A Validated sensitive stability indicating HPLC method for the determination of pimozide and its related impurities in bulk Drug and Formulations
Sai Prudhvi N1,2*, Venkateswarlu B. S.3
1Research Scholar, Vinayaka Missions College of Pharmacy, Salem, India.
2Department of Pharmaceutical Analysis, M.A.M College of Pharmacy, Guntur, India.
3Department of Pharmaceutics, Vinayaka Missions College of Pharmacy, Salem, India.
*Corresponding Author E-mail: saiprudhvi00@gmail.com
ABSTRACT:
Aim: The aim of the present study is to develop a high resolved and validated liquid chromatography method for the separation and quantification of pimozide and its related impurity A and B in bulk and pharmaceutical formulations. Materials and Methods: Separation of impurities and pimozide was achieved on Spherisorb ODS2 column (250mm x 4.6mm, 5μ) using mobile phase composition of methanol, acetonitrile and 0.1M sodium perchlorate in the ratio of 40:30:30 (v/v) at pH 6.1 as mobile phase at a flow rate of 0.8ml/min in isocratic condition. UV detection of the eluents was monitored at a wavelength of 240nm. Results: In the developed conditions, well resolved peaks were observed at a retention time of 7.0, 5.9 and 10.1 min for pimozide, Impurity A and B respectively. Calibration curve was plotted in the concentration range of 75-450 µg/ml for pimozide and 1-6µg/ml for impurity A and B with limit of detection of 2.4µg/mL, 0.016µg/mL and 0.025µg/mL for pimozide, impurity A and B respectively. Forced degradation study confirm that the method can be separate the known and unknown impurities of pimozide and the % degradation was found to be very less in all the stress conditions. Conclusion: The method can suitable for the identification and quantification of impurities A and B along with pimozide in bulk drug and formulations.
KEYWORDS: Pimozide; HPLC Method; Impurity A; Impurity B; Forced degradation.
INTRODUCTION:
The survey of literature for the analysis of pimozide confirms that different analytical methods reported for the estimation of pimozide. One HPLC (6), four UV-visible spectrophotometry methods (7-10) and one HPTLC (11) method was reported for the estimation of pimozide in pharmaceutical formulations. One HPLC with fluorescence detection bio analytical method was reported for the estimation of pimozide in human plasma (12) and Electrooxidation of pimozide was studied using voltammetric and HPLC with electrochemical detection (13).
There is no method reported for the separation and estimation of pimozide and its related impurities. The aim of the impurity profiling is to detect, identify and quantify the organic and inorganic impurities as well as residual solvents in bulk drugs and pharmaceutical formulations. The different pharmacopoeias, such as the British Pharmacopoeia (BP) and the United States Pharmacopoeia (USP) are slowly incorporating limits to allowable levels of impurities present in the API’s or formulations. The estimation of impurity profiles in drug substances and related materials has become one of the most important fields of activity in contemporary pharmaceutical analysis.
The drug pimozide is officially approved by USP and BP and is having 5 known impurities (impurity A to F). Based on the availabulity of impurities, the present work focused on the development and validation of a stability indicating LC method for the separation, qualitative and quantification of related impurities A and B of pimozide in bulk and pharmaceutical formulations. The Molecular structure of pimozide and its impurities A and B were given in figure 1.
Figure 1: Molecular structures of pimozide (c), impurity A (b) and B (c)
MATERIALS AND METHODS:
The separation and quantification of related impurities of pimozide was carried in Waters Spherisorb ODS2 Column (250mm x 4.6mm, 5μ) equipped in isocratic PEAK HPLC instrument a LC 20AT pump for solvent delivery and variable wavelength programmable LC – 7000 UV-detector for detection. The samples were injected using Rheodyne manual inject port and data was analyzed by using PEAK software version 2.0. The standard and samples was weighed in DENVER (SI-234) electronic balance and ultrasonic batch sonicator (1.5 L) was used for the mixing and preparation of mobile phase, standard and samples.
Chemicals and Reagents:
Pimozide active pharmaceutical ingredient (API) and its two impurities A and B were obtained from Intas Pharmaceuticals Limited, Hyderabad, Telangana. The marketed formulation Pimolite© - 2mg was purchased in local pharmacy. Laboratory reagent grade sodium acetate, acetic acid and HPLC grade methanol and water were purchased from Merck chemicals, Mumbai. The membrane filter papers (0.2µ nylon) were purchased from millipore (India).
Preparation of pimozide standard, Impurity A and B solutions:
25mg of standard drug pimozide was weighed accurately and was dissolved in 25mL methanol. Pimozide at a concentration of 1000µg/mL was obtained and the obtained solution is filtered. Pimozide standard solution at a concentration 100µg/mL was prepared by diluting 10mL from 1000µg/mL to 100mL. The same procedure was used for the preparation of impurity A and B separately. Equal volumes of pimozide, Impurity A and B selected known concentrations were mixed to get mixed standard solution. The combined solution of pimozide, Impurity A and B having known concentrations were used for method development and validation study.
Preparation of formulation solution:
The market formulation tablets (n = 10) of pimozide with brand Pimolite© - 2mg was powdered using sterile mortar and pestle. An amount of the tablet powder equivalent to 10mg of pimozide was weighed accurately and was dissolved in 10mL methanol. Then it was filtered and 3mL from the solution was again diluted to 10mL to get a formulation solution having 300µg/mL of pimozide. The solution was used for the estimation of pimozide and its related impurities in pharmaceutical formulations using HPLC developed method.
Method development:
The method development for the identification and quantification of pimozide and its related impurities A and B in pharmaceutical formulations, different method development trails were performed. In the method development, composition of mobile phase, pH of mobile phase, configuration of stationary phase, UV detector wavelength and mobile phase flow rate was studied. In each trail condition, the system suitability parameters were checked and the conditions that produce best results were considered as optimized.
Method validation:
The method was validated for the determination of range of analysis, sensitively, accuracy, precise, rugged robust nature of the method developed for the separation and quantification of pimozide, Impurity A and B. All the method development and validation studies were carried as per the ICH guidelines [14,15].
Force degradation studies:
Forced degradation study was carried for the standard drug pimozide in the develop method to evaluate the effectiveness of the developed method for the separation and identification of known and unknown impurities in the drug [15,16]. 50mg of standard drug pimozide was mixed with 50mL of 0.1N HCl for acid hydrolysis study, 50mL of 0.1N NaOH in base hydrolysis study and 50mL of 3% hydrogen peroxide solution for oxidative degradation study. The solution was incubated for 24 H and then neutralized. The neutralized standard drug was diluted to 300µg/mL and was analyzed in the developed method condition. In photolytic and thermal degradation conditions, standard drug was kept under UV light at 254nm and oven at 600C for 24 hours respectively. Then the standard drug was diluted to 300µg/mL and was analyzed in the developed method condition. The % degradation, number of degradation products formed in the degradation study and the % effectiveness of the method for the separation of degradation products was evaluated.
Formulation analysis:
The formulation solution prepared from the formulation tablets of pimozide (Pimolite© - 2mg) was analysed in the developed method. The % assay of the pimozide in the developed method was calculated in the developed method.
RESULTS AND DISCUSSION:
As there is no stability indicating HPLC method was reported for the quantitative analysis of pimozide and its impurities A and B. The present work aimed to develop a simple, stability-indicating, robust and straightforward enough for routine analyses of pimozide and its impurities A and B in quality control laboratories.
The first step of method development for the quantification of process-related impurities in pimozide drug substance is the appropriate selection of the wavelength to obtain good sensitivity with minimum noise. It was studied by measuring the ultraviolet absorption spectrum of pimozide, impurity A and B. The impurities of pimozide showed significant high UV absorbance at wavelength of 240nm. Hence, the detector wavelength was kept at 240nm throughout the analysis which give best absorption for impurities and significant absorbance for pimozide standard.
The column selectivity for the separation of related substance B and standard drug pimozide was critical because of similar chemical structure and polarities. Preliminary experiments were performed using three different columns including Phenomenex ODS C18 (250 mm × 4.6mm, 5μ), Xbridge Waters C18 (250mm × 4.6 mm, 5μ) and Spherisorb ODS2 (250mm x 4.6mm, 5μ) column. It was noticed that the studied compounds were well retained and separated with comparatively sharp peaks on Spherisorb ODS2 (250mm x 4.6mm, 5μ) column. So, further optimization was carried out on this column.
Several mobile phases with different compositions and polarities like mater, methanol, acetonitrile, phosphate buffer with different pH ranges, acetate buffer with pH ranges and sodium perchlorate at different pH were examined for their efficiency in resolution of pimozide and its impurities A and B. Methanol, acetonitrile and 0.1 M sodium perchlorate in the ratio of 40:30:30 (v/v) at pH 6.1 with 1% perchloric acid as mobile phase was finally selected for subsequent investigations as it yielded the best separation between pimozide and its impurities A and B. The effect of the buffer salt concentration on separation was also investigated by changing its concentration from 0.01 M to 0.3 M and it was found that the tailing factors, theoretical plates and resolution of pimozide and its impurities A and B was highly sensitive to the concentration of sodium perchlorate, and proper peak shapes for pimozide and its impurities A and B was obtained with a sodium perchlorate concentration of 0.1 M. Considering the separation of pimozide and its impurities A and B, run time, column back pressure, the mobile phase flow rate and column oven temperature was fixed at 0.8mL/min and 35◦C respectively.
In the optimized conditions, the standard drug and impurities were well resolved and retained at a retention time of 7.01 min, 5.93 min and 10.06 min for pimozide, Impurity A and Impurity B respectively and clear base line was observed. The method obeys system suitability conditions (Table 1) for both standard and impurities. Figure 2 shows the optimized chromatogram of pimozide and its related compounds A and B in the developed method.
Table 1: System suitability results of pimozide and its impurities in the developed method
|
Parameter |
Pimozide |
Impurity A |
Impurity B |
|
Standard Concentration |
300µg/mL |
4µg/mL |
4µg/mL |
|
Retention time (min) |
7.0 |
5.9 |
10.1 |
|
Peak Area response |
918379 |
49665 |
57981 |
|
Resolution |
4.8 |
--- |
7.6 |
|
Theoretical Plates |
6429 |
4098 |
9680 |
|
Tailing Factor |
0.63 |
0.91 |
0.57 |
Figure 2: Standard chromatogram in the optimized conditions
Accurately correlated calibration range was observed in the concentration range of 75-450µg/mL for pimozide, 1-6µg/mL for impurity A and B. The regression equitation was found to be y = 3021.x – 5283 (R² = 0.999), y = 12951x – 2318 (R² = 0.999) and y = 14556x + 1215 (R² = 0.999) for pimozide, impurity A and B respectively. The calibration curve was found to be linear in the concentration studied for both pimozide and its related impurities studied. The results of linearity study were given in table 2.
Table 2: Linearity results of pimozide and its impurities in the developed method
|
S. No. |
Pimozide |
Impurity A |
Impurity B |
|||
|
Concentration in µg/mL |
Peak Area |
Concentration in µg/mL |
Peak Area |
Concentration in µg/mL |
Peak Area |
|
|
1 |
75 |
214825.4 |
1 |
10930.6 |
1 |
16452.6 |
|
2 |
150 |
438668.5 |
2 |
23675.4 |
2 |
30081.9 |
|
3 |
225 |
687123.3 |
3 |
35659.5 |
3 |
45007.2 |
|
4 |
300 |
918379 |
4 |
49665.7 |
4 |
57981.5 |
|
5 |
375 |
1124945.7 |
5 |
62822.8 |
5 |
74119.2 |
|
6 |
450 |
1343033.1 |
6 |
75295.1 |
6 |
89326.8 |
The repeatability and reproducibility was studied by intraday, interday precision and ruggedness study. The standard solution at a concentration of 300µg/mL of pimozide, 4µg/mL impurity A and B was analysed six times in the same day for intraday precision, six times in three successive days for interday precision and six times for change in analyst for ruggedness study. The % RSD in each changed condition was calculated and was found to be 0.128, 0.405 and 0.443% in intraday precision, 0.258, 0.127 and 0.191 in interday precision and 0.263, 0.519 and 0.586 in ruggedness study for pimozide, impurity A and B respectively. This confirms that the method developed was found to be precise and rugged for the analysis of pimozide, impurity A and B.
The standard concentration of pimozide, impurity A and B were analyzed by change in analytical conditions. The % change was calculated and was found to be within the acceptable limit of less than for pimozide, impurity A and B confirms that the method was found to be robust. The spiked recovery at 50%, 100% and 150% spiked levels at a target concentration of 150µg/mL of pimozide and 2µg/mL of impurity A and B were studied. The % Recovery and the % RSD of recovery in each spike level for pimozide, impurity A and B was calculated (table 3) and was found to be within the acceptable limits confirms that the method was found to be accurate.
Table 3: Accuracy results of pimozide and its impurities in the developed method
|
S. No. |
Compound |
Level |
Concentration in µg/mL |
% Recovery |
% RSD |
|||
|
Target |
Spiked |
Total |
Recovered Mean±SD |
|||||
|
1 |
Pimozide |
50% |
150 |
75 |
225 |
224.65±0.22 |
99.84 |
0.12 |
|
2 |
100% |
150 |
150 |
300 |
298.26±0.42 |
99.42 |
0.14 |
|
|
3 |
150% |
150 |
225 |
375 |
374.18±0.58 |
99.78 |
0.15 |
|
|
4 |
Impurity A |
50% |
2 |
1 |
3 |
2.96±0.020 |
98.77 |
0.67 |
|
5 |
100% |
2 |
2 |
4 |
3.97±0.012 |
99.31 |
0.31 |
|
|
6 |
150% |
2 |
3 |
5 |
4.96±0.007 |
99.32 |
0.15 |
|
|
7 |
Impurity B |
50% |
2 |
1 |
3 |
2.96±0.019 |
98.71 |
0.64 |
|
8 |
100% |
2 |
2 |
4 |
3.96±0.022 |
99.13 |
0.56 |
|
|
9 |
150% |
2 |
3 |
5 |
4.93±0.044 |
98.66 |
0.88 |
|
Values given in table are the mean ± standard deviation (n = 3)
The sensitivity of the method was confirmed by detection and quantification results of pimozide, impurity A and B. The LOD of the method was determined using standard deviation of the response (s) and the slope of the calibration curve (m) values in using the formula (LOD=3.3s/m). The LOQ was calculated from the obtained LOD values (LOQ=3.3×LOD). The limit of detection was found to be 2.4µg/mL, 0.016µg/mL and 0.025µg/mL and quantification limits of 7.96µg/mL, 0.05µg/mL and 0.08µg/mL was observed for pimozide, impurity A and B respectively in the developed method which are found to be very sensitive.
In Acid hydrolysis condition, a very high % degradation of 9.59 % was observed and three additional degradation compounds were observed (Figure 3) along with five additional degradation compounds. The % degradation of 7.67 with four additional degradation compounds was observed in base hydrolysis degradation study (Figure 4). 8.41%, 5.28 and 9.10% of degradation was observed in oxidative degradation (Figure 5), thermal (Figure 6) and UV degradations (Figure 7) respectively. In all these degradation conditions, impurity A and B were detected along with pimozide and the additional degradation compounds formed during the stress study were also effectively separated confirms that the method was found to be effective separation of known and unknown impurities in pimozide. Table 4 shows the forced degradation study results of pimozide in different stress conditions.
Table 4: Forced degradation results of pimozide in the developed method
|
S No |
Condition |
No of additional peaks |
Peak Area |
% recovered |
% degraded |
|
1 |
Acid hydrolysis |
5 + 2 Impurities studied |
830258 |
90.40 |
9.59 |
|
2 |
Base hydrolysis |
4 + 2 Impurities studied |
847936 |
92.32 |
7.67 |
|
3 |
Oxidative degradation |
4 + 2 Impurities studied |
841107 |
91.58 |
8.41 |
|
4 |
Thermal |
2 + 2 Impurities studied |
869825 |
94.71 |
5.28 |
|
5 |
UV |
3 + 2 Impurities studied |
834791 |
90.89 |
9.10 |
Figure 3: Acid hydrolysis chromatogram
Figure 4: Base hydrolysis chromatogram
Figure 5: Oxidative degradation chromatogram
Figure 6: Thermal degradation chromatogram
Figure 7: UV degradation chromatogram
The formulation assay was found to be 98.83% for pimozide in the developed method. In the formulation chromatogram, both the impurities were detected and clear base line was observed. No excepients detected in the formulation chromatogram confirms that the method was suitable for the routine analysis of pimozide. The formulation chromatogram was shown in figure 8.
Figure 8: Formulation chromatogram
The developed method conditions developed for the analysis of pimozide was compared with the previous reported analytical methods. The HPLC method reported by Prachi et al., 2014 was found to having sensitive linearity range of (5-100µg/mL) then the developed method (75-450µg/mL). The reported method was found to be the assay method that can be applied for the analysis of drug in formulations but the present method will suitabale for the analysis of impurities in the formulation and standard drug that suitable high calibration for the detection of impurities which are present in very less amount. The spectrophotometry methods reported by Amal et al., 2015 (7), Sayanna et al., 2014 (8), Lohita et al., 2014 (9) and Prachi et al., 2016 (10) were found to be quantitative methods and in those method separation, identification of impurities in the drug was not possible. The HPTLC method reported by Manjula et al., 2013 (11) and voltammetric, HPLC–EC method reported by Sibel et al., 2002 (13) were found to be the analysis of drugs in formualation and the separation and estimation of impurities was not suitable for these methods. The bio analytical method reported by Kerbusch et al., 1997 (12) was suitable for the analysis of pimozide in human plasma and for pharmacokinetic studies. Hence the method reported here was found to be the only choice for the separation of impurities A and B in pimozide and quantitative analysis of pimozide and its related impurites A and B in pharmaceutical formulations.
CONCLUSION:
A simple and stable RP HPLC method was successfully developed and validated for the separation, qualitative and quantitative analysis of pimozide and its related impurities A and B. All the validation parameters were found to be within the limits and can effectively separate the known and unknown impurities forced during the stress study. The assay utilized a previously unreported set of conditions, including a simple mobile phases in isocratic elution and to effect the separation without using an ion-pair reagent. LOD and LOQ, established by this method, are lesser than earlier reported methods. The method is found to be linear in the specified range, precise and robust. Accuracy of the method is also established for the formulation. Hence the method can be used for the routine analysis of pimozide and its related impurities A and B in furnished products and in formulations.
ACKNOWLEDGEMENTS:
authors sincerely acknowledge thanks to Principal, Teaching and Non-teaching staff of both M.A.M College of Pharmacy and Vinayaka Missions College of Pharmacy for the successful completion of research work.
AUTHOR CONTRIBUTIONS:
Concept – All authors in the manuscript are equitably contributed.
CONFLICT OF INTEREST STATEMENT:
“The authors declared no conflict of interest” in the manuscript.
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Received on 12.12.2019 Modified on 03.02.2020
Accepted on 29.04.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2020; 13(12):6027-6034.
DOI: 10.5958/0974-360X.2020.01051.3