Spectrophotometric determination of folic acid in

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 307 (2002) 316–321 www.academicpress.com

Spectrophotometric determination of folic acid in pharmaceutical preparations by coupling reactions with iminodibenzyl or 3-aminophenol or sodium molybdate–pyrocatechol Padmarajaiah Nagaraja,* Ramanathapura A. Vasantha, and Hemmige S. Yathirajan Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India Received 12 February 2002

Abstract Novel coupling reagents are used for the simple and sensitive spectrophotometric determination of folic acid either in pure form or in its pharmaceutical preparations. The methods are based on the probable diazotization of the p-aminobenzoylglutamic acid obtained after reductive clevage of folic acid, followed by either coupling with iminodibenzyl to give a violet product with kmax of 580 nm or coupling with 3-aminophenol to produce an orange yellow-colored product with kmax of 460 nm. Sodium molybdate and pyrocatechol are used in the third method and the pale red-colored product formed has a kmax of 490 nm. The methods are highly reproducible and have been applied to the determination of folic acid in tablets and the results compare favorably with the official method. Common excipients used as additives in pharmaceutical preparations do not interfere in the proposed methods. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Folic acid; Spectrophotometry; Iminodibenzyl; 3-Aminophenol; Pyrocatechol; Sodium molybdate; Pharmaceuticals

Folic acid (FA)1 or pteroyl-L -glutamic acid, chemically known as N-[4-[[(2-amino-1,4-dihydro-4-oxo-6pteridinyl)methyl]amino]benzoyl]-L -glutamic acid is a water-soluble B vitamin that helps build healthy cells [1]. Excellent reviews and articles concerning various aspects of folic acid have been published [2–7]. A survey of the literature reveals that there are various methods available for the determination of folic acid which include official methods [8–10], liquid chromatography [11,12], highperformance liquid chromatography [13–16], radioassay [17], histochemical [18], flow-injection chemiluminometry [19], fluorimetry [20,21], electroanalytical techniques [22–26], and spectrophotometric methods [27–38]. Most of the spectrophotometric methods reported already suffer from disadvantages such as narrow range of determination, require heating or extraction, long time *

Corresponding author. Fax: +91-821-421263. E-mail address: [email protected] (P. Nagaraja). 1 Abbreviations used: FA, folic acid; IDB, iminodibenzyl; 3-AP, 3aminophenol; Mo–PC, sodium molybdate–pyrocatechol; LOQ, limit of quantification; NEDA, N-(1-naphthyl)ethylenediamine dihydrochloride; p-ABGA, p-animobenzoylglutamic acid.

for the reaction to complete, and instability of the colored product formed. The idea of the present work is to provide simple, sensitive, and rapid spectrophotometric methods for the determination of folic acid in pure form as well as in folic acid tablets. The outstanding feature of the methods is that all the methods are free from interference when excipients are present, particularly some vitamins. In continuation of our work on the spectrophotometric determination of some organic compounds of biological interest and pharmaceutical importance [39–41], the present paper reports sensitive and simple spectrophotometric methods for the determination of folic acid. In all the three methods developed, folic acid was reductive-cleaved and the p-aminobenzoylglutamic acid diazotized, followed by coupling with iminodibenzyl (IDB) or 3-aminophenol (3-AP) or sodium molybdate–pyrocatechol (Mo–PC). The methods offer the advantages of sensitivity and stability. Common additives used as excipients in pharmaceutical preparations and some vitamins do not interfere in the determination of folic acid by the proposed methods.

0003-2697/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 3 - 2 6 9 7 ( 0 2 ) 0 0 0 3 8 - 6

P. Nagaraja et al. / Analytical Biochemistry 307 (2002) 316–321

Materials and methods Chemical and reagents Folic acid, iminodibenzyl, 3-aminophenol, and pyrocatechol were obtained from Sigma (St. Louis, MO). Molybdic acid was purchased from Merck (Darmstadt, Germany). Sodium nitrite (BDH), hydrochloric acid (AR), sulfuric acid (AR), and all other reagents and solvents were of analytical grade. Folic acid tablets were purchased from local sources. Instrumentation A JASCO Model UVIDEC-610 UV-visible spectrophotometer with 1.0 cm matched cells was used for all absorption measurements.

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cooled, the solution was diluted and with 1:1 (v/v) H2 SO4 up to the mark. After the solutions were mixed thoroughly, the absorbances were measured at 580 nm for IDB or at 460 nm for 3-AP or at 490 nm for Mo–PC against the corresponding reagent blanks and the calibration graphs were constructed. Procedure for the assay of tablets Twenty tablets were powdered and mixed thoroughly. An amount equivalent to 50 mg of FA was taken in 0.1 M NaOH and reduced using zinc and HCl. The solution was filtered and diazotized and one of the three recommended procedures given for the pure sample was followed.

Sample preparation

Results and discussion

Folic acid (50 mg) was dissolved in 0.1 M sodium hydroxide solution. This solution was reduced using zinc and concd. hydrochloric acid, filtered, and diluted to 100 ml in a calibrated flask. A solution of 0.5% (w/v) iminodibenzyl in alcohol was prepared. Aqueous solutions of 1% sodium nitrite, 2% sulfamic acid, 0.4% pyrocatechol, and 1% 3-aminophenol were used. Sulfuric acid (1:1, v/v) and hydrochloric acid (5 M) were used for the experiment. An 8% molybdic acid (dissolved in 4 ml of 5 M sodium hydroxide solution and neutralized with hydrochloric acid to obtain a clear solution) solution was freshly prepared. Deionized water was used to prepare all solutions and in all experiments.

The methods involve reductive cleavage of folic acid and the diazotization of formed p-aminobenzoylglutamic acid followed by coupling with IDB or 3-AP or Mo–PC reagents. The violet-colored product formed with IDB has a kmax of 580 nm, orange yellow product with 3-AP has a kmax of 460 nm, and the pale red product with Mo–PC has a kmax of 490 nm. These wavelengths were used for all subsequent measurements. The absorption spectra of the reaction products formed are shown in Fig. 1. The corresponding reagent blanks have practically negligible absorbance at these wavelengths.

Recommended procedure Aliquots of the working standard solution of reduced FA (2.5–200 lg for IDB; 5.0–300 lg for 3-AP or Mo– PC) were transferred into a series of 25-ml calibrated flasks. For the IDB method, 2 ml of 5 M hydrochloric acid was added, cooled, followed by the addition of 1 ml of 2% NaNO2 , and cooled. To this solution, 1.5 ml of 2% sulfamic acid was added along with 2 ml of 0.5% IDB and 3 ml of alcohol. The solution was heated in a boiling water bath for 5 min, cooled and diluted to the mark with 1:1 (v/v) H2 SO4 . For the 3-AP method, 2 ml of 5 M HCl was added, cooled, followed by the addition of 2 ml of 1% NaNO2 , and cooled, and 2 ml of 2% sulfamic acid was added. The solution was mixed with 2 ml of 1% 3-aminophenol, heated in a boiling water bath for 5 min, cooled, followed by the addition of 3 ml of 5 M HCl, swirled, and diluted with water to the mark. For the Mo–PC method, after diazotization as noted above, 2 ml of 8% sodium molybdate was used along with 1.5 ml of 0.4% pyrocatechol. The solution was swirled well, heated in a boiling water bath for 5 min, and

Fig. 1. Absorption spectra of the reaction products; a ! FA–IDB, b ! FA–3-AP, c ! FA–Mo–PC; final FA concentration is 6 lg/ml for b and c (4 lg/ml for a).

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Various concentration and volume ranges for all the reagents were studied. However, the following were the optimum concentration and volume ranges. For the IDB method, it was found that 1–3 ml of 5 M HCl, 1–3 ml of 2% NaNO2 , 1–3 ml of 2% sulfamic acid, and 1–3 ml of 0.5% IDB were necessary for the achievement of maximum color intensity. For the 3-AP method, 1–3 ml of 1% NaNO2 , 1–3 ml of 2% sulfamic acid, 1–3 ml of 1% 3-AP, and 1–5 ml of 5 M HCl were necessary to achieve maximum color intensity. For the Mo–PC method, 1–3 ml of 1% NaNO2 , 1–3 ml of 2% sulfamic acid, 1–3 ml of 8% sodium molybdate, and 1–3 ml of 0.4% pyrocatechol were necessary for the system to stabilize. The excess of nitrite added during diazotization could be removed by the addition of sulfamic acid solution and an excess of sulfamic acid has no effect on color intensity. Excellent results were obtained using 1:1 (v/v) H2 SO4 for dilution compared to other acids and solvents in the IDB and Mo–PC methods. The optical characteristics and precision data are given in Table 1. Limit of quantification (LOQ) is given by the relation 10r=s and limit of detection is 3r=s, where r is the standard deviation of the blank with respect to water and s is the slope of the calibration curve. Naturally, the limit of quantification slightly crosses the lower limit of Beer’s law range. But, limit of detection is well below the lower limit of Beer’s law range. The upper limit of the Beer–Lambert range (Table 1) is determined by a plot of absorbance against concentration at the value of kmax . Beyond this limit (8 lg/ml for the IDB method and 12 lg/ml for the other two methods), the correlation results were really affected. That means, the present studies were carried out according to Beer’s law ranges given in Table 1. Hence, the absorbance meas-

urements were excluded above these limits to keep the relationship linear. Chemical methods for the determination of FA are mainly based on the use of a coupling agent, N-(1naphthyl)ethylenediamine dihydrochloride (NEDA). In the reductive cleavage method of the British Pharmacopoeia [8] one of the products is p-aminobenzoylglutamic acid [42], whereas the oxidative cleavage procedure of the United States Pharmacopoeia [10] gives p-aminobenzoic acid [42]. Both of these products contain an aromatic amino group, which is diazotized using sodium nitrite and hydrochloric acid. Therefore, it is undoubtedly clear that the reductive cleavage of FA gives p-aminobenzoylglutamic acid (p-ABGA), which is diazotized and coupled with IDB or 3-AP or Mo–PC. The probable reaction sequence is shown in Scheme 1. Investigation of the continuous molar variation and the mole ratio method indicates a 1:1 stoichiometry in all the three cases. In the present studies, before coupling the diazotized product with any of the three reagents in order to produce the colored product, it is essential to remove the excess of nitrite by the addition of sulfamic acid. The stability of the product formed by coupling with IDB is about 2 h, while it is 3 h with Mo–PC. In contrast, the coupling product formed with 3-AP is stable for about 13 days. To increase the stability of the products with IDB or Mo–PC beyond the stipulated time was not successful. All three products were stable in the temperature range of 20–60 °C. However, a temperature of 30 °C is preferred for the absorbance measurements. The extent of interference by various excipients (talc, gumacacia, starch, sodium chloride, dextrose, glucose, lactose, carboxymethyl cellulose, magnesium stearate, and sodium alginate) and vitamins (vitamin A, C, B1 ,

Table 1 Optical characteristics and precision data Parameters/characteristics

IDB

AP

Mo–PC

Color kmax (nm) Stability (h) Beer’s law range (lg/ml) Limit of detection (lg/ml) Limit of quantification (lg/ml) Molar absorptivity (M1 cm1 ) Sandell’s sensitivity (lg=cm2 ) Optimum photometric range (lg/ml) Regression equation (y)a Slope (b) Intercept (a) Correlation coefficient (r)b Relative standard deviationc (%) Range of error (at 95% confidence level)

Violet 580 02 0.1–8.0 0.0469 0.1423 7:19 104 0.0066 0.3–7.0

Orange yellow 460 300 0.2–12.0 0.0819 0.2483 4:55 104 0.0105 0.4–10.0

Pale red 490 03 0.2–12.0 0.1571 0.5237 3:15 104 0.0152 0.4–10.0

0.0917 0.0102 0.998 0.2805 0.3893

0.0701 0.011 0.996 0.2272 0.3154

0.0666 )0.0154 0.999 0.2189 0.3038

a

y ¼ bx þ a, where x is the concentration in lg/ml. n ¼ 5. c Five replicates. b

1.21 1.56 1.56 1.23 1.56 1.0 1.00 2.78 1.78 1.5 0.98 0.56 0.99 0.72 0.63 b

Average of five determinations RSD (%). Marketed by: Alembic Chemical Works. c Marketed by: Wyeth Lederle. d Marketed by: Eros Pharma. e Theoretical values for five determinations: Student’s t test, t ¼ 2:78; variance ratio test, F ¼ 6:39.

0.60 0.77 0.72 5:06 0:20 4:97 0:25 1:02 0:20 5:0 0:18 5:01 0:20 0:98 0:20 5:02 0:20 4:98 0:15 1:06 0:15 5 5 1 Folinalb Folvitec Folfitd

3-AP method

Label claim (mg) Tablet

B2 , B6 , and K) was studied in a total volume of 25 ml. The interference was determined by measuring the absorbance of a solution containing 6 lg/ml of FA for the 3-AP and Mo–PC methods (4 lg/ml of FA for the IDB method) and 1200 lg/ml of excipients or vitamins. An error of 2% in the absorbance readings was considered tolerable. The analysis of interference with the excipients or vitamins was conducted from the initial step, i.e., the reduction stage. However, the ratio of excipients and the active ingredient (FA) really does not matter. The percentage recoveries of folic acid varied from 99.4 to 100.7, with the relative standard deviation (%) ranges from 0.2 to 0.4. The significant feature is that some of the vitamins (vitamin B1 , B2 , B6 , and K) do not interfere up to 1200 lg/ml in the present methods. Vitamin C will not interfere up to 200 lg/ml in the IDB and 3-AP methods, while vitamin A can be tolerated up to 400 lg/ ml in the 3-AP and Mo–PC methods. That means,

Table 2 Determination of folic acid in pharmaceutical preparations

Scheme 1. Reaction sequence for the formation of products.

a

Mo–PC method

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4:96 0:22 4:95 0:20 0:98 0:25

IDB method IDB method IDB method

BP method [8]

t valuee Amount of FA founda in mg

3-AP method

Mo–PC method

F valuee

3-AP method

Mo–PC method


320


vitamin C interferes in the Mo–PC method and interference of vitamin A is noted in the IDB method. The reproducibility of the method was checked by five replicate determinations at the 6 lg/ml level of FA (4 lg/ml of FA for IDB) and the relative standard deviation (%) was found to be between 0.22 and 0.28. The applicability of the method for the assay of the folic acid tablets was examined. The results of the assay of available tablets of FA are summarized in Table 2. The analysis of tablets was conducted from the reduction stage. The results are highly reproducible and the assay of tablets was cross-checked by the official method [8] which agrees favorably. The performance of the proposed methods was compared statistically in terms of the Student t test and the Variance ratio F test. At 95% confidence level, the calculated t values and F values do not exceed the theoretical values for the three methods. The theoretical t value was 2.78 (for n ¼ 5) and F value was 6.39 (for n ¼ 5). It is found from Table 2 that there is no significant difference between the proposed methods and the official method, indicating that the proposed methods are as accurate and precise as the official method. The present methods are found to be simple and more sensitive than most of the already reported spectrophotometric methods. The statistical parameters and the recovery study data clearly indicate the reproducibility and accuracy of the methods. Analysis of the authentic samples containing FA showed no interference from common additives and excipients in general. Hence, these methods could be considered for the determination of FA in pure form or in pharmaceutical preparations.

Acknowledgments One of the authors (R.A.V.) thanks the University of Mysore for providing the laboratory facilities and the financial assistance from the UGC, New Delhi, in the form of a fellowship to R.A.V. under the Faculty Improvement Programme is gratefully acknowledged.

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