HPLC METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF RIFABUTIN IN BULK AND CAPSULE DOSAGE FORMHTML Full Text
HPLC METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF RIFABUTIN IN BULK AND CAPSULE DOSAGE FORM
R. Jasmin Sajini *, S. Prema, S. Niveditha, S. Nithya, G. M. Pavithra and V. Nivetha
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai - 600116, Tamil Nadu, India.
ABSTRACT: A selective, accurate, HPLC method was developed by this study for the determination of rifabutin in bulk and capsule dosage form. This method was developed by SHIMADZU LC-2010 HT using C18 column in solvents methanol: acetonitrile: ammonium acetate buffer (50: 45: 05) as mobile phase. At 1.0 ml/min flow rate the mobile phase was pumped, and the sample was detected at 278 nm. For standard rifabutin the retention time was 4.8 min. The method was validated for analytical standards such as linearity, accuracy, precision, and robustness. In a wide range of 5-25 (µg/ml) the linearity was observed. The method was validated, and a recovery study indicates accuracy of this method.
Rifabutin, HPLC, Capsule dosage form, Validation
INTRODUCTION: Rifabutin is a broad-spectrum semisynthetic antibiotic derived from Rifamycin S 1. It acts as anti-aid drug used for treating immunocompromised patients. Rifabutin is used in the first-line treatment for tuberculosis 2. IUPAC name of rifabutin is (9S, 12E, 14S, 15R, 16S, 17R, 18R, 19R, 20S, 21S, 22E, 24Z) 6, 16, 18, 20-tetra hydroxyl 1’isobutyl 14 methoxy 7, 9, 15, 17, 19, 21, 25 hepta methyl spiro[9, 4 (epoxypentadeca [1, 11, 13] trienimino) -2H-furo-[2’, 3’:7, 8] – naphtha [1, 2-d] imidazol-2,4’-piperidin]-5, 10, 26 – (3H, 9H)-trione -16- acetate3. Rifabutin is commonly used as an antitubercular drug. It has bactericidal activity particularly active against mycobacterium species 4. The structure of Rifabutin is depicted in Fig. 1.5
FIG. 1: STRUCTURE OF RIFABUTIN (C46H62N4O11)
MATERIALS AND METHOD: The instrument used for developing a method for HPLC was Shimadzu LC-2010HT equipped with a degassed unit, low-pressure gradient unit, pump unit, ultra-fast autosampler, UV-Vis detector 6, 7. By using a C-18 column Chromatographic separation was attained Isocratic elution was carried out with mobile phase of methanol, acetonitrile and ammonium acetate buffer used as mobile phase. Before injecting the drug, to acquire the saturation the column was equilibrated with mobile phase of stationary phase 8, 9.
Reagent and Materials: Standard rifabutin received from Vivan life sciences Pvt. Ltd., as a gift sample. Capsule dosage form was purchased from (Lupin Pharmaceuticals) containing 150 mg of Rifabutin from local pharmacy in Chennai. All solvents are in HPLC grade methanol and acetonitrile were procured from M/s Merck Ltd., Mumbai, India. Ammonium acetate was obtained from Sisco research laboratories Pvt. Ltd. Maharashtra, India.
Mobile Phase Preparation: Based on the solubility of the rifabutin, the mobile phase was selected. The mobile phase for chromatography is methanol + acetonitrile + ammonium acetate buffer (50:45:05)
Ammonium Acetate Buffer (pH 4.6) Preparation: 38.5 g of ammonium acetate and 35 ml of glacial acetic acid was taken in 500 ml standard flask, then made up the volume with 500 ml of distilled water and adjusted to pH 4.6.
Selection of Analytical UV Wavelength (λmax): To fix wavelength for analysis the prepared stock solution was scanned in ultraviolet spectroscopy over the range of 200-800 nm from resultant spectrum wavelength at 278 nm was chosen as in this range maximum absorption of drug occurs. So, this range is taken to analyze the sample 10.
FIG. 2: λmax OF RIFABUTIN AT 278 nm
Preparation of Standard Stock Solution of Rifabutin: About 10 mg of precisely weighed quantity of the standard drug was added to 100 ml volumetric flask and dissolved with little quantity of distilled water. The resultant solution made up to mark with diluents. Further Samples were prepared in various concentration of 5, 10, 15, 20, 25 µg/ml and used for method development and validation study.
Preparation of Sample: The contents of 20 capsules are removed weighed accurately powder equivalent to 10 mg of rifabutin was diluted with 100 ml mobile phase (methanol: acetonitrile: ammonium acetate buffer [50:45:05]. About 1.5 ml solution was taken in 10 ml volumetric flask, and it was made with 10 ml of mobile phase (1.5 µg/ml). Filtered through microsyringe and used for further studies.
RESULTS AND DISCUSSION:
Method Development: The method was developed by methanol: acetonitrile: ammonium acetate buffer: buffer as a high concentration in mobile phase yield tailing in the peak due to the presence of water in the buffer. During method development, a number of variations have been done with mobile phase in different concentration and 0.8 to 1.2 ml/ml flow rate to give asymmetric peak.
FIG. 3: CHROMATOGRAM OF STANDARD RIFABUTIN (15 µg/ml)
FIG. 4: CHROMATOGRAM OF CAPSULE FORMULATED RIFABUTIN (15 µg/ml)
Linearity: The different concentration varies from 5 to 25 µg/ml were prepared 8, 9. Chromatograms were recorded by injecting 20 µl from each concentration of the solution. All estimation were carried out at triplicate for each concentration 11.
A linear calibration graph (y = 38772x+1747; representing x as peak area and y as concentration respectively) was obtained. The correlation coefficient was found to be 0.983.
TABLE 1: LINEARITY STUDIES OF RIFABUTIN BY HPLC METHOD
|Concentration (µg/ml)||Peak area|
FIG. 5: CALIBRATION CURVE OF RIFABUTIN BY HPLC METHOD
Accuracy: Accuracy was estimated by using a known amount of standard rifabutin added to measured quantity of sample. By calculating peak area ratios the amount of rifabutin were estimated. By the use of three different concentrations equivalent to 80, 100 and 120% of the active ingredient accuracy was evaluated by calculating the recovery of rifabutin with % RSD 12. The percentage of drug present in formulation is reported in Table 2. The result showed that the amount present in capsule dosage form equivalent to label claimed of formulation.
Precision: Both Intraday and Inter day analysis was carried out in the triplicate analysis. Inter day precision did in consequential days by the use of freshly prepared sample. The lowest RSD values prove that ruggedness of the method 13.
Repeatability: The analysis of drug was carried out three times on the same day to find the repeatability of the sample by using 15 µg/ml and the % RSD was calculated for the resultant peak area 14.
TABLE 2: RECOVERY STUDIES OF RIFABUTIN BY HPLC METHOD
|Concentration of STD (ppm)||Concentration of Sample (ppm)||% recovery found||% RSD|
TABLE 3: INTERDAY AND INTRADAY PRECISION STUDIES OF RIFABUTIN BY HPLC
|Concentration||Mean peak area||±SD||% RSD|
|5 µg ml-1||153512.87||829.912||0.54|
|15 µg ml-1||565256.90||15383.58||2.72|
|25 µg ml-1||886511.11||10169.77||1.15|
|5 µg ml-1||153622.60||22373.88||1.45|
|15 µg ml-1||585915.33||15601.08||2.66|
|25 µg ml-1||877082.33||22072.52||2.52|
TABLE 4: REPEATABILITY STUDY OF RIFABUTIN BY HPLC
|Run||Area under the peak|
Robustness: The robustness of the method was evaluated by deliberately varying the chromatographic conditions such as the flow rate was changed to ± 2 ml min-1 and wavelength to about ± 2 nm15.
TABLE 5: ROBUSTNESS STUDY OF RIFABUTIN BY HPLC
|Mean retention time|
CONCLUSION: This present work is precise and validated for the estimation of rifabutin using HPLC with C18 column with UV detection at 278 nm. This method is accurate, can be employed in determination of rifabutin in various dosage forms. Hence, this study is an adaptable method of analysis due to its rapidity and repeatability.
ACKNOWLEDGEMENT: The authors thank Principal, Head of Department of Pharmaceutical Chemistry, Management of Sri Ramachandra Institute of Higher Education and Research, Chennai to provide opportunity to carry out and complete this project successfully.
CONFLICTS OF INTEREST: Nil
- Vyavahare RD: Stability indicating RP-HPLC method for rifampicin in bulk and pharmaceutical dosage form. IJPPR 2017; 10(4): 265-82.
- Wallis RS, Good CE, Riordan MAO, Blumer JL, Jacobs MR, Griffiss JM, Healan A and Salata RA: Mycobactericidal activity of bedaquiline plusrifabutin or rifampin in ex-vivo whole blood cultures of healthy volunteers a randomized controlled trial. WBA of bedaquiline plus rifamycins 2018; 2: 3-12.
- Singh G and Srivastava AK: High - performance liquid chromatography method validation and development strategy for rifabutin. IJPSR 2018; 9(9): 3903-10.
- Aziz DB, Low JL, Wu ML, Gengenbacher, Teo JWP, Dartois V and Dicka T: Rifabutin is active against mycobacterium abscessus complex. Antimicrobial Agents and Chemotherapy 2017; 61(6): 1-10.
- Jaiswal S, Sharma A, Shukla M and Lal J: Simultaneous LC–MS-MS determination of lopinavir and rifabutin in human plasma. Journal of Chromatographic Science 2017; 55(6): 617-24.
- Shah U and Jasani A: UV spectrophotometric and RP- HPLC methods for simultaneous estimation of isoniazid, rifampicin and piperine in pharmaceutical dosage form, Int Jou of Pharm and Pharmaceutical Sci 2014; 6(10): 274-80.
- Bartels H and Bartels R: Determination of Rifabutin by high-performance liquid chromatography using on-line concentration and column switching: J Chromatogr B Biomed Appl 1996; 686(2): 235-40.
- Lau YY, Hanson GD and Carel BJ: Determination of Rifabutin in human plasma by High- performance liquid chromatography with ultraviolet detection. J Chromatogr B Biomed ppl 1996; 676(1): 125-30.
- Gatti G, De Pascalis CR, Miletich F, Casazza R and Bassetti D: Specific high-performance liquid chromato-graphy assay for determination of Rifabutin plasma concentration following Extrelut column extraction, J Chromatogr B Biomed Sci Appl 1999; 728(2): 233-39.
- Ermolenko Y, Anshakova A, Osipova N, Kamentsev M, Maksimenko O, Balabanyan V and Gelperina: Simultaneous determination of Rifabutin and human serum albumin in pharmaceutical formulations by capillary electrophoresis. J Pharmacol Toxicol Methods 2017; 85: 55-60.
- Utkin I, Koudriakova T, Thompson T, Cottrell C, Iatsimirskaia E, Barry J, Vouros P and Gerber N: Isolation and identification of major urinary metabolites of rifabutin in rats and humans. Drug Metab Dispos 1997; 25(8): 963-67.
- Skinner MH, Hsieh M, Torseth J, Pauloin D, Bhatia G, Harkonen S, Merigan TC and Blaschke TF: Pharmacokinetics of rifabutin, Antimicrob Agents Chemother 1989; 33(8): 1237-41.
- Jaiswal S, Sharma A, Shukla M and Lal J: Simultaneous LC-MS-MS determination of lopinavir and rifabutin in human plasma, J Chromatogr Sci 2017; 55(6): 617-24.
- Horne DJ, Spitters C and Narita M: Experience with rifabutin replacing rifampin in the treatment of tuberculosis. Int J Tuberc Lung Dis 2011; 15(11): 1485-89.
- Gonzalez-Montaner LJ, Natal S, Yongchaiyud P and Olliaro P: Rifabutin for the treatment of newly-diagnosed pulmonary tuberculosis a multinational, randomized, comparative study versus rifampicin-rifabutin study group. Tuber Lung Dis 1994; 75(5): 341-47.
How to cite this article:
Sajini RJ, Prema S, Niveditha S, Nithya S, Pavithra GM and Nivetha V: HPLC method development and validation for estimation of rifabutin in bulk and capsule dosage form. Int J Pharm Sci & Res 2020; 11(1): 297-00. doi: 10.13040/IJPSR.0975-8232.11(1).297-00.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
R. J. Sajini *, S. Prema, S. Niveditha, S. Nithya, G. M. Pavithra and V. Nivetha
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai, Tamil Nadu, India.
11 April 2019
21 August 2019
02 September 2019
01 January 2020