Anal. Chem. 1981, 53, 607-609
607
Liquid Chromatographic Determination of Folic Acid in
Multivitamin-Mineral Preparations I. J. Holcomb* and S. A. Fusarl Parke-Davis, Division of Warner-Lambert Company, 201 Tabor Road, Morris Plains, New Jersey 07950
Preparation of the Mobile Phase. To 13 mL of 10% aqueous tetrabutylammonium hydroxide is added, 840 mL of water, 50 mg of ethylenediaminetetraacetic acid disodium salt (EDTA), and 160 mL of acetonitrile. The solution is adjusted by the addition of potassium dihydrogen phosphate to pH 7.6 ± 0.1 by use of a pH meter. The mobile phase was filtered through a 5-jtm Millipore filter with aid of vacuum prior to use. The amount of acetonitrile is adjusted in the mobile phase to give a retention volume for folic acid of 10-16 mL and the amount of acetonitrile is also adjusted in the sample preparation step. Preparation of Lactate-Phosphoric Acid Extractant Solution. Transfer 75 g of calcium lactate, trihydrate, to a 500-mL volumetric flask and add 300 mL of water. Mix. Add 67 mL of concentrated phosphoric acid and mix. Dilute to volume with water. The resulting solution is used in the working standard preparation and the sample preparation. Standard Preparation. A stock standard solution USP Folic Acid Reference Standard (8% water) is prepared by transferring approximately 27 mg, accurately weighed, to a 100-mL low actinic flask and dissolving in 80 mL of 0.1 N sodium hydroxide, to obtain a solution equivalent to about 0.5 mg of folic acid/mL. Dilute to volume with 0.1 N sodium hydroxide. The resulting solution is stable for 3 days with refrigeration when not in use. The working standard solution is prepared by transferring 20 mL of the lactate-phosphoric acid extractant to a 250-mL low actinic volumetric flask, adding 80 mL of water and 5 mL of thioglycerol (10% aqueous solution), and adjusting the pH to 7.0 ±0.1 with 1 N sodium hydroxide using pH paper (Tridicator by Fil-Chem). Transfer exactly 5.0 mL of the stock standard folic acid solution to the flask, add 40 mL of acetonitrile, and dilute to volume with water. Mix. Filter through GF/C glass microfiber paper (Whatman), discarding the first 10 mL, and collect about 8 mL in a glass stoppered flask. The filtrate can be used during a regular work period (8-16 h) for injection into the liquid
Folic acid in multivitamin-mineral preparations is determined by liquid chromatography following pretreatment of the sample with thioglycerol, a calcium lactate-phosphoric acid mixture, and heat. The resulting mixture, after pH adjustment and addition of acetonitrile, is chromatographed on ¿tBondapak C18 using paired ion chromatography. The method of sample pretreatment gives a recovery of 99.2% from a multivitamin-mineral granulation. Statistical data are presented to show an evaluation of the liquid chromatographic system used. Several commercial multivitamin-mineral preparations are assayed and data presented.
Chemical methods for the determination of folic acid in pharmaceutical preparations include the use of spectrophotometry, fluorometry, photometry, and polarography after appropriate sample pretreatment. These methods and the microbiological method of assay have been reviewed by Hashmi (1). The microbiological method is the official method of the Association of Analytical Chemists (2). The photometric method is the USP XIX (3) official procedure and involves the permanganate oxidation of folic acid to p-aminobenzoic acid which is diazotized, coupled with jV-(l-naphthyl)ethylenediamine for photometric determination. Commercial folic acid contains impurities which may cleave to give interference in the official photometric method of assay (4). The use of a reductive cleavage of folic acid results in the formation of p-aminobenzoylglutamic acid which can be reacted with l,2-naphthoquinone-4-sulfonic acid (5) or ninhydrin (6) to form a colored product. The use of a chromatographic separation prior to color development (7) adds considerable selectivity to the photometric methods but also adds more manipulative steps and time. Folic acid has also been determined fluorometrically after thin-layer chromatography (8). Liquid chromatographic methods of assay of folic acid have been developed (4, 9-11) but none have been applied to multivitamin preparations with minerals. This paper presents an application of high-pressure liquid chromatography to the determination of folic acid in several commercial vitamin preparations containing 1 mg or less of folic acid in a unit dose.
chromatograph.
Sample Preparation. The method of sample preparation depends on the type of coating (film coat or sugar coated) and the amount of calcium carbonate present in the unit dose. The film coat is removed from a film-coated tablet by washing with a mixture of dichloromethane and methanol (4:1). The resulting core tablets are allowed to air-dry and an average tablet weight (20 tablets) is determined. The tablets are then ground with a mortar and pestle. A sample weight is taken equivalent to 1 mg of folic acid. A sugar-coated tablet is directly ground with a mortar and pestle after determining the average tablet weight (20 tablets). A sample weight is taken equivalent to 1 mg of folic acid. For tablets that contain less than 350 mg of calcium per unit dose, add 1.7 g of calcium carbonate for 1 mg of folic acid. The sample as prepared is transferred (and calcium carbonate added, if necessary) to a 250-mL low-actinic flask. Thioglycerol, 5 mL of a 10% aqueous solution, is added. The lactate-phosphoric acid extractant is then added along with a magnetic stirring bar, and the mixture is heated at 50 °C with stirring for 30 min. The flask is removed from the heat, and 80 mL of water is added. The resulting mixture is adjusted to pH 7 ±0.1 with 1 N sodium hydroxide by using pH paper. The stirring bar is removed and 40 mL of acetonitrile added. Dilute to volume with water and mix. Filter through a GF/C glass microfiber paper (Whatman), discarding the first 10 mL, and collect about 8 mL in a glassstoppered flask. Procedure and Calculations. The liquid chromatograph was setup and allowed to come to equilibrium with the mobile phase
EXPERIMENTAL SECTION Apparatus. A constant flow pump (Model 6000, Waters
Associates) fitted with an injector (Model U6K, Waters Associates), a detector (Model 1203, Laboratory Data Control) set at 280 nm, and a 10-mV recorder (Linear, Model 385) was used. The column used for development was a gBondapak C18 (Waters Associates) (4.0 mm i.d. X 30 cm, 10 µ particle size). Ten microliters of the standard and sample preparation is injected. Materials. USP Folic Acid Reference Standard, on which the moisture content had been determined by use of the Karl Fischer method, was used as a reference standard material for the assay. Calcium lactate, trihydrate, was USP grade. Tetrabutylammonium hydroxide was used as a 10% aqueous solution (Eastman). Thioglycerol (3-mercapto-l,2-propanediol, Evans Chematics, Inc.) was used as a 10% aqueous solution. Other chemicals were reagent grade substances, The acetonitrile used was glass distilled (Burdick & Jackson Laboratories, Inc.). 0003-2700/81/0353-0607$01.25/0
©
1981 American Chemical Society
608
·
ANALYTICAL CHEMISTRY, VOL. 53, NO. 4, APRIL 1981
Table I. Comparison of Liquid Chromatographic Assay (LCA) Results with Microbiological Assay (MA) Results for Folic Acid sample 1
2 3
4 5
6 7
LCA/MA
X
100 sample
96.2 99.8 93.9 100.5 98.4 104.8 99.2
LCA/MA
X
102.2 104.6 102.5 101.6 98.0 100.7 99.5
8 9
10 11
12 13 14 av
101.2%
flowing at 1 mL/min. Detector sensitivity was set at 0.008. The standard preparation is injected to check the system suitability. A series of five injections should give a standard deviation of no greater than 2% for folic acid and a retention time in the range of 10-16 min, and then sample preparations are injected. The peak height method of quantitation was used. The calibration plot is linear and passes through zero so that the single-point standard method is used for calculations as follows:
folic acid, mg/cap
=
—
hT
X Cr X
250 no.
of dosage units
Figure 1. Chromatogram as obtained for the standard preparation of folic acid in the liquid chromatographic assay (detector sensitivity, 0.008; flow rate, 1 mL/min; FA = folic acid).
where hu and hT represent the peak height of the analyte and the reference standard, respectively, and Cr is the concentration of the reference standard.
RESULTS AND DISCUSSION The current methods for determination of small amounts of folic acid in multivitamin preparations with minerals either lack specificity as with direct photometric chemical methods or require a considerable time before assay results can be obtained as with microbiological methods. The method described is reasonably specific and results can be obtained in a fairly short length of time. A modification of the same mobile phase has been reported (10) to be useful for separation of folic acid derivatives. In our studies, the method has been compared with the microbiological method with excellent results (Table I) showing that the specificity is quite good for folic acid. The time requried for the total assay is reasonably short; one person can complete three samples in duplicate in 4-5 h of working time. Sample Pretreatment. In development of the method, the sample pretreatment was obviously the most critical step. Several liquid chromatographic systems are available for the separation of folic acid as a single peak (9-11). The sample pretreatment is based on the use of a lactate-phosphate mixture (12) (a 1:1 mixture of 3% calcium lactate and 8% trisodium phosphate) was not adequate to obtain good recoveries from real samples although good recovery of a known amount of folic acid was obtained by using the colorimetric method of assay (3). For a more homogenous mixture after contact with a extracting solvent, a higher concentration of phosphoric acid was selected to be used for the initial dispersion of the sample and, to protect the folic acid, a considerable excess of calcium lactate was maintained. In addition, the use of thioglycerol in the sample preparation results in better reproducibility of the method. The type of multivitamin-mineral preparation used in the development of the method contained 11 vitamins and 6 minerals. After the dispersion of the sample in the lactate-phosphoric acid extractant, the sample mixture is diluted and adjusted to pH 7 ± 0.1 with 1 N sodium hydroxide. The adjustment of the pH should be done carefully, mixing the dispersion between additions of sodium hydroxide. If the pH becomes too high, low recoveries may result because of adsorption of folic acid on mineral hydroxides. An amount of acetonitrile is then
f ^
0
2
4
6
Time
8
10
12
14
(Minutes)
Figure 2. Chromatogram as obtained for the sample preparation In the liquid chromatographic assay of a multivitamln-mlneral preparation (detector sensitivity, 0.008; flow rate, 1 mL/min; FA = folic acid).
added to bring the concentration in the final dilution equivalent to that used in the mobile phase. After dilution to volume with water, the mixture is filtered and the filtrate injected. An example of the chromatograms obtained is présented in Figures 1 and 2. The solution from the sample is stable for at least 5 h if kept in a dark place. A standard solution is stable for 24 h at room temperature in a dark place. In samples that do not contain calcium carbonate, the addition of calcium carbonate to the ground tablet is recommended to improve the recovery as will be discussed under recovery studies. The presence of calcium carbonate has no effect on the chromatogram of folic acid. The heating of the folic acid at 50 °C for 30 min in the 2 M phosphoric acid shows no change in the chromatogram of a standard and, therefore, is not necessary in the standard preparation step.
ANALYTICAL CHEMISTRY, VOL. 53, NO. 4, APRIL 1981
Recovery of Folic Acid from a Multivitamin-Mineral Granulation” Using the Liquid Chromatographic Assay Method %
recovered 97.1 100.0 98.6 96.1 99.2
1
2 3
4 5
%
sample 7
8
9
10
“ The granulation contains ten vitamins and seven minerals with tablet excipients. The folic acid represents approximately 0.05% of the total sample.
%of
no. 1
2 3
4 5
6 7
8 9
10
label claim 85 90 95 97 99 101 103
105 110 115
wt of actual folic acid wt of folic acid found
0.351 0.416 0.444 0.469 0.485 0.496 0.512 0.517 0.549 0.586
0.352 0.415 0.448 0.467 0.487 0.492 0.518 0.523 0.549 0.598
mm
sample
1
114.2 113.0 113.0 114.8 115.0
6
2 3 4 5
mm
115.0 114.1 114.0 113.2 av 114.0 RSD 0¡71
7
8 9
Table V. Liquid Chromatographic Assay of Folic Acid in Commercial Multivitamin-Mineral Dosage Forms
folic acid label claim,
Table III. Accuracy Study for the Liquid Chromatographic Assay of Folic Acid
sample
sample
98.5 96.1 102.0 102.4 101.7 99.17
6
peak height,
peak height,
recovered
av
target
609
Table IV. Stability Study of the Chromatographic System for Assay of Folic Acid (ElapsedTime: ~4 m)
Table II.
sample
·
sample
components
mg/unit
1
10 vitamins,
0.4
minerals 10 vitamins, 6 minerals 10 vitamins, 4 minerals 10 vitamins, 4 minerals 10 vitamins, 4 minerals 10 vitamins, 7 minerals 10 vitamins, 7 minerals 10 vitamins, 7 minerals 7 vitamins, 6 minerals 10 vitamins, 5 minerals 7
%
recovery
2
100.3 98.8 100.9 99.6 100.4 99.2 101.2 101.2 100.0 102.0 av 100.5 ±0.5 bias RSD ±0.86
3
Recovery Study. The recovery of folic acid from a multivitamin-mineral mixture was examined over a period of several days. The amount of folic acid added to over 2 g of sample is 1 mg. The recovery data are presented in Table II. The addition of calcium carbonate to the multivitaminmineral mixture was found to be necessary to obtain satisfactory recovery levels; that is, recoveries greater than 98% of theory. In studies where the calcium carbonate is left out, recovery is approximately 90% of theory. In commercial preparations, the assay should be run with and without the addition of calcium carbonate to the sample to determine if the addition is necessary. Accuracy Study. An accuracy study was accomplished to evaluate the system for determination of folic acid. In a series of 10 samples containing folic acid in an amount from 0.85 to 1.15 mg in a final volume of 250 mL, a bias of +0.5 obtained (Table III). Linearity Study. A plot of peak height vs. concentration
was
in the range from 0 to 1.25 mg/250 mL is linear. Stability of the Chromatographic System. A series of nine injections of a standard preparation was made over a 4-h period to determine if the system was stable. For this set of injections, a percent relative standard deviation of ±0.71 was obtained (Table IV). A /wBondapak column used continuously will give satisfactory separations for about 4 months. An adjustment in the amount of acetonitrile concentration may be necessary to obtain a retention time of 10-16 min for folic acid. The columns, of course, will last longer with proper flushing with water and methanol at the end of a series of samples. Commercial Multivitamin-Mineral Preparation. The method has been applied to several commerical preparations. The assay results are presented in Table V. No apparent problems were encountered.
folic acid found,” mg/unit (% label claim) 0.495 (123.8)
4 5
6 7
8 9
10
1.0
0.989 (98.9)
1.0
1.267 (126.7)
0.8
0.934 (116.8)
0.8
1.094 (136.4)
0.4
0.467 (116.8)
0.4
0.491 (122.8)
0.4
0.393 (98.3)
0.4
0.474 (118.5)
1.0
1.06(106.0)
0 Assay values represent the average of two determinations per sample.
ACKNOWLEDGMENT The untiring technical assistance of Ida Semkus is gratefully acknowledged. The authors are indebted to B. Hall and Brian Krause for the microbiological assays.
LITERATURE CITED (1) Hashmi, M. “Assay of Vitamins in Pharmaceutical Preparations”; Wiley: New York, 1973; p 213. (2) "Official Methods of Analysis”, 11th ed.; Assocatlon of Official Agricultural Chemists: Washington, DC, 1970; Section 5, 39.093-39.100. (3) "The United States Pharmacopeia”, 19th rev., Mack Printing Co.: Easton, PA, 1975; p 624. (4) Reif, Van D.; Reamer, J. T.; Grady, Lee T. J. Pharm. Sci. 1977, 66, 1112. (5) Kanjllal, G.; Mahajan, S. N.; Ramana Rao, G. Analyst (London) 1975, 100, 19. (6) Ramana Rao, G.; Mahajan, S. N.; Kanjllal, G.; Mlhan, K. R. J. Assoc.
Off. Anal. Chem. 1977, 60, 531.
(7) Hashmi, M. "Assay of Vitamins In Pharmaceutical Preparations"; Wiley: New York, 1973; p 221. (8) Popova, Y.; Kovacheva, E. Nauchn. Tr. Vlssh Inst. Kranit. Vkusova Prom,, Rovdlv 1969, 16 (Pt. 2), 323-329. Chem. Abstr. 1972, 77,
168685r.
(9) Williams, R. C.; Baker, D. R.; 11, 618. (10) Branfman, A. R.; McComlsh, (11) Chapman, S. K.; Greene, B. 145, 302. (12) Pelletier, O.; Campbell, J. A.
Schmlt, J. A. J. Chromatog. Scl. 1973, M. J. Chromatogr. 1978, 151, 87. C.; Strelffl, R. R. J. Chromatogr. 1978,
J. Pharm. Scl. 1981, SO, 208.
Received for review October 30,1979. Resubmitted November 24,1980. Accepted November 24, 1980. A preliminary description of this work was presented at the Anachem Symposium, Oct 11,1978.
