greater than 0.999 in all cases. Characteristic parameters for regression equations and (r) were given in Table 2. The linearity of the calibration graph was validated by the high value of (r) of the regression.
The specificity of the method was established through the study of resolution factor of drug peaks from nearest resolving peak and also among all other peaks. The specificity of the chromatographic method was determined to ensure separation of CLZ and the internal standard as illustrated in Figure 3 where complete separation of RSV and CLZ was noticed. The HPLC chromatogram recorded for the analyte in capsules (Figure 4) showed almost no peaks within a retention time range of 8 min. The Figures show that CLZ are clearly separated and the peak of analyte was pure and excipients in the formulation did not interfere the analyte. Thus, the HPLC method presented in this study is selective for CLZ.
Limit of detection and limit of quantification
The limit of detection (LOD) and limit of quantification (LOQ) were determined experimentally. LOD was found to be 0.39 μg mL−1 and LOQ was 1.31 μg mL−1 for CLZ showed a good sensitivity of the proposed method.
Table 2: Calibration data for the estimation of CLZ by HPLC.
Optimum concentration range (µg mL-1)
RCLZ/RSV = 0.0252CCLZ+0.7693
Correlation coefficient (r)
Standard deviation of slope
Standard deviation of intercept
LOQ (µg mL-1)
LOD (µg mL-1)
Accuracy and precision
The precision and accuracy of the method were determined by analysis of seven samples for the drug. The proposed method was successfully applied for the analysis of the drug by intra-day (analysis of standard solutions of CLZ in replicates of five in the same day) and the recovery experiments were carried out by spiking the already analyzed samples with seven different concentrations of standard CLZ. The percent recoveries obtained were from 99.17 to 100.07%. The standard deviation, relative standard deviation and relative error % of different amounts tested were determined from the calibration curve. Acceptable repeatability of the results within one day was observed. The accuracy of the method is indicated by the excellent recovery and the precision is supported by the low relative standard deviation, as recorded in Table 3.
Table 3: Accuracy and precision of the determination of CLZ by HPLC.
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. These HPLC condition changes included the concentration of the aqueous component in the mobile phase, the flow rate and the percentage of methanol in the mobile phase. The robustness of the method was studied by using five replicates at a CLZ concentration level of 25 µg mL-1. The degree of reproducibility of the results obtained as a result of small deliberate variations in the method parameters has proven that the method is robust (Table 4).
Table 4: Results of robustness study of CLZ.
Change in methanol percentage
Change in concentration of formic acid
Change in flow rate
4.3. Application of the Assay
The validity of the proposed method for the determination of clorazepate dipotassium was assessed by measuring drug concentration of pharmaceutical dosage forms (Figure 4). The results obtained with the proposed method were compared with the official method  and are shown in Table 5. Mean values were obtained with a Student's t- and F-tests at 95% confidence limits for four degrees of freedom. The results showed comparable accuracy (t-test) and precision (F-test), since the calculated values of t- and F-tests were less than the theoretical data. The proposed method is simple, rapid, accurate, highly sensitive and suitable for the routine quality control without interference from the excipients and additives.
Figure 4: A typical chromatogram of a mixture of CLZ (25 µg mL-1) and RSV (50 µg mL-1) in the mobile phase, prepared from Tranx 5 capsule.
Table 5: Determination of CLZ in capsules by the proposed and official methods.
Official method 
aFive independent analyses. At 95% confidence level t= 2.776 and F= 6.26.
The proposed method for analysis of clorazepate dipotassium in bulk powder and in capsule dosage form based on the use of liquid chromatography with spectrophotometric detection resulted to be simple, accurate and precise. Moreover, the method is fast and feasible. Our method shows satisfactory sensitivity having LOQ 1.31 µg mL-1 and LOD 0.39 µg mL-1. The developed method showed no interference with the formulation excipients. Hence this method can be conveniently used for routine quality control analysis of dipotassium clorazepate in its pharmaceutical formulation.
- The British Pharmacopoeia. Her Majesty’s Stationery Office Ltd, London, 2009.
- Hardman J.G.; Limbird L.E.; Molinoff P.B.; Ruddon R.W.; Gilman A.G.; Goodman and Gilman's The Pharmacological Basis of Therapeutics, 9th ed., McGraw-Hill, 1996.
- United States Pharmacopoeia Convention, Rockville, 34th ed., 2011.
- El-Bayoumi A.A.; Amer S.M.; Moustafa N.M.; Tawakkol M.S., Spectra Densitometric determination of clorazepate dipotassium, primidone and chlorzoxazone each in presence of its degradation product, Journal of Pharmaceutical and Biomedical Analysis, 20(5), 727-735, 1999.
- El-Bardicy M.G.; Bebawy L.I.; Amer M.M., Stability-indicating method for the determination of clorazepate dipotassium. I. via its final degradation products, Talanta, 39(12), 1569-1573, 1992.
- Hanawa T.; Ohta T.; Kimura F.; Tsuchiya T.; Ikoma R.; Uchida T.; Suzuki M.; Nakajima S., Stability of the powdered dosage form prepared by unsealing the capsules: water vapor sorption and discoloration of the powdery contents of clorazepate dipotassium capsules, Drug Development and Industrial Pharmacy, 26(11), 1199-205, 2000.
- Manes J.; Civera J.; Font G.; Bosch F., Spectrophotometric determination of benzodiazepines in pharmaceuticals by ion pairing, Ciencia Industria Farmaceutica, 6(9), 333-338, 1987.
- El-Yazbi F.A.; Abdel-Hay M.H.; Korany M.A., Spectrophotometric determination of some 1,4-benzodiazepines by use of orthogonal polynomials, Pharmazie, 41(9), 639-642, 1986.
- El-Yazbi F.A.; Barary M.H.; Abdel-Hay M.H., Determination of nitrazepa and dipotassium clorazepate in the presence of their degradation products using second derivative spectrophotometry, International Journal of Pharmaceutics, 27(2-3), 139-144, 1985.
- Berzas J.J.; Castaneda G.; Pinilla M.J., Determination of clobazam, clorazepate, flurazepam and flunitrazepam in pharmaceutical preparations, Talanta, 57(2), 333-341, 2002.
- Zevzikoviene A.; Zevzikovas A.; Bertulis A., Investigations of poisonings with benzodiazepine derivatives mixtures by thin-layer chromatography, Medicina (Kaunas), 39(11), 1100-1102, 2003.
- Gil-Agustí M.; Carda-Broch S.; García-Alvarez-Coque M.C.; Esteve-Romero J., Use of micellar mobile phases for the chromatographic determination of clorazepate, diazepam, and diltiazem in pharmaceuticals. Journal of chromatographic science, 38(12), 521-527, 2000.
- Colin P.; Sirois G.; Lelorier J., High-performance liquid chromatography determination of dipotassium clorazepate and its major metabolite nordiazepam in plasma, Journal of Chromatography B: Biomedical Sciences and Applications, 273(2), 367-377, 1983.
- Elrod L.; Shada D.M.; Taylor V.E., High-performance liquid-chromatographic analysis of clorazepate potassium and [the] monopotassium [analogue] in solid dosage forms, Journal of Chromatographic Science, 70(7), 793-795, 1981.
- Brooks M.A.; Hackman M.R.; Weinfeld R.E.; Macasieb T., Determination of clorazepate and its major metabolites in blood and urine by electron capture gas-liquid chromatography, Journal of Chromatography A, 135(1), 123-131, 1977.
- Long G.L.; Winefordner J.D., Limit of Detection. A Closer Look at the IUPAC Definition, Analytical Chemistry, 55(7), 712A–721A, 1983.
- ICH, Q2 (R1), Validation of Analytical Procedure: Text and Methodology, International Conference on Harmonization: Geneva, 2005.
- MILLER J.N.; MILLER J.C., Statistics and Chemometrics for Analytical Chemistry, 6th ed., Ellis Horwood, Chichester, pp. 110, 2010.