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DETERMINATION OF FOLIC ACID IN COMBINATION WITH

IRON IN PHARMACEUTICAL DOSAGE FORMS BY


CHEMICAL AND MICROBIOLOGICAL METHODS

Dissertation Submitted in partially fulfillment of the


requirement for the Degree of Master of Science in

Applied Microbiology
Submitted
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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CERTIFICATE

This 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 Head Signature of the Guide


Dr. G. VIVEKANANDHAN Mr. K. PONMURUGAN

EXAMINERS

1.

2.

DECLARATION

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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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ACKNOWLEDGEMENT
There 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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INTRODUCTION

Folic 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, brewer’s 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 time-
and 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 body’s 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 5-
methyltetrahydrofolate 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 acid’s 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, Alzheimer’s 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 dose-
dependent 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 re-
pigmentation 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 non-
steroidal antiinflammatory drugs appears to increase the body’s 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 methotrexate’s anti-arthritic or anti-
inflammatory 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 non-
toxic. 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 FAECALIS
Prior 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

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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 (γ-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 LITERATURE
This 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

19
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 acid-
ribosomal 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

20
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 0.1–3 mmol/l and 0.0125–0.25 mmol/l for folic acid and 5-
methyltetrahydrofolate, 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 ¼ 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 ion-
pairing 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).

22
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).

23
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).

24
MATERIALS AND METHODS
Folic 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.

25
Formula of Folic Acid Assay Medium
Approximate Formula* Per Liter
• Vitamin Assay Casamino Acids 12.0 g
• Dextrose 40.0 g
• Sodium Citrate 20.0 g
• L-Cystine 0.2 g
• DL-Tryptophan 0.2 g
• Adenine Sulfate 20.0 mg
• Guanine Hydrochloride 20.0 mg
• Uracil 20.0 mg
• Thiamine Hydrochloride 2.0 mg
• Pyridoxine Hydrochloride 4.0 mg
• Riboflavin 2.0 mg
• Niacin 2.0 mg
• Calcium Pantothenate 400.0 μg
• p-Aminobenzoic Acid 200.0 μg
• Biotin 0.8 μg
• Dipotassium Phosphate 1.0 g
• Monopotassium Phosphate 1.0 g
• Magnesium Sulfate 0.4 g
• Sodium Chloride 20.0 mg
• Ferrous Sulfate 20.0 mg
• Manganese Sulfate 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 250°C for at least 1 hour to burn

26
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 25°C
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 35-37°C 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 121°C for 10 minutes.
Procedure

27
Prepare stock cultures of Streptococcus faecalis ATCC 8043 by stab inoculation of
Lactobacilli Agar AOAC. Incubate at 35-37°C 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 35-37°C 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 10µl 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 35-37°C 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

28
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 Inoculating
Standard
rations Folic Acid Microorganism
dilution of Purified Absorbance
of Assay (Streptococcus
Folic Acid water at 540 nm
Stander Medium faecalis ATCC
(2ng/ml)
dilution 8043)
0 0 5 5 0 0
0/D 0 5 5 0 0
0/ 0 5 5 10μl 0.006
0/D 0 5 5 10μl 0.008
1 1 4 5 10μl 0.176
1/D 1 4 5 10μl 0.174
2 2 3 5 10μl 0.517
2/D 2 3 5 10μl 0.518
4 4 1 5 10μl 1.065
4/D 4 1 5 10μl 1.062

29
Table1: Folic acid Standard dilution preparation

Concentrat Test dilution Purified Folic Acid Inoculating Absorbance


ions of of water Assay Microorganism at 540nm
Test Folic Acid Medium (Streptococcus
dilution (2ng/ml) faecalis ATCC
8043)
0 0 5 5 0 0
0/D 0 5 5 0 0
0 0 5 5 10μl 0.009
0/D 0 5 5 10μl 0.007
1 1 4 5 10μl 0.305
1/D 1 4 5 10μl 0.307
2 2 3 5 10μl 0.668
2/D 2 3 5 10μl 0.670
4 4 1 5 10μl 1.328
4/D 4 1 5 10μl 1.330
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%.

30
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

31
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

33
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 µm
in diameter, but sizes may range up to 50 µm 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 high-
pressure 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 variable-
wavelength 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. Multi-
wavelength 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 non-
UV 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 stand-
alone 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).

37
Pump Injector Column Detector Computer

Figure 6: Schematic Diagram of HPLC

38
ANALYTICAL METHOD STANDARIZATION PROTOCOL
Purpose
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) 50 mg
• Folic Acid IP 500 mcg
• Hydroxocobalamin Acetate BP
• Corresponding to Hydroxocobalamin 50 mcg
• Water for Injection IP qs
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
Solvents and Chemicals
Potassium dihydrogen phosphate, acetonitrile, sodium hydroxide. Methanol
Chromatographic System
Wavelength: 283nm.
Column: 4.6 mm x 25 cm C18 Licrospher 100 RP 5µ
Flow rate: About 1.5 ml/min.
Injection Volume: 20µl.
Column oven temperature: 25°c

39
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 µl) 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 PARAMETERS
The 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.

40
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 µl) 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.

41
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 µl) 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.

42
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 20µl 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.

43
ANALYTICAL METHOD STANDARIZATION REPORT
List 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.

44
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.

45
RESULTS
Folic Acid Assay by Microbiological Turbiditmetric Method
Sample Table Report

Conc.(2µg/ml) Abs. S.td JCB 908


0 0.000 0.000
2 0.1750
FOLIC ACID ASSAY 0.3060
4 0.5175 0.6690
1.400
8 1.06425 1.3290
1.200

1.000

0.800
ABS.S.td
ABS.

JCB 908
0.600

0.400

0.200

0.000
0 2 4 6 8 10
CONC.

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

46
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. Conc. Qty. added Area


No. In % %

47
1 20 0.0002% 1.058

2 40 0.0004% 2.772

3 60 0.0006% 3.934

4 80 0.0008% 5.633

5 100 0.001% 7.122

6 120 0.0012% 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%, order

FOLIC ACID ASSAY BY HPLC

5
AREA

Area
4

0
0 20 40 60 80 100 120 140
CONC.

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

48
49
CHROMATOGRAPH

50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
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.

81
DISCUSSION
Assay 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.

82
SUMMARY
Folic 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

83
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.

84
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