Accepted Manuscript Title: Method Development And Validation For The Simultaneous Estimation Of “Albendazole And Praziquantel” In Bulk And Its Synthetic Mixture Author: Shreya R. Shah Suddhasattya Dey Prasannakumar Pradhan H.K. Jain U.M. Upadhyay PII: DOI: Reference:
S1658-3655(13)00077-0 http://dx.doi.org/doi:10.1016/j.jtusci.2013.08.004 JTUSCI 40
To appear in: Received date: Revised date: Accepted date:
3-6-2013 5-8-2013 14-8-2013
Please cite this article as: S.R. Shah, S. Dey, P. Pradhan, H.K. Jain, U.M. Upadhyay, Method Development And Validation For The Simultaneous Estimation Of “Albendazole And Praziquantel” In Bulk And Its Synthetic Mixture, Journal of Taibah University for Science (2013), http://dx.doi.org/10.1016/j.jtusci.2013.08.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Manuscript
METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF “ALBENDAZOLE AND PRAZIQUANTEL” IN BULK AND ITS SYNTHETIC MIXTURE SHREYA R. SHAH1*, SUDDHASATTYA DEY2, PRASANNAKUMAR PRADHAN1 1
Sigma Institute Of Pharmacy, Bakrol, Vadodara, 390019, Gujarat, India
Dr. B.C. Roy College of Pharmacy & Allied Health Sciences, Meghnad Saha Sarani, Bidhan
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H.K.JAIN1 & U.M. UPADHYAY1
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Nagar, West Bengal, Durgapur-713206, India.
*CORRESPONDING AUTHOR: SHREYA R. SHAH
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E-MAIL:
[email protected]
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Contact no: +91 9274201130
ABSTRACT
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A simple, rapid, sensitive RP-HPLC method was developed and validated for simultaneous measurement of Albendazole (ABZ) and Praziquantel (PRQ) along with an IS (Internal Standard: Simvastatin) at single wavelength at 225nm. Chromatographic separation was performed using a Enable C18 column. (250 mm × 4.6 mm, 5 μm: Spinco Biotech Pvt Ltd) and a mobile phase consisting of Acetonitrile: water (60:40 v/v) using 10% orthophosphoric acid for adjusting the pH: 3.2, at a flow rate of 1.0 ml/min. The Calibration curve were linear (r2 ≥ 0.999) over the concentration range of 0.05-8.0 µg/ml.Simvastatin was used as an Internal Standard(1.0µg/ml). The Limit of Quantification (LOQ) was 0.05µg/ml for both ABZ and PRQ. The determination of the two active ingredients was not interfered by the excipients in synthetic mixture. Proposed methods were validated as per ICH guidelines for linearity, accuracy, precision, and robustness for estimation of Albendazole and Praziquantel in bulk and its synthetic mixture form and results were found to be satisfactory. Key words: Albendazole, Praziquantel, Simvastatin (IS), HPLC, UV-detection, Validation.
1. Introduction: Neurocysticercosis is the most common helminthic disease of the nervous system, being considered a serious public health problem in developing countries of Latin America, Asia, and Africa [1–3]. Although restricted to palliative measures, the treatment of neurocysticercosis has advanced over the last 20 years with the use of praziquantel (PZQ) and albendazole (ABZ),
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2. Materials and methods
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drugs considered effective against the cystic larvae [2,4]. ABZ has been found to be more effective than PZQ, but in some patients there is persistence of cysts even after repeated use of ABZ [2]. For these cases, alternative treatment schedules such as the simultaneous use of PZQ and ABZ has been evaluated [4,5]. PZQ combined with ABZ have also been extensively used in human hydatid disease [6–9]. ABZ is extensively metabolized to its active metabolite albendazole sulfoxide (ASOX) that is further metabolized to the inactive albendazole sulfone (ASON) [10]. Due to this extensive metabolism, plasma concentrations of ABZ are usually low and pharmacokinetic studies are developed using ASOX and ASON concentrations [11–15]. PZQ is metabolized to several hydroxylated metabolites [16–18], mainly trans-4hydroxypraziquantel (TRANS), an active metabolite [19]. To evaluate the kinetic disposition of ABZ and PZQ, selective, sensitive and reproducible analytical methods are required for their quantification in plasma samples as well as their metabolites. High-performance liquid chromatography (HPLC) [20–29] and capillary electrophoresis (CE) [30,31] have been used for the development of these methods. The recent technological advances in coupling mass spectrometry to liquid chromatography (LC–MS and LC–MS–MS) brought new insight into quantitative bioanalysis. The use of this technique for the analysis of ABZ metabolites, PZQ and TRANS has been described only for the isolated drugs. Bonato et al. [32] and Chen et al. [33] reported the use of LC–MS–MS for the development of two methods with quantification limits for ASOX of 5.0 and 4.0μg/ml, respectively. Further, Bonato et al. [32] reported a quantification limit of 0.5μg/ml for ASON metabolite. LC–MS–MS was used only for the qualitative analysis of PZQ metabolites [16,34]. This paper describes for the first time a HPLC method for the simultaneous estimation of Albendazole and Praziquantel in bulk and its synthetic mixture. The aim of this work was to develop an HPLC method with ultraviolet detection for the simultaneous determination of Albendazole and Praziquantel in Bulk drug substance and synthetic mixture. The present HPLC method was validated according to the ICH guidelines.
2.1. Chemicals and reagents
Albendazole (Fig. 1) was obtained from Mercury Pharmaceutical Ltd, Vadodara, Gujarat, India, Praziquantel (Fig. 2) was obtained from Micro Labs Ltd., Goa, India and Simvastatin (IS) (Fig 3) was obtained from Dr. Reddy’s Lab, Hyderabad India. Acetonitrile, Methanol and Water (obtained from Merck Specialties private limited, Worli, Mumbai, India.) of HPLC grade were used. All the other reagents (10% ortho phosphoric acid) used for the development of a liquid chromatographic method for simultenous determination of albendazole & praziquantel in bulk & synthetic dosage form was of analytical grade are also obtained from Merck Specialties private limited, Worli, Mumbai, India. 2.2. Chromatographic condition: A high-performance liquid chromatography (Shimadzu, Kyoto, Japan) was composed of a LC20AT Prominence solvent delivery module, a manual rheodyne injector with a 20-µl fixed loop and a SPD-20A Prominence UV–visible detector. Separation was performed on an Enable C18 G column (particle size 5µm; 250mm×4.6mm) preceded by an ODS guard column (10 µm,
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10mm×5mm.) at an ambient temperature. The data acquisition was made with Spinchrom Chromatographic Station® CFR Version 2.4.0.195 (Spinchrom Pvt. Ltd., Chennai, India). The mobile phase consisted of acetonitrile: water in a ratio of 60:40 and PH is adjusted to 3.2 with Orthophosphoric acid, at a flow rate of 1.0 ml/ min. Mowas vacuum filtered and degassed through 0.2 µm pore size polymeric PTFE filters. .
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2.3. Method: 2.3.1. Preparation of standard stock solution of Albendazole, Praziquantel & IS (1000μg/ml): Methanol was used as diluents for the preparation of ABZ & PRQ solutions. 25 mg of both the drugs were weighed and transferred to a 25ml volumetric flask. 15 ml of diluent was added and the solution is sonicated for 15 min. Volume is made up to the mark with diluent to obtain a stock solution of 1000μg/ml. Separately Simvastatin was also preperd diluent to get the working standard solution of 1000μg/ml.
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2.3.2. Preparation of working standard solution Albendazole, Praziquantel & IS (100μg/ml): From the above standard solution 2.5 ml of stock solution was withdrawn and transferred to 25 ml volumetric flask, volume is made up to the mark with diluent to get the working standard solution of 100μg/ml. Separately Simvastatin was also preperd diluent to get the working standard solution of 100μg/ml which was finally diluted to make a final solution of 10 μg/ml.
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2.4. Validation of the Developed Method The developed method was validated by evaluating recovery, linearity, precision, accuracy, quantification limit and stability. Coefficients of variation and relative errors of less than 2% were considered acceptable [36–37].
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2.4.1. Linearity: The linearity was tested in the concentration range of 8, 7, 6, 5, 4, 3, 2, 1, 0.5 & 0.05μg/ml for ABZ & PRQ with a fixed concentration of 1μg/ml diluted from 10µg/ml mentioned in 2.3.2.. The calibration curve was constructed and evaluated by its coefficient of determination (r2). The calibration plot (peak area ratio of ABZ & PRQ to IS versus ABZ & PRQ concentration)was generated by replicate analysis (n = 10) at all concentration levels and the linear relationship was evaluated using the least square method within Microsoft Excel® program. For minimum error with precise, concise and accurate data 10 different concentrations were being taken which gave a wide range of linearity. The coefficient of determination (r2) for ABZ & PRQ were 0.999 & 0.999 for ABZ & PRQ and given in (Fig.4 & 5). 2.4.2. Accuracy: Accuracy of the method was determined by replicate analysis was carried out using the two sets of different standard addition methods at six different concentration levels first one 80%, 100% & 120% and second one 50%, 100% & 150%. and comparing the difference between the spiked value (theoretical value) and that actual found value. 2.4.3. Precision: The precision of the method based on within-day repeatability was determined by replicate analysis of three sets of samples spiked with three different concentrations of ABZ, PRQ (0.05, 1 and 8μg/ml) along with fixed concentration of IS 1µg/ml. The reproducibility (day-to-day
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variation) of the method was validated using the same concentration range as described above, but only a single determination of each concentration was made in three different days. Relative standard deviation (R.S.D.) was calculated from the ratios of standard deviation (S.D.) to the mean and expressed as a percentage.
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2.4.4. Specificity: Specificity study was performed by analyzing standard solution in the presence of an excipient to find was there any interference of excipients in % recovery of ABZ & PRQ. Amount of ABZ & PRQ was spiked with 50%, 100%, and 150% of excipient (talc) and the sample was analyzed for ABZ & PRQ recovery by HPLC along with fixed concentration of IS 1µg/ml.
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2.4.5. Robustness: Robustness of the above method for ABZ & PRQ was carried out the by the slight variation in flow rate, pH and mobile phase ratio along with fixed concentration of IS 1µg/ml. The percentage recovery and % RSD were noted for both the drugs.
3. Result and Discussion
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3.1. Calibration Curve: Linearity of response for ABZ and PRQ for determination of both of them in synthetic mixture, by preparing and injecting mixture of stock solution suitably diluted to achieve concentration of about 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8 µg/ml along with a fixed concentration of IS 1µg/ml. The value of Coefficient of correlation ‘r’, slope and intercept were 0.999, 1.064 & 0.358 for ABZ where as 0.999, 0.169 & 0.060 for PRQ by using UV detection shown in (Fig 6) the absorbance maxima was set at 225nm. The retention times were 4.533 for ABZ, 6.39 for PRQ and 7.8 for IS shown in ( Fig 7). The linear regression data from the calibration curve indicate that the response is linear over the concentration range studied for both the drug. So it can be applied for the determination of ABZ and PRQ in synthetic mixture. 3.2. Analytical method validation:
3.2.1. Linearity: The linearity was tested in the concentration range of 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8µg/ml for ABZ & PRQ with a fixed concentration of IS 1µg/ml and the calibration curve was constructed and evaluated by its correlation coefficient. The linear relationship was evaluated using the least square method within Microsoft Excel® program. The coefficient of determination (r2) for both ABZ and PRQ was 0.999 given in (Table 1 & 2). 3.2.2. Limit of detection and limit of quantification: A limit of detection (LOD) and a limit of quantification (LOQ) were established based on the calibration curve parameters, according to the formula: LOD = 3.3*S.D/Slope and LOQ = 10*S.D/Slope. (Table 2)
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3.2.3. Precision: Precision was measured in terms of repeatability of measurement, performed by injecting the standard solution six times and measure the peak area. The RSD was found to be less than 2.0% for both ABZ & PRQ with a fixed concentration of IS 1µg/ml. This shows that Precision of the method is satisfactory which is shown in (Table 3(a), 3(b), 3(c).) Intermediate Precision: Intermediate Precision with expected results and express as percentage.
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3.2.4. Accuracy: The accuracy of the method was determined by the recovery study carried out using the two sets of different standard addition methods at six different concentration levels first one 80%, 100% & 120% and second one 50%, 100% & 150%. Resulting spiked sample solutions were assayed in triplicate and the results obtained were compared with expected results and express as percentage with a fixed concentration of IS 1µg/ml. The mean % recovery of PRQ and ABZ was found to be in the range 100.81-100.92% & 99.71-100.86% within the acceptance limit at the level of 80%, 100%, 120% given in (Table 4a) and 98.33-100.66 & 98.46-100.73 within the acceptance limit at the level of 50%, 100%, 150% given in (Table 4b).
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3.2.5. Specificity: Specificity study was performed by analyzing standard solution in the presence of an excipient (talc). 10mg each ABZ & PRQ were spiked with 50% (5mg), 100% (10mg), and 150% (15mg) of talc and the samples were analyzed for ABZ & PRQ recovery by HPLC with a fixed concentration of IS 1µg/ml. Acceptance criteria for % interference
