DETERMINATION OF FOLIC ACID IN COMBINATION WITH IRON IN PHARMACEUTICAL DOSAGE FORMS BY CHEMICAL AND MICROBIOLOGICAL METHODSDissertation Submitted in partially fulfillment of the requirement for the Degree of Master of Science in

Applied MicrobiologySubmitted to the Periyar University, Salem- 636 011 By NILKANTHA BANERJEE Reg. No: 07BBC1018

PG AND RESEARCH DEPARTMENT OF MICROBIOLOGY KSR COLLEGE OF ARTS AND SCIENCE TIRUCHENGODE 637215, TAMILNADU.

APRIL - 2009

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CERTIFICATEThis is to certify that to dissertation entitled DETERMINATION OF FOLIC ACID IN COMBINATION WITH IRON IN PHARMACEUTICAL DOSAGE FORMS BY CHEMICAL AND MICROBIOLOGICAL METHODS submitted in part fulfillment of the requirement of the degree of Master of Science in Applied Microbiology to the Periyar University, Salem is a record of bonafide research work carried out by Mr.NILKANTHA BANERJEE under my supervision and guidance and that no part of the dissertation has been submitted for the award of any degree , diploma, fellowship or similar titles of prizes and that the work has not been published in part of full in any scientific or popular journals or magazines .

Signature of the HeadDr. G. VIVEKANANDHAN

Signature of the GuideMr. K. PONMURUGAN

EXAMINERS

1. 2.

DECLARATION2

I hereby declare that the dissertation entitled DETERMINATION OF FOLIC ACID IN COMBINATION WITH IRON IN PHARMACEUTICAL DOSAGE FORMS BY CHEMICAL AND MICROBIOLOGICAL METHODS submitted in part fulfillment of the requirement of the degree of Master of Science in Applied Microbiology to the Periyar University ,Salem is a record of bonafide research work carried out by me under the guidance of Mr.K.PONMURUGAN and has not previously formed the basis for the award of any Degree , Diploma, Fellowship or Similar titles of prizes and that the work has not been Published in part of full in any Scientific or Population Journals or Magazines .

Signature of the Candidate

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Affectionately Dedicated To My Beloved Family, Friends& All Teaching Community

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ACKNOWLEDGEMENTThere are times in life when one feels a sense of accomplishment combined with a sense of gratitude. Writing the acknowledgement page in this project is one among them. This project would have been a distant dream without the grace of almighty. So, first and foremost, I profusely thank God for his blessing and grace, without which my project would not have seen the light of the day. I am pleased to place my profound etiquette to our beloved chairman Lion Dr. K.S. Rangasamy MJF, and my Executive Director Mrs. Kavitha Srinivasan, for providing the infrastructure facilities in the college. I consider it as great privilege to place on record my heartful indebtness to out most reputed principal Dr. N. Kannan, Ph.D., for his valuable advice and concern to students. I also wish to take this Opportunity to express our deep sence of gratitude and heartfelt regards to our Head of the Department Dr. G. Vivekanandan, Ph.D., of Microbiology. I extend my sincere thanks to Dr. LAXMIKANT HARISHCHANDRA BHONSLE, M. Pharm, P.hD., DSM, (DGM-Q.A) & Mr.K.Ponmurugan M.Sc., K.S.R.CAS for his guidance and constant encouragement. I take this opportunity to express my profound gratitude to Mr. Dattatiay Kulkalini, Mr. Pravin Morajkar, Trupti Walke, and Geeta Halmarkar for encouraging the collaboration and for many valuable suggestions. It gives an immense pleasure to thank gratefully and express my profound gratitude to Mr. Pandharinath Talaulikar (DGM-Production) for his able and erudite guidance at every stage of my work and constant monitoring during the course of my project. I sincerely thank him for his sustained and keen interest in my work and extending his whole hearted support in my efforts to complete the project. I own my hearty thanks for his constructive suggestions, untiring patience and strenuous effort in bringing out and finalizing the project in a beautiful and systematic way. I reverentially express my gratitude towards my parents and all other family member for their unwavering support and understanding.

NILKANTHA BANERJEE

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INTRODUCTIONFolic acid, also known generically as folate or folacin, is a member of the B-complex family of vitamins, and works in concert with vitamin B12. Folic acid functions primarily as a methyl-group donor involved in many important body processes, including DNA synthesis. Therapeutically, folic acid is instrumental in reducing homocysteine levels and the occurrence of neural tube defects. It may play a key role in preventing cervical dysplasia and protecting against neoplasia in ulcerative colitis. Folic acid also shows promise as part of a nutritional protocol to treat vitiligo, and may reduce inflammation of the gingiva. Furthermore, certain neurological, cognitive, and psychiatric presentations may be secondary to folate deficiency. Such presentations include peripheral neuropathy, myelopathy, restless legs syndrome, insomnia, dementia, forgetfulness, irritability, endogenous depression, organic psychosis, and schizophrenia-like syndromes. Folic acid is a water-soluble member of the B-complex family of vitamins. Folic acid is composed of three primary structures, a hetero-bicyclic pteridine ring, para-aminobenzoic acid (PABA), and glutamic acid. Because humans cannot synthesize this compound, it is a dietary requirement. Although folic acid is the primary form of folate used in dietary supplements or fortified foods, it comprises only 10 percent or less of folates in the diet. Dietary folic acid, is naturally found in foods, is actually a complex and variable mixture of folate compounds, such as polyglutamate (multiple glutamate molecules attached) conjugate compounds, reduced folates, and tetrahydrofolates. Although folates are abundant in the diet, cooking or processing destroys these compounds. The best folate sources in foods are green, leafy vegetables; sprouts, fruits, brewers yeast, liver, and kidney also contain high amounts of folates.

C19H19N7O6

Figure 1: Biochemical structure of Folic Acid

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(2S)-2-[ [4-[ [ (2 amino 4 oxo - 1,4 dihydropteridin 6 yl ) methyl ] amino ] benzoyl ] amino ] pentanedioic acid. CHARACTERSTICS OF FOLIC ACID Appearance Yellowish or orange, crystalline powder. Solubility Practically insoluble in water and in most organic solvents. It dissolves in dilute acids and in alkaline solutions. Pharmacokinetics Human pharmacokinetic studies indicate folic acid has very high bioavailability, with large oral doses of folic acid substantially raising plasma levels in healthy subjects in a timeand dose-dependent manner. Subsequent to high-dose oral administration of folic acid (ranging from 25-1,000 mg/day), red blood cell (RBC) folate levels remain elevated for periods in excess of 40 days following discontinuation of the supplement. Folic acid is poorly transported to the brain and rapidly cleared from the central nervous system. The primary methods of elimination of absorbed folic acid are fecal (through bile) and urinary, (Schuster et al., 1993).After ingestion, the process of conversion of folic acid to the metabolically active coenzyme forms is relatively complex. Synthesis of the active forms of folic acid requires several enzymes, adequate liver and intestinal function, and adequate supplies of riboflavin (B2), niacin (B3), pyridoxine (B6), zinc, vitamin C, and serine. After the formation of the coenzyme forms of the vitamin in the liver, these metabolically active compounds are secreted into the small intestine with bile (the folate enterohepatic cycle), where they are reabsorbed and distributed to tissues throughout the body. Despite the biochemical complexity of this process, evidence suggests oral supplementation with folic acid is able to increase the bodys pool of the active reduced folate metabolites (such as methyltetrahydrofolate) in healthy individuals (Priest et al., 1999). Enzyme defects, malabsorption or digestive system pathology, and liver disease can result in impaired ability to activate folic acid to the required coenzyme forms in the body. Evidence indicates some individuals have a severe congenital deficiency of the enzyme methyltetrahydrofolate reductase, which is needed to convert folic acid to the 5methyltetrahydrofolate coenzyme form of the vitamin. The existence of milder forms of this enzyme defect is strongly suspected and likely interacts with dietary folate status to determine

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risk for some disease conditions (Ulvik et al., 2001).In individuals with a genetic defect of this enzyme (whether mild or severe), greater dietary exposure to foods rich in folates and supplemental folates in the form of folinic acid or 5-methyltetrahydrofolate might be preferable to folic acid supplementation. Mechanisms of Action Folic acids primary mechanisms of action are through its role as a methyl donor in a range of metabolic and nervous system biochemical processes, as well as being necessary for DNA synthesis. Serine reacts with tetrahydrofolate, forming 5, 10- methylenetetrahydrofolate, the folate derivative involved in DNA synthesis. A methyl group is donated to cobalamin (B12) by 5-methyltetrahydrofolate, forming methylcobalamin. With the help of the enzyme methionine synthase, methylcobalamin donates a methyl group to the amino acid metabolite homocysteine, converting it to the amino acid methionine. Methionine subsequently is converted to S-adenosylmethionine (SAMe), a methyl donor involved in numerous biochemical processes. Deficiency States and Symptoms Folic acid deficiency is considered to be one of the most common nutritional deficiencies. The following may contribute to a deficiency of folic acid: deficient food supply; defects in utilization, as in alcoholics or individuals with liver disease; malabsorption; increased needs in pregnant women, nursing mothers, and cancer patients; metabolic interference by drugs; folate losses in hemodialysis; and deficiencies in enzymes or cofactors needed for the generation of active folic acid. (Halsted, 1989).Absorption of folic acid appears to be significantly impaired in HIV disease, irrespective of the stage of the disease (Revell et al., 1991). Signs and symptoms of folate deficiency include macrocytic anemia, fatigue, irritability, peripheral neuropathy, tendon hyper-reflexivity, restless legs syndrome, diarrhea, weight loss, insomnia, depression, dementia, cognitive disturbances, and psychiatric disorders (Metz et al., 1996).Elevated plasma homocysteine can also indicate a dietary or functional deficiency of folic acid. Clinical Indications Anemia Folic acid has a long history of use in conjunction with vitamin B12 for the treatment of macrocytic anemia. Depending on the clinical status of the patient, the dose of folic acid required to reverse macrocytic anemia varies, but the therapeutic dose is usually 1 mg daily.

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Duration of therapy to reverse macrocytic anemia can be as short as 15 days after initiation of supplementation, or it may require prolonged supplementation. Cervical Dysplasia Research points to an association between folate status in adults and cervical dysplasia (Kwasniewska et al., 1997).however, its role as an efficacious therapeutic intervention is unclear. One report suggests folic acid supplementation (10 mg folic acid for three months) reverses cervical dysplasia in women taking oral contraceptives (Butterworth et al., 1982). In another study, 154 individuals with grade 1 or 2 cervical intraepithelial neoplasia were randomly assigned either 10 mg folic acid or placebo daily for six months. No significant differences were observed between supplemented and unsupplemented subjects regarding dysplasia status, biopsy results, or prevalence of human papilloma virus type-16 infection (Zarcone et al., 1996). It is possible certain subsets of women (perhaps those with an oral contraceptive-induced deficiency) might be more amenable to treatment; however, additional research is required to clarify the therapeutic role of folic acid in cervical dysplasia. Gout There is no evidence demonstrating efficacy of folic acid supplementation in gout. Although some in vitro evidence suggests folate compounds are potent inhibitors of xanthine oxidase activity (Lewis et al., 1984). it appears pterin aldehyde, a photolytic breakdown product of folic acid, and not folic acid itself, is responsible for the observed inactivation of xanthine oxidase (Spector and Ferone, 1984).Available evidence has shown no ability of supplemental folic acid in oral daily doses up to 1,000 mg to significantly lower serum urate concentration, or to decrease urinary urate or total oxypurine excretion in hyperuricemic subjects (Boss and Ragsdale, 1980). Homocysteinemia An abnormally high plasma level of homocysteine, the de-methylated derivative of the amino acid methionine, is an independent risk factor for cardiovascular disease. Elevated plasma homocysteine has been connected to increased risk of neural tube defects and other birth defects, as well as to schizophrenia, Alzheimers disease, cognitive decline, osteoporosis, rheumatoid arthritis, kidney failure, and cancer (Fava et al., 1997).The activated coenzyme form of folic acid (5-methyltetrahydrofolate) is needed for optimal homocysteine metabolism, since it acts as a methyl donor, providing a methyl group to vitamin B12. The methylated form of vitamin B12 (methylcobalamin) subsequently transfers this methyl group to homocysteine.

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The result is a recycling of homocysteine to methionine, resulting in reduction in elevated plasma homocysteine. In healthy subjects even low doses of folic acid can lower homocysteine levels. A dose of 250 mcg daily for four weeks reduced homocysteine an average of 11.4 percent in healthy 18- to 40-year-old women. A dose of 500 mcg daily for the same duration reduced levels an average of 22 percent.32 in a separate study, 650 mcg daily for six weeks resulted in an average plasma homocysteine reduction of 41.7 percent (Fava et al., 1997). In subjects with cardiovascular disease, 800 mcg folic acid daily resulted in an average decrease in homocysteine levels of 23 percent (Landgren et al., 1995). while 2.5 mg daily resulted in an average decrease of 27 percent (Wald et al., 2001).In subjects receiving the higher dose, 94 percent experienced some degree of reduction in homocysteine (Wilcken et al., 1985).Evidence suggests individuals with higher initial homocysteine levels are likely to experience a greater reduction following folic acid supplementation (Wald et al., 2001). In addition to helping reduce blood levels of homocysteine, folic acid may also aid peripheral blood flow by increasing nitric oxide (NO) in vascular endothelial cells. Impaired endothelial NO activity is an early marker for cardiovascular disease, particularly atherosclerosis. In fact, most of the risk factors for atherosclerosis are associated with poor vasodilation due to insufficient NO production. Chronic, unopposed exposure of the vascular endothelium to homocysteine compromises the production of adequate amounts of NO, which leads to injury of the endothelial lining and the initiation/exacerbation of atherosclerosis and/or thrombus formation. Folic acid appears to improve NO synthesis by reducing plasma homocysteine levels, enhancing the availability of key endothelial NO cofactors, and reducing the production of superoxide anions, the net effect of which is improvement of peripheral blood flow (Das, 2003).In a recent doubled-blind, placebo-controlled, crossover study of individuals with coronary heart disease, researchers found supplementation with high-dose folic acid (30 mg per day) improved blood flow to the heart muscle via the coronary arteries. Using positron emission tomography (PET scanning), researchers at Massachusetts General Hospital noted significant improvement in coronary blood flow with folic acid supplementation compared to placebo. The improvement was especially enhanced in areas of the heart that had shown reduced blood flow prior to supplementation. Folic acid supplementation also significantly lowered the study participants blood pressure. The findings from this high-dose folate study demonstrate another significant way this nutrient benefits the cardiovascular system (Tawakol et al., 2005).Although excellent results have been achieved with folic acid monotherapy,

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available evidence suggests an additive effect exists between folic acid and vitamins B6, B12, and betaine with respect to lowering homocysteine levels. Combinations of these nutrients typically produce greater reductions in homocysteine than does folic acid alone (Wilcken et al., 1983).Furthermore, the addition of vitamin C, L-arginine, tetrahydrobiopterin (BH4), and polyunsaturated fatty acids (PUFAs) has been suggested as a means of enhancing the effect of folic acid on endothelial NO production (Das, 2003). Inflammatory Bowel Disease Patients with inflammatory bowel disease (IBD) often have folate deficiencies, caused in part by the drug sulfasalazine, prescribed for IBD but also known to inhibit folate absorption (Lashner et al., 1989). Evidence suggests folic acid supplementation might lower the risk, in a dose-dependent fashion, of colonic neoplasia in patients with ulcerative colitis. A review of 99 ulcerative colitis (UC) patient records found folic acid supplementation was associated with a 62-percent decreased risk of neoplasia compared to patients not taking folate supplements (Lashner. et al., 1989).In another similar study, the files of 98 UC patients disclosed dosedependent protection from neoplasia by folic acid. The relative risk of developing neoplasia was 0.76 for 400 mcg folate and 0.54 for those taking 1 mg folate for at least six months compared to those not supplemented (Lashner et al., 1997). Neuropsychiatric Applications Neuropsychiatric diseases encompass a number of neurological, cognitive, and psychiatric presentations that may be secondary to folate deficiency. Such presentations include dementia, schizophrenialike syndromes, insomnia, irritability, forgetfulness, endogenous depression, organic psychosis, peripheral neuropathy, myelopathy, and restless legs syndrome (Metz et al., 1996).Lower serum and RBC folate concentrations have an association with depression, and deficiency might predict a poorer response to some antidepressant medications ( Fava et al., 1997;Papakostas et al., 2004). Several studies have documented improvement in depression in some patients subsequent to oral supplementation with the coenzyme form of folic acid (methyltetrahydrofolate) at doses of 15-50 mg daily (Passeri et al., 1993).Folic acid (500 mcg per day) significantly improved the antidepressant action of fluoxetine in subjects with major depression (Coppen and Bailey, 2000). Limited evidence implies supplemental folic acid might positively affect morbidity of some bipolar patients placed on lithium therapy ( Coppen et al., 1986). A syndrome characterized by mild depression, permanent muscular and intellectual fatigue, mild symptoms of restless legs,

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depressed ankle jerk reflexes, diminution of vibration sensation in the legs, stocking-type hypoesthesia, and long-lasting constipation appears to respond to folic acid supplementation (5-10 mg per day for 6-12 months), (Botez et al., 1979). Periodontal Disease Folic acid can increase the resistance of the gingiva to local irritants and lead to a reduction in inflammation. A mouthwash containing 5 mg folate per 5 mL of mouthwash used twice daily for four weeks, with a rinsing time of one minute, appears to be the most effective manner of application. The effect of folate on gingival health appears to be moderated largely, if not totally, through a local influence (Thomson and Pack, 1982). Pregnancy Low dietary intake of folic acid increases the risk for delivery of a child with a neural tube defect (NTD). Periconceptional folic acid supplementation significantly reduces the occurrence of NTD (Werler et al., 1993).Supplemental folic acid intake during pregnancy results in increased infant birth weight and improved Apgar scores, along with a concomitant decreased incidence of fetal growth retardation and maternal infections (Scholl et al.,1996). Vitiligo In some individuals, administration of folic acid appears to be a rational aspect of a nutritional protocol to treat vitiligo. Degrees of re-pigmentation ranging from complete repigmentation in six subjects and 80-percent re-pigmentation in two subjects were reported in eight individuals who followed a three-year protocol with a dosage of 2 mg folic acid twice daily, 500 mg vitamin C twice daily, and intramuscular injections of vitamin B12 every two weeks.66 A two-year study using a combination of folic acid, vitamin B12, and sun exposure for treatment of vitiligo reported positive results. One hundred patients with vitiligo were treated, with re-pigmentation occurring in 52 subjects. Total re-pigmentation was seen in six patients and the spread of vitiligo was halted in 64 percent of the patients. Re-pigmentation was most evident on sun-exposed areas (Juhlin and Olsson, 1997). Drug-Nutrient Interactions A number of drugs can interfere with the pharmacokinetics of folic acid. Cimetidine and antacids appear to reduce folate absorption (Russell et al., 1988). Sulfasalazine interferes with folic acid absorption and conversion to the active form ( Lambie and Johnson, 1985).Supplementation with folic acid (15 mg/day for one month) prevents folate deficiency in patients with inflammatory bowel disease treated with sulfasalazine ( Pironi et

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al.,1988).Continuous long-term use of acetaminophen and aspirin, ibuprofen, and other nonsteroidal antiinflammatory drugs appears to increase the bodys need for folic acid (Lambie and Johnson, 1985).Although the mechanism is unclear, anticonvulsants, antituberculosis drugs, alcohol, and oral contraceptives produce low serum and tissue concentrations of folate (Backman et al., 1989). Folic acid reduces elevated liver enzymes induced by methotrexate therapy in rheumatoid arthritis; however, it had no effect on the incidence, severity, and duration of other adverse events (Van Ede et al., 2001). Folic acid supplementation prevents nitric oxide synthase dysfunction induced by continuous nitroglycerin use ( Gori et al., 2001). Anti-seizure medications, including carbamazepine and phenobarbital, appear to utilize folic acid during hepatic metabolism. Folic acid supplementation can increase metabolism of these drugs, thus lowering blood levels of the drugs and possibly resulting in breakthrough seizures. Initiating folic acid therapy after starting these drugs in individuals should be done with caution (Butterworth and Tamura, 1989). The anticonvulsant drugs phenytoin and valproic acid appear to interfere with folate absorption (Goggin et al., 1987).Folic acid supplementation, at a time of day other than when taking an anticonvulsant, may be helpful to prevent deficiency. There is conflicting information regarding the effects of folate supplementation in individuals treated with antifolate medications such as methotrexate (MTX) and 5-fluorouracil (5-FU). There is evidence folic acid might inhibit the activity of these drugs, although in some cases it may increase activity. In fact, the folic acid metabolite, folinic acid (also known as 5-formyltetrahydrofolate and leucovorin), is often used to rescue normal tissue after MTX or 5-FU therapy. Folic acid supplementation does not appear to interfere with methotrexates anti-arthritic or antiinflammatory activity. Since these medications are used to treat a wide range of malignant and nonmalignant disorders, indiscriminate use of folates should be avoided until further investigation is conducted. Nutrient-Nutrient Interactions Some concern exists that supplementation with high doses of folic acid could mask a vitamin B12 deficiency, resulting in neurological injury secondary to undiagnosed pernicious anemia. If there is any possibility of B12-induced anemia in an individual needing folate therapy, dual therapy with B12 and folate should be administered. Some authors have suggested folic acid supplements might interfere with intestinal zinc absorption; however,

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doses as high as 15 mg folic acid daily do not appear to have any significant effect on zinc status in healthy, non-pregnant subjects (Butterworth and Tamura, 1989). Side Effects and Toxicity In doses typically administered for therapeutic purposes, folic acid is considered nontoxic. At doses of 15 mg daily and above, gastrointestinal complaints, insomnia, irritability, and fatigue have been mentioned as occasional side effects. Folic acid is considered safe during pregnancy, with an established recommended intake of 800 mcg daily. Dosage The dose of folic acid required varies depending on the clinical condition. For lowering homocysteine, a minimum dose of 800 mcg daily is generally used. The most common therapeutic dose is in the range of 1-3 mg daily. Doses greater than 10 mg daily have been used in conditions such as cervical dysplasia. Dosages of over-the-counter folic acid supplements are restricted to no more than 800 mcg of folic acid per serving, although prescription forms of folic acid are available in higher doses.

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ENTEROCOCCUS FAECALIS OR STREPTOCOCCUS FAECALISPrior to 1984, Enterococcus was members of the genus Streptococcus, thus Enterococcus faecalis was known as Streptococcus faecalis (schleifer and kilpper-balz, 1984).Enterococcus faecalis formerly classified as part of the Group D Streptococcus system is a Gram positive commensally bacterium inhabiting the gastrointestinal tracts of humans and other mammals (Ryan and Ray, 2004). It is among the main constituents of some probiotic food supplements. A commensally organism like other species in the genus Enterococcus, E. faecalis can cause life-threatening infections in humans, especially in the nosocomial (hospital) environment, where the naturally high levels of antibiotic resistance found in E. faecalis contribute to its pathogenicity (Ryan and Ray, 2004).

Figure 2: Enterococcus faecalis as viewed through a scanning electron microscope Scientific classification

Physiology 15

E. faecalis is a non-motile microorganism and facultatively anaerobic; it ferments glucose without gas production, and does not produce a catalase reaction with hydrogen peroxide. E. faecalis displays gamma hemolysis (-hemolysis). It produces a reduction of litmus milk, but does not liquefy gelatin. Growth of nutrient broth is consistent with being facultatively anaerobic. Pathogenesis E. faecalis can cause endocarditis,as well as bladder, prostate, and epididymal infections; nervous system infections are less common. (Ryan and Ray, 2004; Pelletier, 1996). Antibacterial resistance E. faecalis is resistant to many commonly used antimicrobial agents (aminoglycosides, aztreonam, cephalosporins, clindamycin, the semi-synthetic penicillins nafcillin and oxacillin, and trimethoprim-sulfamethoxazole). Resistance to vancomycin is also becoming more common (Amyes, 2007; Courvalin, 2006). Exposure to cephalosporin is a particularly important risk factor for colonization and infection with enterococci. Vancomycin resistant enterococci (VRE) are usually treated with Linezolid.

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OBJECTIVES To develop Folic Acid assay method with the help of microbiological turbiditmetric method and HPLC method. To compare this two methods and finally to find out which one is more accurate. To determine which method consumes lesser time to perform the Test.

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REVIEW OF LITERATUREThis laboratory has employed experimentally a modification of the method described by Teply and Elvehjem in "The titrimetric determination of 'Lactobacillus casei factor' and 'folic acid' " in which salt solution A and peptone have been omitted from the basal medium and the results of growth are read turbidimetrically after 16 to 18 hours' incubation. Our data indicate that the turbidimetric results are not appreciably affected by the omission of asparagine, alanine, p-aminobenzoic acid, peptone, and salt solution B as suggested by Teply and Elvehjem. However, we have omitted only the salt solution A and peptone as a routine procedure. The laboratory preparation of such a medium containing somany constituents requires a considerable amount of time when large volumes are needed at frequent intervals. Also, considerable fluctuation in the quality of this medium has been encountered because of variations in the quality of some of the constituents, particularly the amino acids and the vitamins. Commercial availability of a standardized dehydrated medium that is quickly and easily prepared is highly desirable in a control laboratory where the completion of large volumes of work in short periods of time is important. Some pantothenic acid assay experiments were performed with a dehydrated "pantothenate assay broth, exp'l" supplied by Difco Laboratories, Inc., Detroit. The protocol supplied with this medium indicated that its composition varied slightly from that described by Skeggs and Wright for the assay of pantothenic acid. The composition of the Skeggs and Wright medium is very similar to that of Teply and Elvehjem. The cardinal difference between the two media is the omission of calcium pantothenate from the former. Other materials present in the folic acid medium but not in the pantothenic acid medium, namely asparagine, alanine, and peptone, are those constituents that Teply and Elvehjem suggested could be omitted (Beryl Capps, 1948). The folic acid content of cow's milk whey was reported by Wright, McMahan, Cheldelin, Taylor, Snell and Williams in 1941. Their data suggested that milk contained only a small amount of folic acid. Microbiological data published by Williams, Cheldelin and Mitchell; by Wright, Skeggs, Welch, Sprague and Mattis; and by Luckey, Briggs, Moore, Elvehjem and Hart; and rat data obtained by Day, Wakin, Zimmerman and McClung support this suggestion. However, precise data for a number of milk samples have not been presented, the suggestion of Wright et al. that milk contains a large amount of "potential folic acid" has not been verified, and data concerning the lability of "folic acid" during the heat processing of

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milk have not been obtained. An investigation originally planned to clarify these three points developed into a critical study of the assay methods for "folic acid" as applied to milk. The results of that study are presented here (Hudson, 1948). The microbiological method for the determination of folate in plant foods uses the growth response of folate-dependent Lactobacillus rhamnosus in extracts that have been enzymatically treated to release the bound vitamin. However, the use of cryoprotected cultures is hampered by low recovery of the microorganism after extended frozen storage times. In this study, growth of L.rhamnosus was enhanced using a microaerophilic enrichment procedure and optimal pH conditions and enzyme reaction times were determined for the release of bound folate in spinach. Optimum pH values for the release of bound folate in spinach samples treated with a-amylase or protease were 3.0 and 4.0, respectively. Although treatment with -amylase had no significant (P > 0.05) effect on measured folate, addition of protease at pH 4.0 significantly (P 0.05) increased the release of folate at an optimum incubation time of 8 h. Therefore, a dual-enzyme treatment (protease and conjugase) is sufficient to determine folate content in spinach (Srilatha Pandrangi1 and Luke Labored, 2004). Hydroxyurea in up to 60 m~ concentration did not inhibit growth or DNA synthesis in nonaerated cultures of Streptococcus faecalis ATCC 8043. In contrast, in cultures a~rated by shaking already 1 mM hydroxyurea decreased the rate of net DNA synthesis and in higher concentrations of the drug the growth of the total cell mass also slowed down and the number of cells per chain increased from 1 2 to 10. The differential rate of DNA synthesis, but not tho growth of tho total cell mass, could be restored almost to the control level by adding thymidine to the medium. Thus there are at least two targets for hydroxyurea in the cells of S. faecalis grown in aerated cultures (Lalzti and Heinonen, 1979). Derivatives of folic acid (pteroylglutamic acid, PCA) have been shown to be essential for the transfer of single carbon fragments in transmethylations and purine biosynthesis. The conversion of folic acid to folinic acid (Citrovorum factor, CF) has been demonstrated in rat liver slices, in chick liver honiogenates, in the rat, and in rnan. The folic acid antagonist aminopterin has been shown to block this conversion in vitro and in vivo by preventing the reduction of folic acid to tetrahydrofolic acid. In man, aminopterin has been reported to increase the urinary excretion of a test dose of folic acid. Aminopterin and other folic acid antagonists have proved to be of temporary value in the management of certain kinds of human cancer, particularly the acute leukemias of childhood's and choriocarcinoma. In the present

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study observations have been made on the fate of intravenously administered folic acid in normal subjects and in patients with leukemia and other neoplasms and on the effect of aminopterin on the metabolism of folic acid (Paul Cond and David Grob, 1957). A six-step biochemical key is presented for the identification of all recognized Enterococcus spp. The key consists of 12 tests, but no more than 6 are needed for the most complicated identification. The reliability of the key has been evaluated with collection type strains and clinical and environmental isolates. This key has fewer tests than those reported in previous studies. There is no commercial kit that includes the whole set of tests. However, some of the tests are included in enzyme activity-based kits that could be used with the proposed key. The key is designed for use in routine applications, especially in environmental and clinical studies with a high number of isolates (Albert Manero and Anicet Blanch, 1999). A strain of Streptococcus faecium (ATCC 8043) which is highly resistant to the antifolic acid compound, amethopterin was gently ruptured by exposing protoplasts of the organism to a hypotonic solution. The crude lysine resulting there from was treated by various chemical and physical techniques designed to separate folic acid reductase from dihydrofolic acid reductase. In the process, the enzyme was purified approximately 160-fold; however, throughout the process, the enzyme preparation maintained the ability to reduce folic acid to tetrahydrofolic acid. Attempts to isolate mutants showing a deficiency in either folic acid reductase or dihydrofolic acid reductase were unsuccessful. Based on these results, it is concluded that folic acid is reduced to tetrahydrofolic acid by one enzyme in S. francium (ATCC 8043). The crude lysine was also subjected to ultracentrifugation. An analysis of the supernatant fluid and the sediment indicated that the reductive activity is located in the soluble fraction of the cell (Eugene Speck and Lewis Affront, 1968). The results of deoxyribonucleic acid-deoxyribonucleic acid and deoxyribonucleic acidribosomal ribonucleic acid hybridization studies demonstrated that Streptococcus faecalis and Streptococcus francium are distantly related to the non-Enterococci streptococci (Streptococcus hooves and Streptococcus equines) of serological group D and to other streptococci. On the basis of our results and those of previous studies, we propose that S. faecalis and S. francium be transferred to the genus Enterococcus (ex Therein and Joshua) nom. rev. as Enterococcus faecalis (Andrews and Harder) comb. nova. and Enterococcus francium (Orla-Jensen) comb. nova., respectively. A description of the genus Enterococcus

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nom. rev. and emended descriptions of E. faecalis and E. francium are given (Karl Schleifer and Renate Kilpper-Balz, 1984). A method is described for the microbiological assay of folic acid activity in serum with Lactobacillus casei as test organism and a modified medium in which the organism gives a greater growth response than in media previously detailed. The results of experiments carried out to validate the use of this medium are shown. In 94 control subjects levels of folic acid activity in the serum ranged from 2 1 to 28 m,tg./ml. (mean 7-8). The values in nine out of 10 patients with megaloblastic anemia due to deficiency of folic acid were [ 0 mpg./ml. or less and one result was 2-0 m,tg./ml. In six patients with megaloblastic anemia associated with pregnancy the results ranged from 0 7 to 4 0 m,g./ml., and in untreated pernicious anemia 28 out of 31 results were within or above the control range and three values were just below the lower limit of normal (Spray, 1964). Folic Acid deficiency in man may be due to a variety of factors. The most important ones are considered to be unusually poor diets, faulty absorption of the vitamin from the intestinal tract, derangement in the intestinal flora resulting in low synthesis of the vitamin and faulty metabolism of the vitamin in the body. Human blood contains several members of the F.A. family of compounds. Therefore, in order to obtain valid information about the F.A. activity in human blood or serum it seems desirable to measure as many forms of the vitamin as possible. Such approach may facilitate detection of the biochemical lesions responsible for, or connected with, the deficiency. In the present paper, various methods modified or developed in our laboratory to achieve this end are described. Most of them have been in use in our laboratory for several years (Grossowlcz et al., 1962). In 1998, the United States introduced mandatory fortification of enriched cereal-grain products with folic acid to reduce the incidence of neural tube defects. As a consequence, substantial amounts of folic acid, the synthetic form of folate, were added to the American diet, and the ability to assess folic acid intake took on greater importance. The purpose of the current study was to separate and quantify folic acid and 5-methyltetrahydrofolate, the most prominent naturally occurring folate in fortified foods, with a reliable and robust method. Folates were heat-extracted from food samples. A trienzyme treatment (a-amylase, rat plasma conjugase, and protease) was applied to the extracts followed by purification by affinity chromatography. Folic acid and 5-methyltetrahydrofolate were separated and quantified by reversed-phase HPLC with fluorescence and UV detection. A gradient elution with phosphate

21

buffer and acetonitrile was used to separate the different forms of folates. The method gave a linear response in a range of 0.13 mmol/l and 0.01250.25 mmol/l for folic acid and 5methyltetrahydrofolate, respectively. These ranges were similar to the expected levels in the samples. The CV of the peak areas of folic acid and 5-methyltetrahydrofolate for 5 commercial wheat flour samples extracted and run separately on the same day was 2.0 and 5.7% and, run over 5 consecutive days, was 7.2 and 7.3%, respectively. Total folate values in 45 samples of fortified food measured by HPLC and by the traditional microbiological assay demonstrated a high correlation (r2 0.986) (Rosalia Poo-Prieto et al., 2006). In cognizance of the difficulties involved in the colorimetric and titrimetric methods for the determination of individual vitamins, this laboratory has been carrying out a series of studies into the use of HPLC for improved analysis of these nutrients. Preliminary studies have been carried out for the determination of four B-vitamins. The present paper reports on further improvements made to enable the simultaneous determination of eight vitamins i.e. B1, B2, B6, B12, and C, niacin, niacinamide and folic acid. Trials were carried out to determine the most suitable chromatographic system include changing the proportion of methanol in the mobile phase, the use of different ion-pairing reagents and other additives such as triethylamine and ammonia. Three sets of HPLC mobile phase systems are proposed to enable successful separation of all eight vitamins in less than 20 minutes, varying slightly with the type of ionpairing reagent and mobile phase additive. This laboratory is currently carrying out trials to determine if the developed methods could be used for the determination of pharmaceutical products and food samples (Khor Swan-Choo and Tee Siong, 1996). A reversed-phase column liquid chromatographic method was developed for the assay of amoxicillin and its preparations. The linear calibration range was 0.2 to 2.0 mg/ml (r = 0.9998), and recoveries were generally greater than 99%o. The high-performance liquid chromatographic assay results were compared with those obtained from a microbiological assay of bulk drug substance and capsule, injection, and granule formulations containing amoxicillin and degraded amoxicillin. At the 99%o confidence level, no significant intermethod differences were noted for the paired results. Commercial formulations were also analyzed, and the results obtained by the proposed method closely agreed with those found by the microbiological method. The results indicated that the proposed method is a suitable substitute for the microbiological method for assays and stability studies of amoxicillin preparations (Mei-Chich Hsu and Pei-Wen Hsu, 1992).

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Taka-diastase and a preparation of hog kidney enzyme have been used routinely to liberate folic acid from its conjugates in the microbiological determination of the vitamin. However, Olson et al.reported recently that taka-diastase and certain proteolytic enzymes are of doubtful value in releasing the vitamin from plant tissues. Hog kidney conjugase also does not completely liberate folic acid in every case with fresh plant materials or plant extracts. It has been demonstrated that, with homogenates of rat liver, autolysis at pH 7.0 results in a rapid increase in the folic acid content, whereas at pH 4.5 neither autolysis of the liver nor digestion of heated samples with hog kidney conjugase causes release of the vitamin. Apparently there are bound forms of folic acid not hydrolyzable by the conjugase preparations now available. According to Luckey et al no one method could be prescribed to attain maximum folic acid values in all types of materials. Charkey et al also suggest that there may be more than one form of the conjugate present in yeast. In spite of the wide-spread occurrence in tissues and organs of enzymes capable of converting the conjugated pteroylglutamic acid to the free acid, there is little information as to whether conjugases differ in respect to their mechanism of action. In this communication are reported the results of certain preliminary observations which suggest that conjugases may vary in their ability to liberate folic acid or folic acid-active substances from natural sources (Sreenivasan, 1948). There is a significant fall in the serum folic acid level during pregnancy, reaching its lowest level at term. This is most pronounced in twin pregnancies. A similar but less spectacular fall occurs in the vitamin B12 concentration. In megaloblastic anemia both folic acid and vitamin B12 levels are lower than in other pregnant women. The degree of megaloblastic change in the bone marrow, as measured by the type and number of megaloblasts, is reflected in the vitamin levels, cases with florid megaloblastosis showing the most marked depression of vitamin B12 and folic acid activity. Although there is a significant difference in the mean folic acid levels between megaloblastic and normoblastic pregnant women, a considerable overlap exists between individual values in the two groups. When the labile folic-acid factor is determined separately the test becomes much more specific. In the present series, all cases of megaloblastic anemia yielded labile-factor levels below 10 mug. per ml. while a similar value was encountered in only one of 35 normal pregnancies. In five women with megaloblastic anemia the vitamin B12 concentration was less than 100, u, ug. Per ml. but rose to normal levels on folic acid therapy alone (Ball and Giles, 1964).

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It is well known that a megaloblastic anemia may develop in patients being treated with barbiturate-like anticonvulsants such as phenytoin sodium (Badenoch, 1954, Hawkins and Meynell, 1954), primidone (Fuld and Moorhouse, 1956), and, very occasionally, other barbiturates (Hobson et al., 1956). The cause of the anemia is uncertain. The patients resemble patients with folic-acid deficiency in that their serum vitamin B12 concentrations are normal, and they fail to respond, or they respond suboptimally, to treatment with vitamin B12, while responding excellently to treatment with folic acid. However, as there is no evidence of malabsorption of folic acid in these patients it has been generally assumed that the drugs act by interfering with the metabolism of folic acid. In this paper we report studies made on the utilization of folic acid in a patient who developed severe megaloblastic anemia while receiving primidone (" mysoline"), (Chanarin et al., 1958).

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MATERIALS AND METHODSFolic Acid Assay by microbiological Turbiditmetric Method Folic Acid Assay Medium is used for determining folic acid concentration by the microbiological assay technique. Summary and Explanation Vitamin assay media are prepared for use in the microbiological assay of vitamins. Three types of medium are used for this purpose: 1. Maintenance Media For carrying the stock culture to preserve the viability and sensitivity of the test organism for its intended purpose. 2. Inoculum Media To condition the test culture for immediate use; 3. Assay Media To permit quantitation of the vitamin under test. They contain all the factors necessary for optimal growth of the test organism except the single essential vitamin to be determined. Folic Acid Assay Medium is used in the microbiological assay of folic acid with Streptococcus faecalis ATCC 8043 as the test organism. Folic Acid Assay Medium is prepared according to the formula described by Capps, Hobbs and Fox, 1 modified with sodium citrate instead of sodium acetate. Principles of the Procedure Folic Acid Assay Medium is a folic acid-free dehydrated medium containing all other nutrients and vitamins essential for the cultivation of Streptococcus faecalis ATCC 8043. The addition of folic acid in specified increasing concentrations gives a growth response that can be measured turbiditmetrically.

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Formula of Folic Acid Assay Medium Approximate Formula* Per Liter Vitamin Assay Casamino Acids Dextrose Sodium Citrate L-Cystine DL-Tryptophan Adenine Sulfate Guanine Hydrochloride Uracil Thiamine Hydrochloride Pyridoxine Hydrochloride Riboflavin Niacin Calcium Pantothenate p-Aminobenzoic Acid Biotin Dipotassium Phosphate Monopotassium Phosphate Magnesium Sulfate Sodium Chloride Ferrous Sulfate Manganese Sulfate 12.0 g 40.0 g 20.0 g 0.2 g 0.2 g 20.0 mg 20.0 mg 20.0 mg 2.0 mg 4.0 mg 2.0 mg 2.0 mg 400.0 g 200.0 g 0.8 g 1.0 g 1.0 g 0.4 g 20.0 mg 20.0 mg 20.0 mg

*Adjusted and /or supplemented as required to meet performance criteria. Precautions Great care must be taken to avoid contamination of media or glassware in microbiological assay procedures. Extremely small amounts of foreign material may be sufficient to give erroneous results. Scrupulously clean glassware free from detergents and other chemicals must be used. Glassware must be heated to 250C for at least 1 hour to burn

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off any organic residues that might be present. Take precautions to keep sterilization and cooling conditions uniform throughout the assay. Quality Control Identity Specifications Folic Acid Assay Medium Dehydrated Appearance Off white to very light beige, free flowing, homogeneous. Solution 3.75% (single strength) or 7.5% (double strength) solution, soluble in purified water upon boiling for 2-3 minutes. Single strength solution is light amber, may have a slight precipitate. Prepared Appearance Very light amber, clear, may have a very slight precipitate. Reaction of 3.75% Solution at 25C pH 6.8 0.2 Cultural Response Folic Acid Assay Medium Prepare the medium per label directions. The medium supports the growth of Streptococcus faecalis ATCC 8043 when prepared in single strength and supplemented with folic acid. The medium should produce a standard curve when tested using a folic acid reference standard at 0.0 to 8.0 ng per 10 ml. Incubate tubes with caps loosened at 35-37C for 18-24 hours. Read the percent transmittance using a spectrophotometer at 540 nm. Directions for Preparation from Dehydrated Product 1. Suspend 7.5 g of the Folic Acid Assay Medium in 100 ml of purified water. 2. Heat with frequent agitation and boil for 2-3 minutes. 3. Dispense in 5 ml amounts into tubes, evenly dispersing the precipitate. 4. Add standard or test samples. 5. Adjust the tube volume to 10 ml with purified water. 6. Autoclave at 121C for 10 minutes. Procedure

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Prepare stock cultures of Streptococcus faecalis ATCC 8043 by stab inoculation of Lactobacilli Agar AOAC. Incubate at 35-37C for 24-48 hours. Store tubes in the refrigerator. Make transfers at monthly intervals. Prepare the inoculum for assay by subculturing a stock culture of Streptococcus faecalis ATCC 8043 into a tube containing 10 ml of Lactobacilli Broth AOAC. After incubation at 35-37C for 18-24 hours, centrifuge the cells under aseptic conditions and decant the supernatant. Wash the cells three times with (2500 rpm for 15 minutes) 10 ml of sterile 0.85% saline. After the third wash, dilute the cell suspension 2:100 with sterile 0.85% saline. Use 10l of this latter suspension to inoculate each of the assay tubes. It is essential that a standard curve be set up for each separate assay. Autoclaving and incubation conditions that influence the standard curve readings cannot always be duplicated. The standard curve is obtained by using folic acid at levels of 0.0, 1, 2, 4, 6, and 8 ng per 10 ml assay tube. Turbiditmetric readings should be made after incubation at 35-37C for 18-24 hours. Refrigerate tubes for 15-30 minutes to stop growth before reading at 540 nm. Prepare the folic acid stock solution required for the standard and Test (Capps et al., 1948). Curve as follows Standard dilution preparation 1. Dissolve 50 mg dried Folic Acid USP Reference Standard or equivalent in about 30 ml of 0.01(N) NaOH and 300 ml purified water. 2. Adjust to pH 7.5 0.5 with diluted HCL solution. Add purified water to give a volume of 500 ml. 3. Add 2 ml of the solution from step 2 to 50 ml purified water. Adjust the pH to 7.5 0.5 with HCL solution. Dilute to 100 ml with purified water to give a stock solution containing 2 ng folic acid per ml. Prepare the stock solution fresh daily. Prepare the standard solution for the assay by diluting 1 ml of this stock solution in 1 liter with purified water. This solution contains 2 ng folic acid per ml. Use 0.0, 0.5, 1, 2, 3, and 4 ml per assay tube. Following incubation, place the tubes in the refrigerator for 15-30 minutes to stop growth. The growth can be measured by a turbiditmetric method and the curve constructed from the values obtained. The most effective assay range is between the levels of 2 and 10 ng folic acid per 10 ml tube. Test dilution preparation 1. Dissolve 1ml test sample of 500 ml purified water. Adjust the pH to 7.5 0.5 with HCL solution. The Test sample Folic Acid concentrations 500 mcg per ml

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2. Prepare the Test solution for the assay by diluting 1 ml of this Test solution in 500 ml with purified water. This solution contains 2 ng folic acid per ml. 3. Use 0.0, 0.5, 1, 2, 3, and 4 ml per assay tube.

1

2

3

4

Figure 3:1-50mg Std. folic acid /ml, 2-2ng std folic acid/ml, 3-1mcg/ml test folic acid conc., 4-2ng test folic acid conc. /ml. Concent rations of Stander dilution 0 0/D 0/ 0/D 1 1/D 2 2/D 4 4/D Standard dilution of Folic Acid (2ng/ml) 0 0 0 0 1 1 2 2 4 4 5 5 5 5 4 4 3 3 1 1 Purified water Inoculating Folic Acid Assay Medium 5 5 5 5 5 5 5 5 5 5 Microorganism (Streptococcus faecalis ATCC 8043) 0 0 10l 10l 10l 10l 10l 10l 10l 10l 0 0 0.006 0.008 0.176 0.174 0.517 0.518 1.065 1.062 Absorbance at 540 nm

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Table1: Folic acid Standard dilution preparation Concentrat Test dilution ions of Test dilution 0 0/D 0 0/D 1 1/D 2 2/D 4 4/D of Folic Acid (2ng/ml) 0 0 0 0 1 1 2 2 4 4 5 5 5 5 4 4 3 3 1 5 5 5 5 5 5 5 5 5 Purified water Folic Acid Assay Medium Inoculating Microorganism (Streptococcus faecalis ATCC 8043) 0 0 10l 10l 10l 10l 10l 10l 10l 0 0 0.009 0.007 0.305 0.307 0.668 0.670 1.328 1.330 Absorbance at 540nm

1 5 10l Table2: Folic acid Test dilution preparation

*D= Duplicated Expected Results 1. Prepare a standard concentration response curve by plotting the response readings against the amount of standard in each tube. 2. Determine the amount of vitamin at each level of assay solution by interpolation from the standard curve. 3. Calculate the concentration of vitamin in the sample from the average of these volumes. Use only those values that do not vary more than 10% from the average. Use the results only if two-thirds of the values do not vary more than 10%.

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Figure 4: The UV spectrophotometer with absorbance at 540 nm Limitations of the Procedure 1. The test organism used for inoculating an assay medium must be cultured and maintained on media recommended for this purpose. 2. Aseptic technique should be used throughout the assay procedure. 3. The use of altered or deficient media may cause mutants having different nutritional requirements that will not give a satisfactory response. 4. For successful results of these procedures, all conditions of the assay must be followed precisely.

FOLIC ACID ASSAY BY HIGH-PRESSURE LIQUID CHROMATOGRAPHY (HPLC) METHOD

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Figure 5: Dionex HPLC High-pressure liquid chromatography (HPLC), sometimes called high-performance liquid chromatography, is a separation technique based on a solid stationary phase and a liquid mobile phase. Separations are achieved by partition, adsorption, or ion-exchange processes, depending upon the type of stationary phase used. HPLC has distinct advantages over gas chromatography for the analysis of organic compounds. Compounds to be analyzed are dissolved in a suitable solvent, and most separations take place at room temperature. Thus, most drugs, being nonvolatile or thermally unstable compounds, can be chromatographed without decomposition or the necessity of making volatile derivatives. Most pharmaceutical analyses are based on partition chromatography and are completed within 30 minutes. As in gas chromatography, the elution time of a compound can be described by the capacity factor, which depends on the chemical nature of the analyte, the composition and flow rate of the mobile phase, and the composition and surface area of the stationary phase. Column length is an important determinant of resolution. Only compounds having different capacity factors can be separated by HPLC. Apparatus A liquid chromatograph consists of a reservoir containing the mobile phase, a pump to force the mobile phase through the system at high pressure, an injector to introduce the sample into the mobile phase, a chromatographic column, a detector, and a data collection device such as a computer, integrator, or recorder. Short, small-bore columns containing densely packed particles of stationary phase provide for the rapid exchange of compounds between the mobile

32

and stationary phases. In addition to receiving and reporting detector output, computers are used to control chromatographic settings and operations, thus providing for long periods of unattended operation. Pumping Systems HPLC pumping systems deliver metered amounts of mobile phase from the solvent reservoirs to the column through high-pressure tubing and fittings. Modern systems consist of one or more computer-controlled metering pumps that can be programmed to vary the ratio of mobile phase components, as is required for gradient chromatography, or to mix isocratic mobile phases (i.e., mobile phases having a fixed ratio of solvents). However, the proportion of ingredients in premixed isocratic mobile phases can be more accurately controlled than in those delivered by most pumping systems. Operating pressures up to 5000 psi or higher, with delivery rates up to about 10 ml per minute are typical. Pumps used for quantitative analysis should be constructed of materials inert to corrosive mobile phase components and be capable of delivering the mobile phase at a constant rate with minimal fluctuations over extended periods of time. Injectors After dissolution in mobile phase or other suitable solution, compounds to be chromatographed are injected into the mobile phase, either manually by syringe or loop injectors, or automatically by auto samplers. The latter consist of a carousel or rack to hold sample vials with tops that have a pierce able septum or stopper and an injection device to transfer sample from the vials to a loop from which it is loaded into the chromatograph. Some auto samplers can be programmed to control sample volume, the number of injections and loop rinse cycles, the interval between injections, and other operating variables. A syringe can be used for manual injection of samples through a septum when column head pressures are less than 70 atmospheres (about 1000 psi). At higher pressures an injection valve is essential. Some valve systems incorporate a calibrated loop that is filled with test solution for transfer to the column in the mobile phase. In other systems, the test solution is transferred to a cavity by syringe and then switched into the mobile phase.

Columns

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For most pharmaceutical analyses, separation is achieved by partition of compounds in the test solution between the mobile and stationary phases. Systems consisting of polar stationary phases and nonpolar mobile phases are described as normal phase, while the opposite arrangement, polar mobile phases and nonpolar stationary phases, and are called reverse-phase chromatography. Partition chromatography is almost always used for hydrocarbon-soluble compounds of molecular weight less than 1000. The affinity of a compound for the stationary phase, and thus its retention time on the column, is controlled by making the mobile phase more or less polar. Mobile phase polarity can be varied by the addition of a second, and sometimes a third or even a fourth, component. Stationary phases for modern, reverse-phase liquid chromatography typically consist of an organic phase chemically bound to silica or other materials. Particles are usually 3 to 10 m in diameter, but sizes may range up to 50 m or more for preparative columns. Small particles thinly coated with organic phase provide for low mass transfer resistance and, hence, rapid transfer of compounds between the stationary and mobile phases. Column polarity depends on the polarity of the bound functional groups, which range from relatively nonpolar octadecyl silane to very polar nitrile groups. Liquid, nonbound stationary phases must be largely immiscible in the mobile phase. Even so, it is usually necessary to presaturate the mobile phase with stationary phase to prevent stripping of the stationary phase from the column. Polymeric stationary phases coated on the support are more durable. Columns used for analytical separations usually have internal diameters of 2 to 5 mm; larger diameter columns are used for preparative chromatography. Columns may be heated to give more efficient separations, but only rarely are they used at temperatures above 60 because of potential stationary phase degradation or mobile phase volatility. Unless otherwise specified in the individual monograph, columns are used at ambient temperature. Ion-exchange chromatography is used to separate water-soluble, ionizable compounds of molecular weight less than 1500. The stationary phases are usually synthetic organic resins; cation-exchange resins contain negatively charged active sites and are used to separate basic substances such as amines, while anion-exchange resins have positively charged active sites for separation of compounds with negatively charged groups, such as phosphate, sulfonate, or carboxylate groups. Water-soluble ionic or ionizable compounds are attracted to the resins, and differences in affinity bring about the chromatographic separation. The pH of the mobile phase,

34

temperature, ion type, ionic concentration, and organic modifiers affect the equilibrium, and these variables can be adjusted to obtain the desired degree of separation. In size-exclusion chromatography, columns are packed with a porous stationary phase. Molecules of the compounds being chromatographed are filtered according to size. Those too large to enter the pores pass unretained through the column. Smaller molecules enter the pores and are increasingly retained as molecular size decreases. These columns are typically used to measure aggregation and degradation of large molecules. Detectors Many compendia HPLC methods require the use of spectrophotometer detectors. Such a detector consists of a flow-through cell mounted at the end of the column. A beam of UV radiation passes through the flow cell and into the detector. As compounds elute from the column, they pass through the cell and absorb the radiation, resulting in measurable energy level changes. Fixed, variable, and multi-wavelength detectors are widely available. Fixed wavelength detectors operate at a single wavelength, typically 254 nm, emitted by a low-pressure mercury lamp. Variable wavelength detectors contain a continuous source, such as a deuterium or highpressure xenon lamp, and a monochromatic or an interference filter to generate monochromatic radiation at a wavelength selected by the operator. The wavelength accuracy of a variablewavelength detector equipped with a monochromatic should be checked by the procedure recommended by its manufacturer; if the observed wavelengths differ by more than 3 nm from the correct values, recalibration of the instrument is indicated. Modern variable wavelength detectors can be programmed to change wavelength while an analysis is in progress. Multiwavelength detectors measure absorbance at two or more wavelengths simultaneously. In diode array multi-wavelength detectors, continuous radiation is passed through the sample cell, then resolved into its constituent wavelengths, which are individually detected by the photodiode array. These detectors acquire absorbance data over the entire UV-visible range, thus providing the analyst with chromatograms at multiple, selectable wavelengths and spectra of the eluting peaks. Diode array detectors usually have lower signal-to-noise ratios than fixed or variable wavelength detectors, and thus are less suitable for analysis of compounds present at low concentrations. Differential refractometer detectors measure the difference between the refractive index of the mobile phase alone and that of the mobile phase containing chromatographed

35

compounds as it emerges from the column. Refractive index detectors are used to detect nonUV absorbing compounds, but they are less sensitive than UV detectors. They are sensitive to small changes in solvent composition, flow rate, and temperature, so that a reference column may be required to obtain a satisfactory baseline. Fluorometric detectors are sensitive to compounds that are inherently fluorescent or that can be converted to fluorescent derivatives either by chemical transformation of the compound or by coupling with fluorescent reagents at specific functional groups. If dramatization is required, it can be done prior to chromatographic separation or, alternatively, the reagent can be introduced into the mobile phase just prior to its entering the detector. Potentiometric, voltametric, or polarographic electrochemical detectors are useful for the quantitation of species that can be oxidized or reduced at a working electrode. These detectors are selective, sensitive, and reliable, but require conducting mobile phases free of dissolved oxygen and reducible metal ions. A pulseless pump must be used, and care must be taken to ensure that the pH, ionic strength, and temperature of the mobile phase remain constant. Working electrodes are prone to contamination by reaction products with consequent variable responses. Electrochemical detectors with carbon-paste electrodes may be used advantageously to measure nanogram quantities of easily oxidized compounds, notably phenols and catechols. New detectors continue to be developed in attempts to overcome the deficiencies of those being used. Data Collection Devices Modern data stations receive and store detector output and print out chromatograms complete with peak heights, peak areas, sample identification, and method variables. They are also used to program the liquid chromatograph, controlling most variables and providing for long periods of unattended operation. Data also may be collected on simple recorders for manual measurement or on standalone integrators, which range in complexity from those providing a printout of peak areas to those providing chromatograms with peak areas and peak heights calculated and data stored for possible subsequent reprocessing. Procedure The mobile phase composition significantly influences chromatographic performance and the resolution of compounds in the mixture being chromatographed. For accurate

36

quantitative work, high-purity reagents and HPLC grade organic solvents must be used. Water of suitable quality should have low conductivity and low UV absorption, appropriate to the intended use. Reagents used with special types of detectors (e.g., electrochemical, mass spectrometer) may require the establishment of additional tolerances for potential interfering species. Composition has a much greater effect than temperature on the capacity factor, k. In partition chromatography, the partition coefficient, and hence the separation, can be changed by addition of another component to the mobile phase. In ion-exchange chromatography, pH and ionic strength, as well as changes in the composition of the mobile phase, affect capacity factors. The technique of continuously changing the solvent composition during the chromatographic run is called gradient elution or solvent programming. It is sometimes used to chromatograph complex mixtures of components differing greatly in their capacity factors. Detectors that are sensitive to change in solvent composition, such as the differential refract meter, are more difficult to use with the gradient elution technique. The detector must have a broad linear dynamic range, and compounds to be measured must be resolved from any interfering substances. The linear dynamic range of a compound is the range over which the detector signal response is directly proportional to the amount of the compound. For maximum flexibility in quantitative work, this range should be about three orders of magnitude. HPLC systems are calibrated by plotting peak responses in comparison with known concentrations of a reference standard, using either an external or an internal standardization procedure. Reliable quantitative results are obtained by external calibration if automatic injectors or auto samplers are used. This method involves direct comparison of the peak responses obtained by separately chromato graphing the test and reference standard solutions. If syringe injection, which is irreproducible at the high pressures involved, must be used, better quantitative results are obtained by the internal calibration procedure where a known amount of a no interfering compound, the internal standard, is added to the test and reference standard solutions, and the ratios of peak responses of drug and internal standard are compared. Because of normal variations in equipment, supplies, and techniques, a system suitability test is required to ensure that a given operating system may be generally applicable. The main features of system suitability tests are described below (Indian Pharmacopoeia 2007, British Pharmacopoeia 2008 and The United States Pharmacopeia 2008).

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Pump

Injector

Column

Detector

Computer

Figure 6: Schematic Diagram of HPLC

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ANALYTICAL METHOD STANDARIZATION PROTOCOLPurpose The purpose of this protocol is to standardization the analytical methods of the test parameters of Jectocos Plus injection for the test for folic acid and determined with appropriate accuracy and precision. . Composition of Injection The following composition is of Jectocos plus Injection. Composition Each ml contains: Iron Sorbitol Citric Aid Complex Corresponding to Fe (total) Folic Acid IP Hydroxocobalamin Acetate BP Corresponding to Hydroxocobalamin Water for Injection IP 50 mcg qs 50 mg 500 mcg

Method of Analysis Assay of Folic Acid Equipments The following equipments shall be used for the validation studies. All the Equipments shall be calibrated as per schedule. HPLC: QC/I/LC/003 (Dionex). Analytical Balance: QC/I/BL/003 (Sartorius BP210S) Millipore Water Purification System: QC/I/WP/001(Mili-Q) Standards: Folic Acid Potassium dihydrogen phosphate, acetonitrile, sodium hydroxide. Methanol Chromatographic System Wavelength: 283nm. Column: 4.6 mm x 25 cm C18 Licrospher 100 RP 5 Flow rate: About 1.5 ml/min. Injection Volume: 20l. Column oven temperature: 25c

Solvents and Chemicals

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Preparation of Mobile Phase A mixture of 93 volumes of 0.05 M potassium dihydrogen phosphate and 7 volumes of acetonitrile adjusted to pH 6.0 with 5 M sodium hydroxide. Placebo Preparation Take 4 ml of without Folic Acid content Injection (The Test solution concentration of Folic Acid is 0.05mg/ml) in 10 ml volumetric flask Make the volume with 0.1 M sodium hydroxide. Take 5ml dilute Test solution in 100ml volumetric flask Make the volume with mobile phase Standard Preparation Weigh standard Folic Acid 20mg in 100 ml volumetric flask Make the volume with 0.1 M sodium hydroxide. Take 5ml dilute standard Folic Acid solution in 100ml volumetric flask Make the volume with mobile phase. Procedure Separately inject equal volumes (about 20 l) of the standard solution and the test solution in the chromatograph, record the chromatograms and measure the responses for the major peaks.

LIST OF STANDANDISATION PARAMETERSThe following matrix is developed to define various validation parameters required for each analytical tests of Jectocos plus injection.

Key A: Specificity B: Precision C: Accuracy D: Linearity E: Limit of Detection F. Limit of Quantization G Range. H. Roundness.

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Specificity of the procedure Specificity as the ability to assess unequivocally the analyte in the presence of components that may be expected to be present such as matrix and internal standard components, degradation products, impurities. In the case of Analyte Folic Acid. Preparation 0.1 (M) NaOH solution 4gm NaOH solution dissolve in 1 lit of Mili-Q water for preparation of 0.1 (M) NaOH Procedure Separately inject six replicate equal volumes (about 20 l) of the standard Folic acid in the chromatograph, record the chromatograms and measure the responses for the major peaks. Acceptance Criteria The relative standard deviation should not be more than 2% of response of major peaks produced by six replicate Injections. Accuracy The Accuracy of an analytical method is the closeness of the test results obtained by that method to the true value. The accuracy of an analytical method should be established across its range. Spiking 20%, 40%, 60%, 80%, 100%, and 120% of gave Test Solution preparation 100 %. Preparation of 20% Test Solution Take 4ml of Folic Acid content Injection (The Test solution concentration of Folic Acid is 0.05mg/ml) in 10 ml volumetric flask Make the volume with 0.1 M sodium hydroxide. Take 1ml dilute Test solution in 100ml volumetric flask Make the volume with mobile phase. Preparation of 40% Test Solution Take 4ml of Folic Acid content Injection (The Test solution concentration of Folic Acid is 0.05mg/ml) in 10 ml volumetric flask Make the volume with 0.1 M sodium hydroxide. Take 2ml dilute Test solution in 100ml volumetric flask Make the volume with mobile phase. Preparation of 60% Test Solution Take 4ml of Folic Acid content Injection (The Test solution concentration of Folic Acid is 0.05mg/ml) in 10 ml volumetric flask Make the volume with 0.1 M sodium hydroxide. Take 3ml dilute Test solution in 100ml volumetric flask Make the volume with mobile phase.

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Preparation of 80% Test Solution Take 4ml of Folic Acid content Injection (The Test solution concentration of Folic Acid is 0.05mg/ml) in 10 ml volumetric flask Make the volume with 0.1 M sodium hydroxide. Take 4ml dilute Test solution in 100ml volumetric flask Make the volume with mobile phase. Preparation of 100%Test Solution Take 4ml of Folic Acid content Injection (The Test solution concentration of Folic Acid is 0.05mg/ml) in 10 ml volumetric flask Make the volume with 0.1 M sodium hydroxide. Take 5ml dilute Test solution in 100ml volumetric flask Make the volume with mobile phase. Preparation of 120% Test Solution Take 4ml of Folic Acid content Injection (The Test solution concentration of Folic Acid is 0.05mg/ml) in 10 ml volumetric flask Make the volume with 0.1 M sodium hydroxide. Take 6ml dilute Test solution in 100ml volumetric flask Make the volume with mobile phase. Procedure Separately inject 2 replicate equal volumes (about 20 l) of the Diluents, placebo test solution with all impurities with in the chromatograph, record the chromatograms and measure the responses for the major peaks. Acceptance Criteria Recovery should be 95% to 105% with respect to the added percentage The test results with respect to test concentration should be linear and co- relation coefficient should not be less than 0.997. Record y Intercept = mx + C Slope = 0.9992 Co- relation coefficient Preparation of 100% Standard Solution Weigh standard Folic Acid 20mg in 100 ml volumetric flask Make the volume with 0.1 M sodium hydroxide. Take 5ml dilute standard Folic Acid solution in 100ml volumetric flask Make the volume with mobile phase. Limit of 40% quantitated (LOQ) The quantitated limit is a characteristic of quantitative in diual impurities for low levels of compounds in sample matrices, such as impurities in bulk drug substances and degradation products in finished pharmaceuticals. It is the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions.

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The quantitated limit is expressed as the concentration of analyte (e.g., percentage, parts per billion) in the sample. Procedure Inject 6 conjugative standard injections in to the chromatograph of 20l each calculate the Relative Standard Deviation. Conclusion Evaluate the lowest possible concentration of Folic Acid at which the given analytical method is observed to be accurate and precise. Limit of 20% detection (LOD) The detection limit is a characteristic of limit tests. It is the lowest amount of analyte in a sample that can be detected, but not necessarily quantitated, under the stated experimental conditions. Thus, limit tests merely substantiate that the amount of analyte is above or below a certain level. The detection limit is usually expressed as the concentration of analyte (e.g., percentage, parts per billion) in the sample. Ruggedness The ruggedness of the analytical method is the degree of reproducibility of the test results obtained by the analysis of the same sample under a variety of conditions. Carry out the experiments precision by using two analysts to prove that the change of operational and environmental variables of the method has no significant effect in the reproducibility of the test results. Calculate the relative standard deviation between the results obtained by two analysts using a minimum of six determinations at 100% of test Concentration. Acceptance Criteria The Analytical results should be reproducible. The relative Standard deviation should not be more than 2% when Analysis carried out by two different chemists on same Instrument. Evaluation of results Result of Analytical method of jectocos plus injection for different analytical parameters Specificity, precision, accuracy, Linearity, Range, LOD, LOQ and Ruggedness. Should be discussed such as Results are within prescribed standard, acceptable limit. The analytical method is found to be specific, precise accurate Linear Within range and Reproducible for folic acid in method is applied in our routine activities in laboratories.

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ANALYTICAL METHOD STANDARIZATION REPORTList of Standardization parameters The following matrix is developed to define various Standardization parameters required for each analytical test.

Key A: Specificity B: Precision C: Accuracy D: Linearity E: Limit of Detection F. Limit of Quantization G Range. H. Roundness Folic acid Specificity Equipment: Analyte: folic acid in iron preparation. Formulation matrix: Placebo. Blank: Acetonitrile. WS purity: 91.86 % w/w.

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By analyzing individually Blank Acetonitrile diluting solution, placebo, a sample solution of folic acid P with standards and test preparation with standard in test solution, each analyte found specific, pure without any interference. It is observed that there is no any interference with principal analyte folic acid and standard known impurities from the placebo, blank, sample matrix. Acceptance criterion No apparent interference observed between the peaks and selectively separated from one another and also from formulation matrix (placebo) and impurity peak as well as active ingredient the chromatographic peaks are distinct form each other. Precision Prepare six samples individually, analyze and calculate the RSD It is observed that the six individual preparations of sample solution at 100 % concentration of standard folic acid in precision and internal precision study, the results are within the specified RSD limit.

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RESULTSFolic Acid Assay by Microbiological Turbiditmetric Method Sample Table Report

Conc.(2g/ml) 0 2 4 1.400 81.200 1.000 0.800 0.600 0.400 0.200 0.000 0 2

Abs. S.td 0.000 0.1750 FOLIC ACID ASSAY 0.5175 1.06425

JCB 908 0.000 0.3060 0.6690 1.3290

ABS.

ABS.S.td JCB 908

4 CONC.

6

8

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Graph.1: The graph showing microorganism turbidity absorbance vs. concentration

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Table3: Jectocos plus Injection Study Test Data NOTE: Conc. from graph *Folic Acid Limitations 500 to 750 Calculation Assay= (Mean conc. per ml) x (500/1) x (500/1) x (1/1000) = 2.5979 x (500/1) x (500/1) x (1/1000) = 649.475 mcg per ml

Folic Acid assay by High-Pressure Liquid Chromatography (HPLC) Method Linearity Spiking 20 %, 40%, 60 %, 80%, 100% and 120 % of folic acid to placebo will perform the linearity test. Analyze and record the area of each concentration of folic acid. Plot a calibration curve of concentration verses area of the peak. Calculate the co-relation coefficient. Folic acid Sr. No. Conc. In % Qty. added % Area

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1 2 3 4 5 6

20 40 60 80 100 120

0.0002% 0.0004% 0.0006% 0.0008% 0.001% 0.0012%

1.058 2.772 3.934 5.633 7.122 8.565

It is observed that in folic acid linearity study, the areas at 283 nm of from 20 to 120 concentrations well correlates and is within the specified limits. Acceptance criteria RSD should not be more than 2.0 % RSD Observed: 0.757%, orderFOLIC ACID ASSAY BY HPLC9 8 7 6 AREA 5 4 3 2 1 0 0 20 40 60 CONC. 80 100 120 140 Area

Graph.2: Graph showing folic acid area v/s concentration. Acceptance criteria Co-relation coefficient is not less than 0.9992 * The specified limits is 0.997

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Range The range of an analytical method is the interval between upper and lower level of folic acid. To demonstrate the range it is to be determined with a suitable level of Precision, Accuracy and Linearity. The range is expressed in percentage. For establishment of range the linearity concentration 20%, 100%, and 120% of folic acid is used. Evaluation of Results Analytical Method Validation subjected for different analytical parameter such as specificity, precision, internal precision, accuracy, linearity, range and ruggedness. The results are within prescribed standard, acceptable limit. The analytical method is found to be specific, precise, accurate, linear within range and reproducible for folic acid. The method is standard and valid and can be used as routine regular analytical method in our laboratory. Ruggedness The ruggedness of the analytical method is the degree of reproducibility of the test results obtained by the analysis of the same sample under a variety of conditions. Carry out the experiments precision by using two analysts to prove that the change of operational and environmental variables of the method has no significant effect in the reproducibility of the test results. Calculate the relative standard deviation between the results obtained by two analysts using a minimum of six determinations at 100% of test Concentration. Limit of detection Using present given analytical method Folic acid can be determined up to 20% of standard concentration in parental formulation. Limit of quantitated Concentration is in parental formulation with precise & Accuracy.

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DISCUSSIONAssay of folic acid by turbiditmetric method using Streptococcus faecalis as a test organism was done with standard concentration of Folic Acid 2mmcg, 4mmcg & 8mmcg. Turbidity was measured at 540nm of all concentrations of folic acid in duplicate & inoculate sample as blank. The graph was plotted of concentration v/s turbidity & it was observed that the turbidity is linear with the concentration of folic acid. The test sample assured to have a concentration of 500mcg/ml was serially diluted to have 2mmcg concentration & turbidity of test sample was measured. From turbidity measurements the concentration of the test was obtained from the graph. It was observed that the results of sample tested were satisfactory & were within the specified limits. The assay can be satisfactorily used to assay samples of folic acid with combination of Iron in solid & in liquid dosage forms. Determination of Folic acid by HPLC method in iron preparation is developed & standardized by using Analytical method Validation parameters like, specificity, precision accuracy linearity range ruggedness & limit of detection & limit of qualification is used. This method may be used in routing analysis for qualification & for qualitative purpose for Folic acid in pharmaceutical products.

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SUMMARYFolic acid, also known generically as folate or folacin, is a member of the B-complex family of vitamins, and works in concert with vitamin B12. Folic acid functions primarily as a methyl-group donor involved in many important body processes, including DNA synthesis. Therapeutically, folic acid is instrumental in reducing homocysteine levels and the occurrence of neural tube defects. It sometimes plays a key role in preventing cervical dysplasia and it also protects against neoplasia in ulcerative colitis. Folic acid also shows promise as part of a nutritional protocol to treat vitiligo, and it may reduce inflammation of the gingiva. Further, certain neurological, cognitive, and psychiatric presentations may be secondary to folate deficiency. Such presentations include peripheral neuropathy, myelopathy, restless legs syndrome, insomnia, dementia, forgetfulness, irritability, endogenous depression, organic psychosis, and schizophrenia-like syndromes. Low dietary intake of folic acid increases the risk for delivery of a child with a neural tube defect (NTD). Periconceptional folic acid supplementation significantly reduces the occurrence of NTD. Supplemental folic acid intake during pregnancy results in increased infant birth weight and improved Apgar scores, along with a concomitant decreased incidence of fetal growth retardation and maternal infections. The dose of folic acid required varies depending on the clinical condition. For lowering homocysteine, a minimum dose of 800 mcg daily is generally used. The most common therapeutic dose is in the range of 1-3 mg daily. Doses greater than 10 mg daily have been used in conditions such as cervical dysplasia. Dosages of over-the-counter folic acid supplements are restricted to no more than 800 mcg of folic acid per serving, although prescription forms of folic acid are available in higher doses. Folic acid assay by microbiological turbidimetric method using the organism streptococcus faecalis ATCC 8043 strain used. The organism uses the folic acid for the enhancement of its growth. The taste and standard of folic acid concentration 0, 1,2,4,6 and 8 ng per 10ml assay tube. As the concentration of the folic acid increases there is an increase in the absorbance thus we conclude that the absorbance gets doubled at each level. The absorbance taken by UV spectrophotometer at 540 nm. Folic acid assay by HPLC method the folic acid is identified from the iron and folic acid combination. By using 0.05 M potassium dihydrogen phosphate as a mobile phase using the column C18 and the wavelength is 283 nm. The standard average folic acid area 7.86 and

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the retention time is 1.87 minute. The test sample average area is 7.12 and the retention time is 1.81 so our test sample is showing that the folic acid is identified. The total time taken is 5 minute for one injection. In these two tests the microbiological assay method is more sensitive and HPLC method is more accurate and the time taken is less. Thus it can be told that pharmaceutical companies can implement these method to improve there quality.

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REFERENCES1. Zettner, A., Boss, G.R., Seegmiller, J.E. (1981) A long-term study of the absorption of large oral doses of folic acid. Ann. Clin. Lab. Sci. 11:516-524. 2. Schuster, O., Weimann, H.J., Muller. J. (1993) Pharmacokinetics and relative bioavailability of iron and folic acid in healthy volunteers. Arzneimittelforschung. 43:761-766. 3. Gregory, J.F., 3d, Bhandari, S.D., Bailey, L.B. (1992) Relative bioavailability of deuterium-labeled monoglutamyl tetrahydrofolates and folic acid in human subjects. Am. J. Clin. Nutr. 55:1147- 1153. 4. . Levitt, M., Nixon, P.F., Pincus, J.H., Bertino, J.R. (1971) Transport characteristics of folates in cerebrospinal fluid; a study utilizing doubly labeled 5-methyltetrahydrofolate and 5- formyltetrahydrofolate. J. Clin. Invest. 50:1301- 1308. 5. Priest, D.G., Schmitz, J.C., Bunni, M.A. (1999) Accumulation of plasma reduced folates after folic acid administration. Semin Oncol. 26:S38-S41. 6. Yates, J.R., Ferguson-Smith, M.A., Shenkin, A. (1987) Is disordered folate metabolism the basis for the genetic predisposition to neural tube defects? Clin Genet.31:279-287. 7. Lussier-Cacan, S., Xhignesse, M, Piolot, A. (1996) Plasma total homocysteine in healthy subjects sexspecific relation with biological traits. Am. J. Clin. Nutr. 64:587593. 8. Kluijtmans, L.A., Van, den, Heuvel, L.P., Boers, G.H. (1996) Molecular genetic analysis in mild hyperhomocysteinemia: a common mutation in the methylenetetrahydrofolate reductase gene is a genetic risk factor for cardiovascular disease. Am. J. Hum.Genet.58:35-41. 9. Whitehead, A.S., Gallagher, P, Mills, J.L. (1995) A genetic defect in 5, 10 methylenetetrahydrofolate reductase in neural tube defects. Q.J.M. 88:763- 766. 10. Ulvik, A, Evensen, E.T., Lien, E.A. (2001) Smoking, folate and methylenetetrahydrofolate reductase status as interactive determinants of adenomatous and hyperplastic polyps of colorectum. Am. J. Med. Genet. 101:246-254. 11. Halsted, C.H. (1989) The intestinal absorption of dietary folates in health and disease. J. Am. Coll. Nutr.8:650-658. 12. Revell, P, ODoherty, M.J.,Tang, A, Savidge, G.F. (1991) Folic acid absorption in patients infected with the human immunodeficiency virus. J. Intern. Med. 230:227-231.

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13. Botez, M.I. (1976) Folate deficiency and neurological disorders in adults. Med Hypotheses. 2:135- 140. 14. Audebert, M, Gendre, J.P., Le, Quintrec, Y. (1979) Folate and the nervous system (authors transl). Sem. Hop.55:1383-1387. 15. Young, S.N., Ghadirian, A.M. (1989) Folic acid and psychopathology. Prog. Neuropsychopharmacol Biol. Psychiatry. 13:841-863. 16. Metz, J, Bell, A.H., Flicker, L. (1996) The significance of subnormal serum vitamin B12 concentration in older people: a case control study. J. Am. Geriatr. Soc. 44:13551361. 17. Quinn, K, Basu, T.K. (1996) Folate and vitamin B12 status of the elderly. Eur. J. Clin. Nutr. 50:340-342. 18. Fin