Comparative in-vitro Quality Evaluation of Cephalexin

Comparative in-vitro Quality Evaluation of cephalexinA COMPARATIVE IN-VITRO QUALITY EVALUATION OF DIFFERENT BRANDS OF COMMERCIALLY functional CEPHALEXIN IN KARACHI (PAKISTAN)SHAHBAZ AHMED NOORAbstractApproximately 600 generic drug applications (ANDAs) approved each year, 65 % of prescriptions are for generic indication from oncology to insomnia without reiterateing clinical trials .Every generic molecule references a previously approved drug.cephalexin Monohydrates was first sold by Eli Lilly in 1972. The availability of many brands of cephalexin 500 mg abridgements in Pakistan market today place health supervise providers in a difficult situation of choice of a equal brand or alternate drop possibility .The objectives of this study was to carry out pharmaceutical equivalence studies on different brands of cephalexin 500mg capsules available in topical anesthetic market of Karachi. The invitro parameters include on the whole pharmacopeia and non-pharmacopeia tests that were dis resultant role rate( in 0.1 N Hydrochloric Acid at 37 C ) ,weight uniformity , disintegration time , moisture pith ( with the help of suitable Karl Fischer Titrator ) , appearance (observed physically before and after scattering of the shell) , credit ( compare manually retention time in HPLC chromato thou of sample with chromatogram of reference standard ) and HPLC tab of cephalexin (as describe below based on HPLC assay method AM1172) were as well analyzed. Along with the inappropriate intake of antimicrobials drugs, substandard antibiotic medicines may also contribute in resistance development against susceptible organism.The study will be helpful to evaluate the efficacy and justification of generic substitution of cephalexin 500 mg capsule brands in Karachi (Pakistan)INTRODUCTIONAntibiotic, being the wonder drugs, are widely prescribed in the developing countries. In reality, 25 to 40 % prescriptions contain one or more antibiotic (Public Sector studies, 1990-1993)Antibi otic resistance can be developed through its wide and inappropriate use without following any standard guidelines. This Alarming situation has been addressed in several scientific journals (Glass et al.,1980 Farrar , 1985 Saha et al., 2003Lina et al.,2007Hassan et al.,2009)Post trade surveillance include complete procedures undertaken to recover more data and information about a carrefour after it had been granted marketing authorization and made available for public use, this data use for product improvement ,development standard and regulations. Regulatory agencies rely on limited information get from clinical trials and from scientific literature as guide to granting to marketing authorization of medicine for public use. Post-marketing surveillance of approved dosage forms is to assess the quality, trenchantness and safe use of medicine to large population.To Assess the standard of a product, invitro dissolution test is widely used because , for any solid dosage forms, gastro intestinal absorption first requires dissolution of the tablet or capsules that librates the drug into solution (Goodman Gilmans The Pharmacology basis of Therapeutics,2006)The dissolution characteristics of a drug from the dosage form depend on many factors including its formulation and manufacturing process (Augsburger et al., 1983).CephalexinChemically, Cephalexin is (6R, 7R)-7-(R)-2-Amino-2-Phenylacetamido-3-Methyl-8-oxo-5-thia-1-azabicyclo 4.2.0 oct-2-ene-2-carboxylic superman monohydrate is a first generation cephalosporins for oral administration which is bactericidal, and mainly used in the treatment of various bacterial infections caused by gram +ve and gram -ve microorganisms. .( Ralph R et.al 1970).Excellent oral absorption and lack of serum binding of cephalexin compensates significantly for the lower in vitro activity.( Warren E. Wick et.al 1967).Antibacterial Spectrum of Cephalexin Cephalexin appears to be a highly effective antibiotic. It possesses the qualities of rapid oral absorption, production of high drug levels in the blood and urine, and near absence of side effects. It is effective against infections due to gram-positive cocci infections, with the exception of Enterococcus, and for most infections caused by E. coli and Klebsiella. It is useful in the treatment of a wide variety of infections in infants and children, and particularly worthy with susceptible infections in patients who do not require parenteral antimicrobial therapy. (R.C. Rudoy, M.D.1977)The first-generation cephalosporins, epitomized by cephalothin and cefazolin, have good activity against gram-positive bacteria and relatively retiring activity against gram-negative microorganisms. Most gram-positive cocci (with the exception of enterococci, methicillin-resistant S. aureus, and S. epidermidis) are susceptible. Most oral cavity anaerobes are sensitive, but the B. fragilis group is resistant. Activity against Moraxella catarrhalis, E. coli, K. pneumoniae, and P. genus Mirabilis is good (Goodman Gilmans The Pharmacology basis of Therapeutics,2006)Literature SurveyA ) Helio et.al in 2007 In this study, they observed the antibacterial spectrum of the orally prescribe cephalosporin (cefaclor, cefdinir, cefpodoxime, cefprozil, cefuroxime axetil, cephalexin) and amoxicillin/clavulanate, as these drugs were widely prescribed for community acquired respiratory tract infections. Narrowest zone was of Cefaclor and Cephalexin against evaluated pathogens. boilers suit, the 3rd Generation of cephalosporin is more useful in respiratory tracts and other infections when administered orally, as compared with older generation or amoxicillin alone.B ) Chalermporn et.al in 2005 ,they study for rapid and sensitive identification of cephalosporins drugs such as cefoxitin , cefazolin , cephalexin, cefadroxil , cefaclor and cefoperazone, a flow injection (FI) method was used. This method was applied successfully for the identification of cefoxitin, cefazolin, cephale xin, cefadroxil , cefaclor and cefoperazone. The method was successfully applicable for the identification of cefoxitin ,cefazolin , cephalexin and cefadroxil in the pharmaceutical formulation with a sample throughput of 90h1.C ) Rist et.al in 2002, they studied on Oral Cephalexin compared with mupirocin calcium thresh about with obeisance to safety and efficacy in secondarily septic eczema treatment. The adverse effect most common , they observed was diarrhea and nausea, effective clinical dose of mupirocin cream was apply three time daily as compared to cephalexin orally four times daily in secondarily infected eczma as topical regimen was preferred by the patient that enhance patient compliance.D ) Lennart Venemalm et.al in 2001, worked for the diagnosis of allergy against -lactams, prazinone-polylysine conjugate was able to bind with antibodies IgE in sera from patient with suspected allergy to cephalosporins. This information may be used for diagnosis of allergy against -la ctams.E ) Yagnesh P. Patel et.al in 1998 They studied, modern data on the isocratic assay of five antimicrobial drugs ,five different assay were performed to one single piece, antibiotic used in this study were cephalexin and cefaclor (cephalosporins), isoniazid and pyrazinamide (anti-tubercular drugs) and minocycline (tetracycline series) . Overall they concluded that any one of the drug could be used as internal standard for other four drugs.F ) Godfried Owusu-Ababio et.al in 1995 Sustained oust of cephalexin ciprofloxacin, was evaluated in open chemostat sytem. They concluded that chemostat system is convenient for sustained release drug testing as a function of formulation parameter.G ) Anne H. et.al in 1992 In this study, the Cefaclor uptake was observed concluded that it was sodium-independent, but proton-dependent and also the energy dependent , dipeptides inhibited uptake of -lactam antibiotic but amino acid had no effect on uptake , the overall data from this study conc luded that a single proton-dependent dipeptide transport carrier support the orally absorbed antibiotic uptake into Caco-2 cell shows correlation for the use of Caco-2 cell for cellular model of the intestinal proton-dependent dipeptides transporters for study purpose.H ) R. Wise et.al in 1991 Cefibuten, invitro activity was studied against 572 bacterial strain and was compared with orally administered -lactams. Cefibuten being more active against Enterobacteriaceae. There was less activity of cefibuten cefexime against Staphylococci and reduce activity against streptococcus pneumonia.I ) Luis J et.al in 1982 They studied, Acid degradation of cephalexin in presence of formaldehyde, produce 2-Hydroxy-3-phenyl-6-methylpyrazine.In 5M HCL this product gives well defined reduction expandlength with half wave potential of -0.45 vs. SCE. The wave was diffusion controlled irreversible, linear relation with the cephalexin conc. was shown with diffusion current and cephalexin in plasma c an be determined through it.J ) Ralph R et.al in 1970 They performed to detect various crystal forms of compounds, the employment of solubility versus solvent composition diagram. This appears to be a convenient sensitive method for detecting current crystalline phase.Aim of ProjectThe objective of this study is to carry out pharmaceutical equivalence studies on different brands of cephalexin 500mg capsules available in local market of Karachi.The invitro parameters include all pharmacopeia and non-pharmacopeia tests that are dissolution rate, weight uniformity, disintegration time, moisture content, appearance, Identification and HPLC assay of cephalexin will also be analyzed.The susceptibility test of drug will also be evaluate by ICLS ( Formerly NCCLS ) Reference Disk Diffusion (Kirby-Bauer) method against different clinical isolates of Staphylococcus aureus (03), E. coli (03), Klebsiella pneumonia (03), Pseudomonas aeruginosa (02),Importance of studyThis is a growing concern t hat if a health care provider, providing the substitute of the generic is also the Bioequivalent. For the health care providers to use these brands interchangeable, the bioequivalence of these brands have to be ascertained, this mean that there should be continued post marketing monitoring of the medicines.EXPERIMENTTESTING surgical processTest 1Cephalexin Monohydrate (As base) modus operandi Perform HPLC assay of Cephalexin as describe below based on HPLC assay method AM1172.Test 2IdentificationProcedure Compare manually retention time in HPLC chromatograms of sample with chromatograms of reference standards.Test 3Moisture ContentProcedure Three times separately the moisture content in the powder of the product with the help of suitable Karl Fischer Titrator will be determine and then calculate their mean.Test 4AppearanceProcedure From the 20 capsules take 10 capsules and observe physically before and after opening of the shells.Test 5Weight VariationFrom 20 capsules take 10 random capsules from composite sample and note individual weight of filled capsules. Calculate their average, minimum, maximum and COV%.Test 6DisintegrationProcedure Prepare a 0.6% v/v solution of HCl (Add carefully 16.2ml of 37% HCl into 500ml of distilled water and then make the solution up to 1000ml with distilled water) and fill the vessel of a suitable disintegration test apparatus with this solution up to recommended height. Maintain the temperature between 35 C and 39 C. Place 1 capsule in each of the 6 tubes of the basket and run the equipment. Start the stopwatch and note the time when all capsules are disintegrated. If 1 or 2 capsules fail to disintegrate, repeat the test on 12 additional capsules. The requirements of the test are met if not less than 16 of the 18 capsules tested have disintegrated.Test 7 decompositionProcedure According to USP 2007 page 1691.HPLC ASSAY OF CEPHALEXIN BASED ON HPLC ASSAY AM1172 (1.01) FORCEPHALEXIN CAPSULES.500 MG / CAPSULES.I.PRINCIPLET he sample is dissolved in water and the water-insoluble component removed by filtration. An aliquot of the filtrate is diluted with water and examined by reverse phase HPLC.II.APPARATUS1. A suitable high performance liquid chromatographic system equipped with a variableWavelength UV Detector.2. A suitable analytical balance with tolerance of +/- 0.1 mg.3. A suitable Ultrasonic bath.4. sufficient Syringes such as BD, 10ml.5. Analytical Column 150 4.6 mm. i.d stainless blade column packed with S5 ODS2 or equivalent column.6. Pre-Column 50 4.6 mm .i.d stainless steel column packed with 80mPorasil or equivalent is fitted between the pump and injector.7 Detector Condition 254nm range 0.5 aufs8. Pump Condition track down rate 1.0 ml / minute.9. Injection bulk 20l10. pH meter.11. Mobile phase See reagents.B) REAGENTSCephalexin analytical reference standard methanol HPLC gradeAcetonitrile HPLC grade1- Hydroxybenzotriazole, Reagent grade.Triethylamine ( T.E.A ) analytical gradeOrthop hosphoric acid, analytical grade1- Pentanesulfonic acid, sodium salt, Reagent grade(8) Mobile PhaseDissolve 1g Pentanesulfonic acid sodium salt (Monohydrate) and 15ml of Triethylamine in 850 ml of distilled water, adjust the pH to 3.0 with Orthophosphoric acid. Add 50 ml of Methanol and 100 ml of Acetonitrile, Mix well and degas before use.Note premeditation should be taken when degassing the mobile phase to avoid evaporating the mixture.Internal standard solutionDissolve 1 gm of 1- Hydroxybenzotriazole in 1000ml of mobile phase, mix well.C) PROCEDUREThis procedure contains the minimum recommended sample and standard Preparation for assays performed manually. Alternate weights and volumes may be substituted if necessary, as long as they are documented and provide nominally equivalent concentration of the analyte.PREPARATION OF REF. STANDARD SOLUTIONAccurately weigh approximately 32, 40 and 48 mg of Cephalexin reference Standard in to into three separate 200ml volumetric flasks resp ectively, pipette 30 ml of the internal standard solution in to each flask and swirl to dissolve, Dilute to volume with distilled water and mix well. SAMPLE PREPARATIONTake 20 random capsules from composite sample and note individual weight content of each capsule and determine average content weight of 20 capsules. Accurately weigh about 500 mg sample from composite sample of weight content of 20 capsules in 500ml volumetric flask and record the weight, add water to dissolve and make up the volume with water up to 500 ml mix well and diffuse. Take 20ml of above dilution in 100ml volumetric flask, add 30ml Internal Standard and then add water upto 100ml mix well and filter through 0.45m filter paper.d) CHROMATOGRAPHYMake 20 l injection of a standard to ensure the chromatography system complies with the system suitability criteria. The peak pursue should not be greater than 1.5, peak efficiency should be greater than 4000 plates. The resolution between the two peaks should be grea ter than 2.5. A typical chromatogram is attached. demarcation line OF CEPHALEXIN MONOHYDRATE CONTENT.CEPHALEXIN THEORY LABEL 500MG / CAPSULE 500MG / CAPSULE (As Cephalexin anhydrous) ( As Cephalexin anhydrous)Acceptance spring 475 525mg per capsule (95 105 %).Regulatory Limit 462.5 550 mg per capsule (92.5 110 %) (British Pharmacopeia)REFERENCES(Augsburger LL,Shangraw RF,Giannini RP, Shah VP, Prasad VK and Brown D (1983) Thiazides VIII Dissolution Survey of marketed Hydrochlorothiazides tablets. J.Pharma Sci.,72(8)876-881)Anne H. Dantzig, Linda B. Tabas, Linda Bergin , Cefaclor uptake by the proton-dependent dipeptide transport carrier of human intestinal Caco-2 cells and coincidence to cephalexin uptake Biochimica et Biophysica Acta (BBA) Biomembranes, tidy sum 1112, Issue 2, 9 December 1992, Pages 167-173.Chalermporn Thongpoon, Boonsom Liawruangrath, Saisunee Liawruangrath, R. Alan Wheatley, Alan Townshend , Flow injection chemiluminescence determination of cephalosporins in pharmaceutical preparations using tris (2,2-bipyridyl) ruthenium (II)-potassium permanganate system , Analytica Chimica Acta, Volume 553, Issues 1-2, 30 November 2005, Pages 123-133.(Farar WE (1985) Antibiotic resistance in developing countries. The Journal of Infectious sicknesss,152(6)1103-1106 )(Glass RI, Huq I, Alim ARM and yunus M (1980) Emergence of multiply antibiotic-resistant vibro cholerae in Bangladesh . 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Journal of Clinical Microbiology,41 (12)5582-5587)Warren E. Wick, Cephalexin, a New Orally Absorbed cephalosporin Antibiotic , Appl Environ Microbiol. 1967 July 15(4) 765-769).Yagnesh P. Patel, Nehal Shah, Indravadan C. Bhoir, M. Sundaresan, Simultaneous determination of five antibiotics by ion-pair high-performance liquid chromatography Journal of Chromatography A, Volume 828, Issues 1-2, 18 December 1998, Pages 287-290.1 Page