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Qualitative and Quantitative Analysis of Various Pharmacophore 2011, Vol. 2 (3), 149-157 ISSN 2229 – 5402 Pharmacophore (An International Research Journal) Available online at http://www.pharmacophorejournal.com/ Original Research Paper QUALITATIVE AND QUANTITATIVE ANALYSIS OF VARIOUS CONSTITUENTS OF VACCINES BY USING ANALYTICAL TECHNIQUES Naresh Singh Gill*, Poonam Kumari Rayat College of Pharmacy, Railmajra, District Nawanshahar, Near Ropar, Punjab-144533, India ABSTRACT The present study deals with the various analytical techniques like ultra-violet spectrophotometer, high performance liquid chromatography and AxSYM system for the quality control of various vaccines. Ultra-violet spectrophotometer was used to analysis the thiomersal content in Diphtheria tetanus pertussis (DTP) vaccine 0.0107%, protein content in Rabies vaccine 0.657%, and phenol content in Diphtheria tetanus pertussis vaccine 0.230% respectively. High performance liquid chromatography was used to determine the molecular size distribution of the meningococcal vaccine 97.50% and the AxSYM system was used to estimate the in-vitro potency assay of hepatitis B 0.9769%. Formalin content in Diphtheria tetanus pertussis (DTP) vaccine 2.0% was estimated by qualitative analysis. It was confirmed that results were found after quantitative and qualitative analyzing the constituents of vaccine samples with the analytical techniques employed in the present study accurate, efficacious and rapid rather than the other previous method. Keywords: Analytical techniques, AxSYM system, HPLC, UV-spectrophotometer, Vaccine. INTRODUCTION colorimetric titration and simpler spectrophotometer. But UV-spectrophotometer Analytical chemistry is the science of making method of analysis is more economic and quantitative measurements and it is the simpler (IP, 2007). Under computer controlled chemistry discipline concerned with the instrumentation, UV-spectrophotometer playing chemical composition of materials. Analytical a very important role in the analysis of vaccine chemistry mainly divided into two group‟s samples (Greenlief, P, 2001). qualitative analysis and quantitative analysis (Helmenstin, AM, et.al, 2009). Spectroscopy High-performance liquid chromatography is a (quantitative analysis) was a new field of study chromatographic technique that can separate a for the thiomersal (preservative), protein mixture of compounds and is used in (binding agent), phenol (preservative) content in biochemistry and analytical chemistry to the vaccine (Plotkin, SA, 2006). Indian identify, quantify and purify the individual pharmacopoeia described assay method using components of the mixture. It is basically a http://www.pharmacophorejournal.com/ 149 Naresh Singh Gill et al. / Pharmacophore 2011, Vol. 2 (3), 186-194 highly improved form of column The main focus of the present study, an attempt chromatography. Instead of a solvent being was made to perform the qualitative and allowed to drip through a column under gravity, quantitative analysis of various constituents of it is forced under high pressures up to 400 vaccines using analytical techniques. atmospheres to makes it much faster (Snape, MD MATERIALS AND METHODS and AJ Pollard, 2005). The molecular size of polysaccharide vaccines is an important pysico- Chemicals chemical parameter which correlates with Formaldehyde, phenyhydrazine hydrochloride, immunogenicity (Lloyd, RS and JW Dolan, potassiumferrocyanide, formalin standard, 2007). Many other method used for the hydrochloride, nitric acid, aqueous solution of determination of size distribution to the thiomersal , solution of ammonium acetate, polysaccharides vaccine i.e gel filtration and ion dithizone, chloroform, stock standard: bovine exchange chromatography but the latest model- serum albumin of concentration 5 mg/ml, 560 HPLC is the most important than the other working standard, biuret reagent, 4- perivious method because it shows the sharp aminoantipyrine, borate buffer, standard phenol peak , have high resolution power, superior solution concentration 0.25%, sodium refractive index detector and rapid monitoring dihydrogen phosphate, sodium chloride, sodium techniques (Richmond P et.al, 1999). azide, dextran, acetone, MQW ( Milli Q Water), The AxSYM is an immunochemical automated Anti-HBs (Mouse, Monoclonal, IgM) coated analyzer made by Abbott Laboratories. It is used microparticles in phosphate buffer with protein for serology tests and therapeutic drug stabilizers, minimum concentration 0.15% monitoring, and uses antibodies to alter the preservative: sodium azide, anti-biotin (Rabbit), deflection of polarized light. It can also be used alkaline phosphatase conjugates in tris buffer to monitor hormone level and some cardiac with (Rabbit) IgG, minimum concentration markers such as troponin (Jilg, W et.al, 1984). 0.03µg/ml preservative: sodium azide, The main purpose of this system to estimate the biotinylated anti-HBs (Goat, IgG) in tris buffer in-vitro potency assay of hepatitis B vaccine. containing animal sera (Goat, Calf, Rabbit, Appearance and use blood samples and reagents Mouse), preservative: sodium azide, Quinolone, are placed in separate carousels on the right of PBS + 2% BSA. The entire chemicals are the machine. This instrument is used in medical obtained from the Central Research Institute, laboratories by trained medical personnel. It can Kasauli, distt. Solan, Himachal Pradesh. process about 100 samples an hour Instrument (Shipchandler, MT and Moore, EG, 1995). Potency of the vaccine determined by two UV-spectrophotometer manufacturer by Jainco method in-vivo and other by in-vitro or analytical Inks & Chemicals Karnataka, HPLC model-560 technique i.e. AxSYM system. The in-vivo with PDA and RI detector manufacturer by method is a time-consuming and labor intensive central drug house Pvt. Ltd. New Delhi, , process. The animal ethics committee makes the AxSYM system manufacturer by Abbott rule to avoiding the animal death. In-vivo Laboratories , solvent filtration unit with studies are very pain full, So that the new receiving flask, Laboratory Vortex Mixer, Bio method established for the estimation of hepatitis Safety Cabinet, pipettes and tips obtained from B potency. This analytical technique is most E- Merck Pvt. Ltd. Mumbai. important, accurate, precious, efficient and Vaccine specific than the in-vivo and previous potency assay study (Karimzadeh, H et.al, 2010). http://www.pharmacophorejournal.com/ 150 Naresh Singh Gill et al. / Pharmacophore 2011, Vol. 2 (3), 186-194 DTP, Rabies, Typhoid, Meningococcal, used as a vaccine preservative (COX, NH and A Hepatitis B. The entire vaccines are Forsyth, 1988). Estimation of thiomersal content authenticated from The Central Research test performed on the DTP vaccine by using UV Institute Kasauli, distt. Solan, Himachal Pradesh. Spectrophotometer. In this method Separating All the vaccines stored at the temperature 2-8°C. funnels were washed with concentrated nitric acid and rinsed with tap and distilled water. Two Estimation of Formalin Content by Qualitative Method aliquots of a standard 0.01% aqueous solution of thiomersal (0.5 and 1.0 ml) and two samples of Formalin or formaldehyde is used as a the test solution (1ml) were added to individual detoxification agent who converts the toxin of separatory funnels. The volume was adjusted to the antigen into toxoid (Plotkin, SA, 2006). 10 ml with 1% solution of ammonium acetate at Estimation of formalin content test performed on pH 6.0. Ten milliliters of a 1 in 10 dilution of a the DTP, vaccine by using qualitative analysis. fresh solution of 0.01% dithizone in chloroform In this method formalin is added to the sample as was added to each funnel. The contents were a detoxification agent who converts the toxin of shaken vigorously for 45 seconds. The the antigen into toxoid and the left free formalin chloroform layer was separated carefully and is to be tested. Phenyl hydrazine hydrochloride spectrophotometer was set at 490 nm using the from a complex with the free formalin and the diluted dithizone solution and a scan were taken potassium ferrocyanide acts as an indicator of the test solution from 470- 520 nm.The which gives color different shades on the transmission at 520 nm was plotted against the addition of conc. hydrochloride. thiomersal concentration of the standards on Estimation of Thiomersal Content by Chemical semi- logarithmic paper and the concentration of Method Using UV-Spectrophotometer thiomersal in the sample was determined. The Thiomersal is an organomercurial salt complex thiomersal content can be calculated by of sodium ethyl mercuriothiosalicylate following calculation. containing ethylmercury that has been widely O.D. of Test Thiomersal content = × Conc. of Standard Thiomersal O.D. of Standard Protein Estimation by Biuret Method Using test 1ml of distill water were added in a test- UV-Spectrophotometer tube marked as „Blank‟. Various concentration Estimation of the protein content test performed (4mg/ml, 3mg/ml, 2mg/ml and 1mg/ml) of the on the Rabies vaccine by biuret method using standard were prepared from the stock standard UV spectrophotometer (Stoscheck, CM, 1990). solution (5mg/ml) and take 1ml each of various Under alkaline conditions, substances containing standard concentration in a test-tube marked as two or more peptide bonds from a purple „Standard‟. Prepare 1:10 dilution of the sample complex with copper salts in a reagent. The (0.9 ml of distilled water and 0.1 ml of vaccine concentration of more protein in the sample is sample) were prepared and taken the 1ml each of determined
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