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COMPARATIVE STUDY OF CHROMATOGRAPHIC, SPECTROPHOTOMETRIC AND NON AQUEOUS TITRIMETRIC METHODS FOR DETERMINATION OF PROTEASE INHIBITOR IN TABLETS © 2014 A. Behera*, D. G. Sankar**, S. K. Moitra*, S. C. Si*
*School of Pharmaceutical Sciences, Siksha 'O' Anusandhan University Bharatpur, Ghatikia, Bhubaneswar, Orissa, 751003, India 1E-mail: anindita02@gmail.com **Pharmaceutical Analysis and Quality Assurance Division, College of Pharmaceutical Sciences, Andhra University
Visakhapatnam, Andhra Pradesh, 530003, India Received 23.03.2012; in final form 12.03.2013
The paper describes HPLC, UV spectrophotometric and non aqueous titrimetric method for the estimation of poorly water soluble protease inhibitor i.e. Ritonavir in raw material and tablet dosage form. HPLC analysis was carried out in a C18 column using acetonitrile, methanol, and buffer in the ratio 60 : 20 : 20 (v/v/v) at 240 nm. For the spectrophotometric determination methanolic solution of ritonavir was reacted with 3-Methyl Benzothiazolin-2-one hydrazone (MBTH). The oxidative coupled green coloured chromogen was analysed at 633 nm. Non aqueous titration was carried out using perchloric acid as titrant and the end point was determined using crystal violet as indicator. The three methods were validated and statistically evaluated to correlate the difference between the methods for estimation of Ritonavir in pharmaceutical dosage form.
Keywords: Ritonavir, HPLC method, spectrophotometry, 3-methylbenzothiazolin-2-one hydrazone, non aqueous titration, statistical analysis.
DOI: 10.7868/S0044450214070020
Ritonavir (RTV) is a selective, competitive and reversible inhibitor of the human immunodeficiency virus protease enzyme. Chemically it is (5S, 8S, 10S, 11s)-10-hydroxy-2-methyl-5-(1-methylethyl)-1-[2-(1-methylethyl)-4-thiazolyl]-3, 6-dioxo-8, 11-bis-(phenylmethyl)-2,4,7,12-tetraazatridecan-13-oic acid 5-thiazolyl methyl ester [1] (Scheme 1).
Scheme 1. Chemical structure of Ritonavir (RTV).
RTV is official in Indian [1], British [2] and US [3] pharmacopoeias. RTV selectively inhibits liver enzyme Cytochrome P450 which helps increasing the
bioavailability of other protease inhibitors such as Ata-zanavir Sulfate or Lopinavir in dual protease therapy [4]. From the literature review, it was found that RTV has been determined by HPLC in biological samples and pharmaceutical dosage forms [5—15] singly or in combination with other antiretroviral drugs, and Sudha et al. developed an HPTLC method in single dosage form [16], and Sulebhavikar et al. developed an HPTLC method in combined dosage form with Lopinavir [17]. Garren et al. studied the bioavailability of generic Ritonavir and Lopinavir tablet in dog model [18]. Rao et al. reported stress degradation study by LC-MS method [19].
RTV is used as a booster for the protease inhibitors like Atazanavir and Lopinavir in second line treatment ofAIDS. The official methods of assay include the liquid chromatographic method employing peak reagents (sodium hexane sulfonate) and use of buffer at specific column temperature. Heine et al. [8] reported the retention time of RTV in biological sample at 34 min, which cannot be always applicable for routine quality control of raw materials and dosage forms. So some easier, accurate and precise methods are being developed such as spectrophotometric and chromato-graphic ones for analysis of raw materials and dosage
forms. As RTV is classified as Class IV-like drug under BCS classification [20], a non aqueous titrimetric method is developed using perchloric acid as titrant in presence of glacial acetic acid and crystal violet to detect the endpoint. The three methods are validated according to ICH guidelines [21] and the results from the methods are compared statistically using analysis of variance (ANOVA).
EXPERIMENTAL
Reagents and materials. Reference standard and raw material of RTV were procured from Matrix Laboratories (Hyderabad, India) as gift sample. Methanol, acetonitrile, water, triethylamine and orthophos-phoric acid were of HPLC grade; methanol, ferric chloride, 3-methyl benzothiazolin-2-one hydrazone, perchloric acid, glacial acetic acid, potassium hydrogen phthalate and acetic anhydride were of analytical grade. Crystal violet was of laboratory grade. For spectrophotometry study double distilled water was used. Two brands of tablets (Ritomune, Cipla and Viriton, Ranbaxy; 100 mg) were purchased from local market.
Instrumentation and analytical conditions. The chromatographic system consisted of a JASCO (Japan) chromatograph equipped with an LC-Net II/ADC, an MU-2010 Plus PDA detector, a PU-2089 Plus quaternary pump, an online degasser and a Rheodyne model 7725 injector valve with 20 ^L sample loop. The chromatograph is coupled with Chrompass software (v. 1.7.403.1). Separation of RTV was done on a HiQ-Sil C18HS (250 x 4.6 mm, particle size 5 ^m, Kyatech, Japan) under reverse-phase partition chromatograph-ic conditions.
For spectrophotometry a Jasco model V-630 UV-vis-ible spectrophotometer with 1.0 cm matched cells was used. The spectrophotometer is coupled with Spectra Manager software. The RTV reacts with MBTH to form the green coloured chromogen.
The nonaqueous titration was carried out by 0.1 M perchloric acid as titrant prepared in glacial acetic acid—acetic anhydride and standardized by potassium hydrogen phthalate, previously dried at 120°C for 2 h, as per IP method. The end point was determined by crystal violet indicator [22].
Procedure for preparation of standards, calibration and analysis. HPLC. Working stock solution of 1000 ^g/mL of RTV was prepared by dissolving 25 mg of reference standard in 25 mL of methanol. The stock solution was diluted further with methanol to obtain working dilutions of concentrations 500, 400, 300, 200, 100 and 50 ^g/mL. The prepared samples were also filtered through a 0.45 ^m nylon membrane filter before injection. The injection volume was 10 ^L. Mixture of acetonitrile—methanol—buffer 60 : 20 : 20 (v/v/v) was used as mobile phase. The buffer was prepared by adding 2 mL of triethylamine in 1000 mL of water and the pH was adjusted to 6.2 ± 0.05 by orthophosphoric
acid. The flow rate was 1 mL/min, and the assay run time was 10 min. Absorbance was measured at 240 nm. The standard calibration curve was plotted as absor-bance vs concentration.
Commercially marketed tablets of RTV, Ritomune (100 mg, Cipla) and Viriton (100 mg, Ranbaxy) were purchased. The samples were prepared by extraction with methanol. Twenty tablets were weighed carefully, average weight was calculated and equivalent amount of solid content was weighed accurately. Weighed amount was dissolved in methanol to prepare the sample solution of concentration 1000 ^g/mL. Desired dilution of sample solution was prepared. The sample was filtered through Whatman filter paper no. 41, then through 0.45 ^m membrane filter before injection.
Spectrophotometry. Standard working solution of RTV was prepared by dissolving 2.5 mg of reference standard in 25 mL of methanol to prepare a solution of 100 ^g/mL. Aliquots of the working standard solution of RTV (10—60 ^g/mL) were transferred into 10 mL calibrated volumetric flask; 1.5 mL of 1% FeCl3 was added, shaken well, kept aside for 5 min. Then 2 mL of 0.5% MBTH solution was added and shaken well, which resulted in formation of a green coloured chro-mogen. The volume was made up by double distilled water. The absorbance of the chromogen was measured at 633 nm against the reagent blank, and standard calibration curve was plotted.
For analysis of tablet dosage form, twenty tablets were accurately weighed. An equivalent amount of 10 mg of the drug was taken and dissolved in methanol; volume was made up to 100 mL. Then the sample solution was filtered through Whatman filter paper no. 41. The sample solution of desired dilution was prepared from the stock solution and treated in the same manner with ferric chloride and MBTH as with the standard, and the green coloured chromogen was measured at 633 nm for quantification.
Nonaqueous titration. The reference standard of 0.1 g was weighed accurately and dissolved in 25 mL of glacial acetic acid. It was swirled well to dissolve the drug completely. Two drops of crystal violet indicator was added. The solution was titrated with standard 0.1 M perchloric acid until the colour changed from blue to emerald green. The titration was repeated until three concordant readings were found. The result was compared with a blank determination.
For analysis of tablets, twenty tablets were weighed accurately and triturated to a powder. An amount of tablet powder containing 0.1 g ofRTV was weighed accurately; 25 mL of glacial acetic acid was taken in a dry conical flask, the tablet powder added to it and kept aside for 2 h for complete extraction of RTV. The solution was sonicated for 10 min, filtered, and two drops of crystal violet were added. The solution was titrated with standard 0.1 M perchloric acid until the colour changed from blue to emerald green. The titration was repeated until three concordant readings were
AU 4400 4000 3600 3200 2800 2400 2000 1600 1200 800 400 0
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Fig. 1. Representative chromatogram of RTV in acetonitrile-methanol-buffer 20 : 20 : 20 (v/v/v) showing retention time at 5.55 ± 0.02 min.
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found. The result was compared with a blank determination.
Method validation. The optimized chromatographic and spectrophotometric methods were validated according to ICH Q2 (R1) guidelines. The validation parameters were linearity, accuracy, precision, limit of detection and limit of quantification, robustness and specificity. Linearity was checked by triplicate injection of HPLC dilutions and measuring absorbances of ion complexes of MBTH in triplicate at 633 nm. Calibration curve was plotted by six point least square method and applied for regression analysis. The accuracy of the methods was studied by
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