Available on line at Association of the Chemical Engineers AChE www.ache.org.rs/CICEQ Chemical Industry & Chemical Engineering Quarterly 16 (2) 139−148 (2010) CI&CEQ PAVAGADA TITRIMETRIC AND SPECTROPHOTOMETRIC JAGANNATHAMURTHY DETERMINATION OF DOXYCYCLINE HYC- RAMESH LATE USING BROMATE-BROMIDE, METHYL KANAKAPURA BASAVAIAH MYSORE RANGANATH ORANGE AND INDIGO CARMINE DIVYA One titrimetric and two indirect spectrophotometric methods are described for the NAGARAJU determination of doxycycline hyclate (DCH) in bulk drug and in its formulations. RAJENDRAPRASAD The methods use bromate-bromide, methyl orange and indigo carmine as rea- KANAKAPURA gents. In titrimetry (method A), DCH is treated with a known excess of bromate- BASAVAIAH VINAY -bromide mixture in acid medium and the residual bromine is back titrated iodo- metrically after the reaction between DCH and in situ bromine is ensured to be Department of Studies in Chemistry, complete. In spectrophotometric methods, the excess of bromine is estimated by Manasagangothri, University of treating with a fixed amount of either methyl orange (method B) or indigo car- Mysore, Mysore, India mine (method C) and measuring the change in absorbance either at 520 or 610 nm. Titrimetric method is applicable over 1-8 mg range and the calculations are SCIENTIFIC PAPER based on a 1:2 (DCH:bromate) stoichiometric ratio. In spectrophotometry, the calibration graphs were found to be linear over 0.25-1.25 and 1.0-5.0 µg mL-1 for UDC 543.422.3:615.33 method B and C, respectively, with corresponding molar absorptivity values of × 5 × 4 -1 -1 DOI 10.2298/CICEQ091208020R 2.62 10 and 6.97 10 L mol cm . The accuracy and precision of the assays were determined by computing the intra-day and inter-day variations at three dif- ferent levels of DCH. Key words: doxycycline; determination; dyes; spectrophotometry; phar- maceuticals. Doxycycline hyclate (DCH): (4S,4aR,5S,5aR,6R,12aS)-4-(dimethylamino)- -3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo- -1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carbo- xamide monohydrochloride, compound with ethyl alcohol (2:1), monohydrate, (Figure 1) is a broad spectrum antibiotic, with activity Figure 1. Molecular structure of DCH. against a wide range of gram-positive and gram-nega- tive bacteria. It has been used for the treatment of for the determination of DCH in pharmaceutical dosage infectious diseases caused by rickettsiae, mycoplas- forms including fluorimetry [5], phosphorimetry [6], li- mas and chlamydiae [1]. DCH is widely used in medi- quid chromatography [7-11], thin layer chromategraphy cine and veterinary practice. As a result, DCH residue [12], sequential injection chromatography [13], doxycy- can occur in food products of animal origin [2]. cline opto-sensors [14,15], ion selective electrodes- The drug is official in the British Pharmacopoeia based potentiometry [16] and capillary electrophoresis (BP) [3] and the United States Pharmacopoeia (USP) [17]. Few visible spectrophotometric methods based on [4], which describes HPLC methods for the determi- the different reaction mechanisms are found in the li- nation of DCH either in raw material or in pharmaceu- terature for the assay of DCH. These include FIA-spec- tical formulations. Several methods have been reported trophotometry with copper carbonate [18], chloramine-T [19] and 4-aminophenazone/potassium hexacyanofer- rate(III) [20] and also based on colour reactions with Correspondening author: K. Basavaiah, Department of Chemis- thorium (IV) [21], sodium cobaltnitrite [22] and uranyl try, University of Mysore, Manasagangothri, Mysore-570 006, India. acetate [23]. Besides, kinetic spectrophotometry using E-mail: [email protected] Cu(II)/H2O2 [24] and multivariate calibration method Paper received: 8 December, 2009 [25] have also been reported by different workers. Paper revised: 5 March, 2010 Paper accepted: 18 March, 2010 139 P.J. RAMESH et al.: TITRIMETRIC AND SPECTROPHOTOMETRIC DETERMINATION OF… CI&CEQ 16 (2) 139−148 (2010) The chromatographic techniques are most widely titrimetric and spectrophotometric methods that would used. Although the procedures are specific, most of the overcome many of the problems encountered in the described methods are time consuming and require reported methods. This work describes one titrimetric multistage extraction procedures. On the other hand, and two spectrophotometric methods for the determi- the reported spectrophotometric methods suffer from nation of DCH in pharmaceuticals based on the bromi- one or the other disadvantage such as poor sensitivity, nation reaction using bromate-bromide mixture and by use of organic solvent, scrupulous control of experi- employing two dyes, methyl orange and indigo carmi- mental variables and special equipment (Table 1). Ti- ne. The methods were successfully applied to the de- trimetry and spectrophotometry are well established termination of DCH in two different brands of tablets techniques, and owing to their speed, fair selectivity, with good accuracy and precision and without detec- reduced costs and versatility of application, they can be table interference by excipients. The accuracy was fur- considered to be advantageous alternatives to sophisti- ther ascertained by placebo blank and synthetic mix- cated and expensive techniques normally used in phar- ture analyses and also by recovery experiments via the maceutical analysis. A complexometric titration method standard-addition procedure and the methods were to has been reported for the determination of DCH [26]. be simple, accurate and easy to apply to routine ana- The method employs spectrophotometric titration of lysis. DCH with Mg2+ and Ca2+ in aqueous tris-buffer. The tit- rations were performed at two fixed pH values (pH 7.0 EXPERIMENTAL and 8.5) and the consistent sets of UV-Visible ab- sorption and fluorescence spectra were recorded at Apparatus various times. Since the method utilized buffers, pH is A Systronics model 106 digital spectrophotome- very crucial and a sophisticated instrument to measure ter with 1-cm matched quartz cells was used for all fluorescence response is required. By considering these absorbance measurements. drawbacks, the present work is aimed at developing Table 1. Comparison of the performance characteristics of the present spectrophotometric methods with the published methods λ LOQ No. Reagent/s used Methodology max Linear range Remarks Ref. nm µg mL-1 1 Copper carbonate Complex colour measured 395 10.0-80.0 - FIA assembly required and least 18 mg mL-1 sensitive Chloramine-T Oxidation of drug in alkaline 525 From 5.37×10-5 - 19 medium and red coloured to 7.16×10-4 product measured mol L-1 4-Aminophenazone and Colour of the dye measured 520 - - FIA assembly required and the pH 20 potassium hexacya- dependent noferrate(III) 2 Thorium(IV) Yellow complex measured 398 0.4-3.2 µg mL-1 - pH dependent and narrow linear 21 range 3 Sodium cobaltnitrite Colour forming reaction 243 0.01-0.03 - Heating required. Less sensitive 22 and acetic acid mg mL-1 4 Uranyl acetate-DMF 1:1 Complex formation 405 0-135 - Requires organic solvent, 23 -1 medium reaction µg mL less sensitive 5 Cu(II)/H2O2-alkaline Degradation study 510 2.97-17.78 1.89 Use of buffers, scrupulous control of 24 medium µg mL-1 experimental variables and special equipment for kinetic measurement required 6 DMF/NaOAc-AcOH Partial least squares 277- 1.7-42 - Require special equipment, Use of 25 buffer (pH 4.5) multivariate calibration 349 µg mL-1 organic solvent, pH dependant method 7 KBrO3-KBr /HCl and Bromination of drug and 520 0.25-1.25 0.07 Highly sensitive, non-stringent Present -1 methyl orange determination of unreacted µg mL optimum conditions used, simple work Br2 with methyl orange instrument employed. KBrO3-KBr /HCl and Bromination of drug and 610 0.5-5.0 0.27 -1 indigo carmine determination of unreacted µg mL Br2 with indigo carmine 140 P.J. RAMESH et al.: TITRIMETRIC AND SPECTROPHOTOMETRIC DETERMINATION OF… CI&CEQ 16 (2) 139−148 (2010) Reagents and materials the flask. The liberated iodine was titrated with 0.03 M All the reagents used were of analytical-reagent sodium thiosulphate to a starch end point. A blank tit- grade and distilled water was used throughout the in- ration was run under identical conditions. The stoi- vestigation. chiometry was calculated using the formulae: A bromate-bromide solution equivalent to 5 mM ml of bromate×molarity of bromate n = KBrO3-50 mM KBr was prepared by dissolving accu- ml of DCH titrated×molarity of DCH rately weighed 418 mg of KBrO3 (S.d. Fine Chem Ltd, Mumbai, India) and 3 g of KBr (Merck, Mumbai, India) and the amount of the drug in the measured aliquot in water and diluting to the mark in a 500 mL cali- was calculated from: brated flask and this solution was used in titrimetric VMwR work. For use in spectrophotometric study, a 1000 μg mg= -1 n mL KBrO3 solution containing a large excess of KBr was prepared by dissolving 100 mg of KBrO3 and 1 g where V = volume of bromate reacted; Mw = relative of KBr in water and diluting to the mark in a 100 mL molecular mass of the drug; R = molar concentration calibrated flask. This was diluted stepwise to get 10 of bromate; n = number of moles of bromate reacting μg mL-1 and 30 μg mL-1 bromate solutions for use in with each mole of the drug. method B and C, respectively. Spectrophotometry (method B). Different aliquots -1 A 0.03 M sodium thiosulphate (Na2S2O3) (S.d. (0.0-2.5 mL) of 5 μg mL DCH solution were accura- Fine Chem Ltd, Mumbai, India) solution was prepared tely measured into a series of 10 mL calibrated flasks in water and standardized [27]. Hydrochloric acid and the total volume was adjusted to 2.5 ml with wa- (S.d. Fine Chem, Mumbai, India, Sp. gr. 1.18) (2 and ter.