A simple, selective, linear, precise, and accurate RP-HPLC method was developed and validated for rapid assay of Pizotifen in pharmaceutical dosage form. Isocratic elution at a flow rate of 1.0 mL/min was employed on Chromosil C18 (250 mm × 4.6 mm, 5
Pizotifen (4-(1-methyl-4-piperidylidine)-9,10-dihydro-4H-benzo-
(a) Structure of Pizotifen. (b) RP-HPLC Chromatogram of standard solution (c) On
Abounassif et al. [
The reference sample of Pizotifen (API) was obtained from V.V MED, Hyderabad. The Formulation was procured from the local market. acetonitrile, methanol, ammonium dihydrogen phosphate, triethylamine, and orthophosphoricacid used were of HPLC grade and purchased from Merck Specialties Private Limited, Mumbai, India.
Peak HPLC containing LC 20 AT pump, variable wavelength programmable UV/VIS detector, and Rheodyne injector were employed for the investigation. The chromatographic analysis was performed on a Chromosil C18 column (250 mm × 4.6 mm, 5
Proper selection of the stationary phase depends upon the nature of the sample, molecular weight, and solubility. Pizotifen, the selected drug was analyzed by reverse phase columns. Among C8 and C18, Chromosil C18 column (250 mm × 4.6 mm, 5
For analysis 100 ppm standard solution was prepared in mobile phase. Required concentrations were obtained from 100 ppm standard solution by proper dilution. pH of the mobile phase was adjusted to 5.8 with orthophosphoric acid and filtered through 0.45
The formulation tablets of Pizotifen (Migralin—0.725 mg) were crushed to give finely powdered material. With powder we prepared 70 ppm solution in mobile phase and then filtered it through Ultipor N66 Nylon 6,6 membrane sample filter paper.
For developing the method, a systematic study of the effect of various factors was undertaken by varying one parameter at a time and keeping all other conditions constant. Method development consists of selecting the appropriate wavelength and choice of stationary and mobile phase. The following studies were conducted for this purpose.
The spectrum of diluted solutions of Pizotifen in mobile phase was recorded separately on UV spectrophotometer. The peak of maximum absorbance wavelength was observed. The spectra of the Pizotifen showed that a wavelength was found to be 230 nm. RP-HPLC chromatogram of standard solution is given in Figure
The proposed method was validated as per ICH guidelines. The parameters studied for validation were specificity, linearity, precision, accuracy, robustness, system suitability, limit of detection, limit of quantification, and solution stability.
The specificity of the method was performed by comparing the chromatograms of blank, standard, and sample. It was found that there is no interference due to excipients in the tablet formulation and also that there is good correlation between the retention times of standard and sample. The specificity results are shown in Table
Specificity study.
Name of the solution | Retention time in min |
---|---|
Blank | No peaks |
Pizotifen | 2.01 |
Linearity was performed by preparing mixed standard solutions of Pizotifen at different concentration levels including working concentration mentioned in experimental condition, that is, 25 ppm. Twenty microliters of each concentration was injected in duplicate into the HPLC system. The response was read at 230 nm, and the corresponding chromatograms were recorded. The regressions of the plots were computed by least square regression method. Linearity results are presented in Table
Linearity results.
Level | Concentration of Pizotifen in ppm | Mean peak area |
---|---|---|
Level-1 | 5.0 | 30333.1 |
Level-2 | 10.0 | 61990.7 |
Level-3 | 15.0 | 88759.2 |
Level-4 | 20.0 | 121043.1 |
Level-5 | 25.0 | 148510.7 |
Level-6 | 30.0 | 186164.2 |
6119.996 | ||
Slope intercept correlation coefficient | 966.4 | |
0.998 |
Precision of the method was performed as intraday precision and interday precision. To study the intraday precision, six-replicate standard solution of Pizotifen was injected. The percent relative standard deviation (% RSD) was calculated, and it was found to be 1.4, which is within the acceptable criteria of not more than 2.0. Results of system precision studies are shown in Table
Results of Precision Study (Intraday).
Sample | Conc (PPM) | Injection no. | Peaks area | RSD (acceptance criteria 2.0) |
---|---|---|---|---|
Pizotifen | 25 ppm | 1 | 152526.6 | |
2 | 151615.4 | |||
3 | 152127.8 | 1.40 | ||
4 | 153481.7 | |||
5 | 150705.3 | |||
6 | 156830.7 |
For interday precision six-replicate standard solution of Pizotifen was injected on the third day of sample preparation. The percent relative standard deviation (% RSD) was calculated, and it was found to be 1.07, which is within the acceptable criteria of not more than 2.0. Results of system precision studies are shown in Table
Results of precision study (interday).
Sample | Conc (PPM) | Injection no. | Peaks area | RSD (acceptance criteria 2.0) |
---|---|---|---|---|
Pizotifen | 25 | 1 | 146180.5 | |
2 | 142312.7 | |||
3 | 143153.8 | 1.07 | ||
4 | 145268.1 | |||
5 | 143858.6 | |||
6 | 145253.9 |
A known amount of standard drug was added to the fixed amount of preanalyzed tablet solution. Percent recovery was calculated by comparing the area before and after the addition of the standard drug. The standard addition method was performed at 25%, 50%, and 75% level. The percent recovery and % RSD were calculated, and results are presented in Table
Mean percentage recovery.
Level | Amount of Pizotifen spiked (ppm) | Amount of Pizotifen recovered (ppm) | % recovery | Mean % recovery | % RSD | Mean % RSD |
---|---|---|---|---|---|---|
25% | 5 | 4.97 | 99.4 | |||
5 | 5.02 | 100.4 | ||||
5 | 4.98 | 99.6 | 0.53 | |||
50% | 10 | 10.01 | 100.1 | |||
10 | 9.99 | 99.9 | 99.92994 | 0.2603 | ||
10 | 9.98 | 99.8 | 0.15 | |||
75% | 15 | 14.99 | 99.93 | |||
15 | 15.02 | 100.13 | 0.101 | |||
15 | 15.01 | 100.06 |
Pizotifen at 6 ppm concentration was analyzed under these changed experimental conditions. It was observed that there were no marked changes in chromatograms, which demonstrated that the developed method was robust in nature. The results of robustness study are shown in Table
Robustness.
Condition | Mean area | % assay | % difference |
---|---|---|---|
Unaltered | 144805.5 | 100.0 | 0.0 |
Flow rate at 0.8 mL/min | 144720.3 | 99.94 | 0.06 |
Flow rate at 1.2 mL/min | 143951.2 | 99.41 | 0.59 |
Mobile phase: | |||
Acetonitrile : Methanol | |||
92%08% | 144902.5 | 100.06 | 0.06 |
88%12% | 144763.4 | 99.97 | 0.03 |
pH of mobile phase at 6.0 | 144275.2 | 99.63 | 0.37 |
pH of mobile phase at 5.6 | 144358.7 | 99.69 | 0.31 |
System suitability was studied under each validation parameter by injecting six replicates of the standard solution. The system suitability parameters are given in Table
System suitability parameters.
Parameter | Tailing factor | Theoretical plates |
---|---|---|
Specificity study | 1.34 | 4964.61 |
Linearity study | 1.17 | 4590.18 |
Precision study | 1.48 | 4875.34 |
For this study six replicates of the analyte at lowest concentration were measured and quantified. The LOD and LOQ of Pizotifen are given in Table
LOQ and LOD.
Parameter | Measured volume |
---|---|
Limit of quantification | 65 ng/mL |
Limit of detection | 20 ng/mL |
Various combinations of orthophosphosph acid, triethylamine, ammonium dihydrogen phosphate, acetonitrile, and methanol were tested as mobile phase. The concentrations of acetonitrile and methanol were optimized to give symmetric peak with short runtime. A system suitability test was applied to representative chromatograms for various parameters. Ten-point graph was constructed covering a concentration range 5–30 ppm (three independent determinations were performed at each concentration). Linear relationship between the peak area signals of Pizotifen and the corresponding drug concentrations was observed. The calibration curve was obtained for a series of concentration in the range of 05–30 ppm and it was found to be linear. The data of regression analysis of the calibration curves are shown in Table
The stability of Pizotifen in standard and sample solutions was determined at ambient temperature (20 ± 10°C). The solutions were checked in triplicate after three successive days of storage, and the data were compared with freshly prepared samples. In each case, it could be noticed that solutions were stable for 48 hrs, as during this time, the results did not decrease below 98%. This denotes that Pizotifen is stable for at least 2 days at ambient temperature. The system suitability parameter values are within the limits. The statistical evaluation of the proposed method revealed its good linearity, reproducibility, and its validation for different parameters.
A validated RP-HPLC method has been developed for the determination of Pizotifen in tablet dosage form. The proposed method is simple, rapid, accurate, precise, and specific. Its chromatographic runtime of 6 min allows the analysis of a large number of samples in short period of time. Therefore, it is suitable for the routine analysis of Pizotifen in pharmaceutical dosage forms. So it could be used for the rapid and reliable determination of Pizotifen in tablet formulations.