ISSN:0003-2654    

DOI:10.1039/AN97500FX001

出版商:RSC    

年代:1975

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN97500BX003

出版商:RSC    

年代:1975

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN97500BP009

出版商:RSC    

年代:1975

数据来源: RSC

摘要:

Analyst, Janmry, 1975, Vol. 100, pp. 19-24 19 Colorimetric Determination of Folic Acid in Pharmaceutical Preparations Geeta Kanjilal, S. N. Mahajan and G. Ramana Rao Qzdity ControE Laboratories, Indian Drugs and Pharmaceuticals Limited, Hyderabad-500037, India A colorimetric method, based on colour development with 1 , 8-naphtho- quinone-4-sulphonic acid, sodium salt, has been developed for the determina- tion of folic acid. The method depends on the availability of the primary amino group that is liberated on reduction of folic acid. The colour produced has an absorbance maximum at 485 nm and obeys Beer’s law in the concen- tration range 11-7 to 58.7 pg ml-1. The method has been used successfully for assaying folic acid, tablets containing folic acid alone or in combination with iron and vitamin B,,, and injections of folic acid.Many methods for the determination of folic acid have been described in the literature. Among these, a polarographic method1 is official in the U.S.S.R. Pharmacopoeia. A fluori- metric procedure2 has found application in determining folic acid in natural extracts and yeast while a microbiological m e t h ~ d , ~ involving the use of Stre$tococcus faecalis, is useful for assays of multi-component formulations containing this vitamin. Chemical methods are based on the use of a coupling agent, N-1-naphthylethylenediamine hydrochloride (Bratton - Marshall’s reagent). In the reductive cleavage method of the British Pharma~opoeia,~ one of the products is $-aminobenzoylglutamic acid,5 whereas the oxidative cleavage procedure of the United States Pharmacopeia6 gives p-aminobenzoic acide5 Both of these products contain an aromatic amino group, which is diazotised using sodium nitrite and hydrochloric acid.Before coupling the diazotised product with Bratton - Marshall’s reagent in order to produce a violet colour, it is essential to remove the excess of nitrous acid by the addition of ammonium sulphamate. 1 ,2-Naphthoquinone-4-sulphonic acid, sodium salt (Folin’s reagent) has been used as a chromogenic agent for a number of compounds containing a free amino group.’ Therefore, it seemed to be logical to examine the application of this reagent to the determination of folk acid after liberation of the amino group by use of one of the official methods mentioned pre- viously.Use of this reagent offers the advantage of eliminating some of the steps involving the use of a pipette that are required for colour development in the official methods. The method that was developed, using 1,2-naphthoquinone-4-sulphonic acid, sodium salt, is described below. It consists in producing the colour with folic acid in sample and standard solutions simultaneously, measuring the absorbances at 485 nm and calculating the concen- tration of the vitamin from these values. Experimental Apparatus Colorimeter. This was a Spectronic-20 (Bausch and Lomb Inc., Rochester, New York 14602, U.S.A.). Reagents 1,2-Na~hthoqui~tone-4-sul~honic acid, sodium salt, solution. Dissolve 150 mg of laboratory- reagent grade material in 50 ml of water and filter the solution.Prepare fresh daily. Zinc dust (zinc powder). Potassiuum hydrogen phthalate sohtion, 0.2 M. Prepare this solution from analytical-reagent Sodium hydroxide soluutions, 0.1 M and 0.2 M. Prepare these solutions from analytical- grade potassium hydrogen phthalate by dissolving it in boiled and cooled water. reagent grade sodium hydroxide by dissolving it in boiled and cooled water.20 .Analyst, Vol. 100 Prepare a phthalate buffer solution of pH 5 by mixing 500ml of the potassium hydrogen phthalate solution with 238.5 ml of 0.2 M sodium hydroxide solution and making the volume up to 2 litres with boiled and cooled water. Adjust the pH of the solution with use of a pH meter by adding either 0.2 M potassium hydrogen phthalate solution or 0.2 M sodium hydroxide solution.XAN JILAL, MAHAJAN AND RAO : COLORIMETRIC DETERMINATION Bu$zr solution. Calcium lactate solution, 3 per cent. m/V. Trisodium Phosphate solution, 8 per cent. m/V. Lactate - phosphate bufer solution. Mix equal volumes of the trisodiunn phosphate and calcium lactate solutions. Standard Preparation Weigh out accurately about 140 mg of folic acid A.S. and transfer them into a 100-ml calibrated flask. Dissolve the acid in, and dilute it with, sodium hydroxide solution (0.1 M). Sample Preparation Folic acid. Proceed as under Standard Preparation. Tablets of folic acid. Weigh out not less than twenty tablets, grind them to a fine powder and transfer an accurately weighed portion of the powder, equivalent to about 140 mg of folic acid, into a 100-ml beaker. Triturate with 50 ml of 0.1 M sodium hydroxide solution, filter and collect the filtrate in a 100-ml calibrated flask. Wash the residue and the filter with three 10-ml portions of 0.1 M sodium hydroxide solution and dilute the filtrate and washings to the mark with the same reagent.Tablets of folic acid with iron. Weigh out not less than fifty tablets, grind them to a fine powder and transfer an accurately weighed portion of the powder, equivalent to about 25mg of folic acid, into a 500-ml Erlenmeyer flask. Add 250ml of lactate-phosphate buffer to the flask by means of a 50-ml pipette and then heat the mixture on a water-bath at 50 "C, shaking the flask thoroughly. A black, flocculent precipitate separates out in 1 hour. Filter, and collect the filtrate after rejecting the first 20ml; use the filtrate for the deter- mination of folic acid.Dilute a suitable volume of the injection with 0.1 M soclium hydroxide solution so as to give a concentration of folic acid of about 140mg per 10C)ml. Determination Transfer 20-ml aliquots of sample solution, in duplicate, to 100-ml Erlenmeyer flasks, add 15 ml of 4 N hydrochloric acid and 2 g of zinc dust, shake the flasks frequently during a period of 20 minutes, and filter the mixtures into 100-ml calibrated flasks. In each instance make the volume up to the mark with water by repeated washing of the reaction flask and filtering through the original filter. Protect the filtrate from direct light. Treat the standard solution and blank (20ml of water) simultaneously in the same manner. Transfer, by use of a pipette, 5 ml of each filtrate into 50 ml calibrated flasks, add 2 ml of 1,2-naphthoquinone- 4-sulphonic acid, sodium salt, reagent to each flask and mix the contents.After 10 minutes make up the volume with buffer solution and measure the absorbances of' the sample and standard solutions at 485 nm against the reagent blank. In the instance of folic acid with iron tablets, take 50ml of the filtrate and proceed with the reduction as described. Take 20 ml of the reduced solution for colour development, so that the final concentration of folic acid is within the experimental range, and proceed as described above. Calculations Injections of folic acid. At A , The amount of folic acid (mg) in the sample taken = - x S x D where At is the average absorbance of the test solution, A , the average absorbance of the standard solution, S the amount of folk acid (mg) present in 1 ml of standard solution, and D the dilution factor.Results and Discussion The orange-coloured solution was found to show maximum absorbance at a wavelength of 485 nm; its absorption spectrum is given in Fig. 1. A 2-ml volume of a 0-3 per cent.January, 19 75 OF FOLIC ACID IN PHARMACEUTICAL PREPARATIONS 21 I I I I I I I 1 375 400 425 450 475 500 525 550 575 Wavelength/nm Fig. 1. Absorption spectrum of the orange coloured complex obtained by the proposed method. solution of the 1,2-naphthoquinone-4-sulphonic acid, sodium salt, reagent was found to be the optimum for colour development. Buffer solutions of pH 2 to 7 were tried, but at pH 6 and 7 precipitation was observed, therefore the graph of pH versus absorbance is plotted only up to pH 5.4 in Fig.2. Maximum absorbance is observed at pH 5. 1 2 3 4 6 PH Fig. 2. Effect of pH on the absorbance of t h e colour. The colour obeys Beer's law (Fig. 3) in the concentration range from 11.7 to 58-7 pg ml-1 of folic acid. However, the slope of the straight-line graph was not always reproducible, hence with each experiment a standard was run simultaneously. The colour is stable for several hours at room temperature but fades on heating. The addition of sodium thiosulphate and formaldehyde has been recommended by earlier workers8 in an attempt to reduce the absorbance of blanks of 1,2-naphthoquinone-4-sulphonic acid, sodium salt, reagent but the reduction obtained was not significant.The addition of these reagents was, therefore, excluded in the proposed method. The repeatability of the method was checked by assaying several aliquots from the same solution, while the precisions of the proposed and official4 methods were determined by taking different amounts of the same batch for determination. The results summarised in Table I show that the method is marked by good repeatability and its precision is comparable with that of an official method.' The results from recovery studies confirm the accuracy of the proposed method (Table 11). The addition of the sample at various concentrations was achieved by adding different aliquots of a standard solution of folic acid. The proposed method was applied to the analysis of several samples of pure folic acid, its tablets and injections.The same samples were also assayed by the official4 and micro-22 KANJILAL, MAHA JAN AND RAO : COLORIMETRIC DETERMINATION Analyst, VoZ. 100 Co n cent ra t i o n lpg m I-’ Fig. 3. Relationship between the concentration of folic acid and absorbance of the coloured solution at 485 nm. biological methods. With injections “official method” refers to that of the United States Pharmacopeia as injections of folic acid are not included in the British Pharmacopoeia. An examination of the results summarised in Tables I11 and IV reveals that the proposed procedure gives results comparable with the two commonly used methods if the inherent error in the respective methods is considered. The method was also applied to the analysis of individual dosage forms and the results met the requirements of the U.S.P.for uniformity of drug content. The results are presented in Table V. TABLE I REPEATABILITY AND PRECISION STUDIES OF THE PROPOSED METHOD FOR FOLIC ACID Precision, per cent. Experiment Repeatability, number per cent. 1 99.04 2 99.04 3 98-77 4 98-77 Mean 98.91 s.d. 0-16 I Proposed method 98.77 98.19 99.10 100.20 99-06 0.84 Offici2 method 98.04 98-93 99.10 100.90 99.24 1.20 In the instance of formulations containing iron, the tablet powder was extracted with lactate - phosphate buffer9 instead of sodium hydroxide. Whereas laboratory-prepared injections could be assayed satisfactorily the assay values for two commercial injection samples were repeatedly found to be below the value claimed on the label (Table 111).Both samples gave high blanks and failed to conform to the British Pharmacopoeia test for free amines in folic acid.4 The presence of free amines in these injections was further demonstrated by a thin-layer chromatographic test as described in TABLE I1 RECOVERY EXPERIMENTS FOR FOLK ACID Amount of folic acid/mg - Recovery, Sample Added Recovered per cent. A 26.82 26.65 99.38 B 28.39 28.35 99.86 C 30-15 30-04 99.63 D 33.35 32-85 98.42 E 34-14 34.73 101.70Jamary, 1975 OF FOLIC ACID IN PHARMACEUTICAL PREPARATIONS TABLE I11 FOLIC ACID DETERMINATION BY THE PROPOSED AND OFFICIAL METHODS Purity limits for folic acid (C,,H,,N,O,): U.S.P. 98 to 102 per cent., B.P. Values for pure folic acid samples are expressed in per cent., for folic acid in (minimum) 95 per cent.tablets in mg per tablet and in injections in mg ml-1. Folic acid 1 . . .. .. . . 98.4 98.1 2 .. .. .. .. 98.9 100.4 3 .. .. .. .. 99.5 100.5 Sample Proposed method Official method Tablets of folic acid 1" . . ,. 5.38 5.42 2* . . .. 5.29 5-25 3t I . .. 1.00 - 4t . . .. 1.01 - Injections of folic acid I$ . . .. 14.56 14.47 2; .. .. 14-82 14.85 35 . . .. 12.96 12-39 45 . . .. 12.88 12-03 * Each tablet contains 5 mg of folic acid. t Each tablet contains 1 mg of folk acid, 100 mg of iron(I1) fumarate # Prepared injection: each 1 ml contains 15 mg of folk acid and 0-5 per 5 Commercial sample of injection: composition same as under $. and 10 pg of vitamin B,. cent, of phenol. 23 the British Pharmacopoeia. After development, the chromatogram was sprayed with 1,2-naphthoquinone-4-sulphonic acid, sodium salt, which turned the free amine spots obtained from the sample orange, whereas no orange-coloured spots appeared with folk acid A.S.TABLE IV COMPARISON OF FOLIC ACID CONTENT BY THE PROPOSED AND MICROBIOLOGICAL METHODS Values expressed as in Table 111. Microbiological Sample Proposed method method Tablets 1" . . .. . . . . 5-29 5-27 2" .. .. .. .. 4-73 4-92 Injections 1:. . . . . .. 14.66 14.80 2: . . . . .. 14.82 14.92 .. .. .. 14-84 14.61 :; .. .. .. 12.90 13-23 *, # and 5: See notes under Table 111. Ascorbic acid was found to interfere in the present method, as it was observed that when this vitamin was added to folic acid solution no colour development took place. From the results presented in the tables, it is clear that the proposed method can be applied to the analytical control of folic acid, its tablets, including compound tablets with iron and vitamin BI2, and its injections.TABLE V VERIFICATION OF UNIFORMITY OF CONTENT IN FOLIC ACID TABLETS BY PROPOSED METHOD Tablet 1 2 3 4 5 6 7 8 9 10 Sample A*/mg per tablet 5-16 5.54 5.71 5.65 4-75 5-32 5.53 5.09 5.70 4.52 Sample B*/mg per tablet 4-88 4.84 4.77 4-91 4-84 4.98 4-91 5-21 4.67 5.21 * Both of the commercial tablets are labelled 5 mg per tablet of folic acid. U.S.P. limits for content uniformity are 85 to 115 per cent.24 KANJILAL, MAHAJAN AND RAO The authors thank Dr. L. K. Behl, Managing Director of Indian Drugs and Phanna- ceutieals Ltd., for his encouragement, and the Director, National Chemical Laboratory, Poona, for the supply of a culture of Stre$tococcus fuecalis that was used in microbiological determinations. The authors also thank Mr. S. Raghuveer for help in microbiological deter- minations. 1. 2. 3. 4. 5. 6. 7. 8. 9. References “State Pharmacopoeia of U.S.S.R.,” Tenth Edition, Ministry of Health, Moscow, 1966, p. 49. Allfrey, V., Teply, L. J., Greffen, C., and King, C. G., J . Bid. Chem., 1949, 178, 465. “Official Methods of Analysis of the Association of Official Analytical Chemists.” Eleventh Edition, Association of Official Analytical Chemists, Washington, D.C., 1970, p. 786. “British Pharmacopoeia 1973,” H.M. Stationery Office, London, 1973, p. 210. Freed, M., Editor, “Methods of Vitamin Assay,” Third Edition, Interscience Publishers, New York, 1966, p. 227. “The United States Pharmacopeia,” XVIIIth Revision, Mack Publishing Co., Easton, Pa., 1970, p. 270. Snell, F. D., and Snell, C. T., “Colorimetric Methods of Analysis,” Third Edition, Volume 4, D. Van Nostrand Company Inc., Princeton, New Jersey, U S A . , 1954, p. 32. Panalks, T., and Campbell, J. A., Analyt. Chem., 1961, 33, 1038. Pelletier, O., and Campbell, J. A., J . Pharm. Sci., 1961, 50, 208. Received Mavch 6th, 1974 Accepted June 18tle, 1974

ISSN:0003-2654    

DOI:10.1039/AN9750000019

出版商:RSC    

年代:1975

数据来源: RSC