A sensitive and precise RP-HPLC method has been developed for the simultaneous estimation of clidinium bromide (CDB) and chlordiazepoxide (CDZ) in pure and pharmaceutical formulations. The separation was achieved on a Nucleodur C8 (
Chlordiazepoxide (7-chloro-
A high-performance liquid chromatographic system consisted of Hitachi (Japan) Model L-2000 equipped with a binary pump (model L-2130, flow rate range of 0.000–9.999 mL min−1), degasser, and a column oven (model L-2350, temperature range of 1–85°C). All samples were injected (10
Working reference standards of clidinium bromide (CDB), chlordiazepoxide (CDZ), and almotriptan (ALT) were supplied by MSN Laboratories Ltd., Centaur Pharmaceuticals PVT. Ltd., and SMS Pharmaceuticals Ltd., (India), respectively. HPLC grade methanol, acetonitrile, and water were purchased from Labscan (Ireland) and analytical reagent grade ammonium acetate (Merck) was used to prepare the mobile phase. Tablets were purchased from Syrian market, containing clidinium bromide 2.5 mg and chlordiazepoxide 5 mg per tablet.
Chromatographic separation was performed on a reversed-phase Nucleodur column C8 (
Individual stock standard solutions of clidinium bromide (2000.0
To construct the calibration curve five replicates (10
Twenty tablets were accurately weighted and finely pulverized. In the case of capsules, the contents of twenty capsules were completely evacuated from shells. An appropriate portion of this powder, equivalent to five tablets content of CDB and CDZ, was placed in a 25 mL volumetric flask with 20 mL of methanol. The solution was sonicated for 15 min and diluted to volume with methanol to obtain solution of CDB (500
The HPLC method was validated in terms of precision, accuracy, and linearity according to ICH guidelines. Assay method precision was determined using five independent test solutions. The intermediate precision of the assay method was also evaluated using different analysts on three different days. For intra day precision, different concentrations of CDB and CDZ were analyzed five times on the same day whereas for inter day precision the same drug concentrations were analyzed on three different days, and the percentage RSD of area was calculated. The accuracy of the assay method was evaluated with the recovery. Linearity test solutions were prepared as described in Section
The optimized compositions were used for the analysis of all solutions individually as well as in combination. The mobile phase used initially was composed of ammonium acetate (0.1 M) and methanol. However, to achieve the optimum resolution, a small portion of acetonitrile was added in the mobile phase until obtaining good results. The chromatographic conditions were optimized for separation of drugs by varying methanol, strength of buffer solution, pH, proportion of acetonitrile, and flow rate. During the optimization of the method, different columns (Nucleodur C8,
Plots of the retention time versus methanol or acetonitrile percentage in the mobile phase of ALT, CDB, and CDZ.
An increase in the percentage of methanol and acetonitrile decreases the retention of compounds, ALT, CDB, and CDZ. Increasing methanol percentage to more than 50% CDB peak is eluted with the solvent front, while at methanol percentage lower than 35% the elution of CDZ peak is seriously delayed. Also increasing acetonitrile percentage to more than 35% CDB peak is eluted with the solvent front, while at acetonitrile percentage lower than 20% the elution of CDZ peak is seriously delayed. The effect of pH in the chromatographic elution of the compounds was also investigated by changes the pH values of the aqueous component of the mobile phase from 4.0 to 6.0. A satisfactory separation and peak asymmetry for the drugs was obtained with mobile phase consisting of ammonium acetate (0.1 M, pH 5.0 adjusted with acetic acid)-methanol-acetonitrile (30 : 40 : 30, v/v/v), pumped at a flow rate 1.0 mL min−1 at 25°C. Quantitation was achieved with UV detection at 218 nm based on peak area. A representative chromatogram is shown in Figure
A typical chromatogram of a mixture of ALT (100
The selectivity of the HPLC method is illustrated in Figure
A typical chromatogram of a mixture of ALT (100
In the system suitability tests, five replicate injections of freshly prepared working standard solutions of CDB (300.0
System suitability parameters.
Parameter | Almotriptan | Clidinium bromide | Chlordiazepoxide |
---|---|---|---|
Theoretical plates ( |
1711 | 4174 | 6598 |
Resolution factora ( |
— | 2.59 | 5.19 |
Tailing factor ( |
1.03 | 1.43 | 1.16 |
Capacity factor ( |
2.19 | 2.90 | 4.20 |
% RSD for seven injections | 0.31 | 0.28 | 0.27 |
The calibration curves for CDB and CDZ were linear over the concentration range of 2.5–300.0
Sensitivity and regression parameters.
Parameter | Clidinium bromide | Chlordiazepoxide |
---|---|---|
Optimum concentration range ( |
2.5–300.0 | 3.0–500.0 |
Regression equation* |
|
|
Correlation coefficient ( |
0.9999 | 0.9999 |
Standard deviation of slope | 0.0019 | 0.0025 |
Standard deviation of intercept | 0.0135 | 0.0504 |
Regression equation** |
|
|
Correlation coefficient ( |
0.9999 | 0.9999 |
Standard deviation of slope | 1.1 × 10−5 | 2.8 × 10−5 |
Standard deviation of intercept | 5.9 × 10−4 | 8.1 × 10−4 |
Limit of quantification, LOQ ( |
0.294 | 0.403 |
Limit of detection, LOD ( |
0.088 | 0.121 |
**Regression equation for the ratio of peak area of drug to that of I.S. versus concentration of drug in
The precision and accuracy of the method were evaluated by analysis of seven samples for drugs mixture. Intraday assay variation was evaluated by injecting these samples in replicates of five in the same day. Interday assay variation was evaluated by injecting these samples in replicates of five on 4 different days from 1 to 10 days after preparation (Table
Accuracy and precision of within- and between-run analysis for the determination of clidinium bromide and chlordiazepoxide by HPLC.
Nominal concentration ( |
Intra-day ( |
Inter-day ( |
||||
---|---|---|---|---|---|---|
Mean ± SD |
RSD (%) | Recovery (%) | Mean ± SD |
RSD (%) | Recovery (%) | |
Clidinium bromide | ||||||
| ||||||
2.50 |
|
3.17 | 100.80 |
|
2.79 | 100.40 |
6.50 |
|
2.09 | 102.77 |
|
2.53 | 103.38 |
25.00 |
|
1.99 | 102.52 |
|
1.09 | 102.96 |
50.00 |
|
1.47 | 101.72 |
|
0.77 | 101.22 |
75.00 |
|
1.27 | 101.04 |
|
0.56 | 101.51 |
150.00 |
|
0.60 | 100.61 |
|
0.49 | 101.12 |
300.00 |
|
0.14 | 100.64 |
|
0.57 | 100.93 |
| ||||||
Chlordiazepoxide | ||||||
| ||||||
3.00 |
|
2.94 | 102.00 |
|
2.63 | 101.33 |
12.00 |
|
1.15 | 101.08 |
|
1.98 | 100.83 |
25.00 |
|
0.92 | 100.48 |
|
1.15 | 100.80 |
60.00 |
|
0.89 | 100.96 |
|
0.95 | 101.63 |
125.00 |
|
0.35 | 100.04 |
|
0.53 | 100.34 |
250.00 |
|
0.22 | 100.03 |
|
0.35 | 99.98 |
500.00 |
|
0.18 | 105.59 |
|
0.21 | 103.97 |
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. Robustness of the method was investigated under a variety of conditions including changes of pH of the mobile phase, flow rate, and percentage of acetonitrile and methanol in the mobile phase. The standard solution is injected in five replicates and sample solution of 100% concentration is prepared and injected in triplicate for every condition and % R.S.D. of assay was calculated for each condition. 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
Results of robustness study.
Factor | Level | Mean % assay ( |
% RSD of results | ||
---|---|---|---|---|---|
Clidinium bromide | Chlordiazepoxide | Clidinium bromide | Chlordiazepoxide | ||
pH of mobile phase | 5.1 | 100.3 | 100.5 | 1.32 | 1.25 |
4.9 | 100.1 | 100.7 | 0.86 | 0.69 | |
Flow rate (mL/min) | 0.9 | 99.9 | 100.1 | 0.97 | 1.04 |
1.1 | 100.2 | 100.8 | 0.58 | 0.53 | |
% of acetonitrile | 25 | 99.7 | 100.04 | 1.24 | 0.95 |
35 | 100.6 | 101.05 | 0.47 | 0.73 | |
% of methanol | 35 | 100.4 | 100.6 | 0.86 | 0.49 |
45 | 100.6 | 100.9 | 0.79 | 0.61 |
Stability studies were carried out at laboratory temperature for 10 days to find potential stability problems of the drug in the formulations. Samples were analyzed at intervals of 0, 1, 5, and 10 days. The results obtained are given in Table
Stability study for the drug in different formulations.
Producta | Time (days) | Amount foundb (mg) | % Recovery | % ±RSD | |||
---|---|---|---|---|---|---|---|
CDB | CDZ | CDB | CDZ | CDB | CDZ | ||
0 | 2.51 | 5.04 | 100.40 | 100.80 | 1.91 | 0.22 | |
Ribax capsules | 1 | 2.52 | 5.05 | 100.80 | 101.00 | 0.98 | 0.49 |
5 | 2.50 | 5.02 | 100.00 | 100.40 | 0.32 | 0.74 | |
10 | 2.49 | 5.01 | 99.60 | 100.20 | 1.05 | 0.86 | |
| |||||||
0 | 2.56 | 5.03 | 102.40 | 100.60 | 0.49 | 0.29 | |
Laberax tablets | 1 | 2.55 | 5.05 | 102.00 | 101.00 | 0.53 | 0.36 |
5 | 2.51 | 5.04 | 100.40 | 100.80 | 0.82 | 0.40 | |
10 | 2.52 | 5.01 | 101.80 | 100.20 | 0.75 | 0.29 | |
| |||||||
0 | 2.58 | 5.01 | 103.20 | 100.20 | 1.13 | 0.31 | |
Librax tablets | 1 | 2.55 | 5.00 | 102.00 | 100.00 | 0.97 | 0.91 |
5 | 2.53 | 4.99 | 101.20 | 99.80 | 0.68 | 0.87 | |
10 | 2.54 | 4.98 | 101.60 | 99.60 | 0.81 | 0.95 |
bFive independent analyses.
The developed method was successfully applied to analyze CDB and CDZ in marketed tablet formulations. The assay results are shown below for the average of five determinations of the four tablets. The performance of the proposed methods was assessed by comparison with the official method [
Determination of CDB and CDZ in pharmaceutical formulations by the proposed method and official method.
Sample | Clidinium bromide | Chlordiazepoxide | ||
---|---|---|---|---|
% Recoverya ± S.D. | ||||
Proposed method | Official method | Proposed method | Official method | |
Ribax (2.5 mg CDB and 5.0 mg CDZ/capsule) | ||||
| ||||
|
|
|
|
|
|
1.96 | 2.03 | 2.12 | 1.89 |
|
1.47 | 1.67 | ||
| ||||
Laberax (2.5 mg CDB and 5.0 mg CDZ/tablet) | ||||
| ||||
|
|
|
|
|
|
1.28 | 1.92 | 1.73 | 1.83 |
|
1.47 | 1.70 | ||
| ||||
Librax (2.5 mg CDB and 5.0 mg CDZ/tablet) | ||||
| ||||
|
|
|
|
|
|
2.04 | 2.18 | 1.60 | 1.74 |
|
1.17 | 1.23 |
aFive independent analyses.
bTheoretical values for
A simple, specific, precise, and sensitive RP-HPLC method has been developed and validated for quantitative determination of clidinium bromide and chlordiazepoxide in raw materials and pharmaceutical preparations with a limit of detection of 0.088 and 0.121
There is no kind of financial gain between the authors and the mentioned corporations and identities inside the paper.