Analysis of Various Vitamins in Multivitamin Tablets

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1 High Performance Liquid Chromatography Analysis of Various Vitamins in Multivitamin Tablets Effective August 24, 2007, the US Food and Drug Administration (FDA) issued a regulation (21 CFR Part 111) that established the minimum current Good Manufacturing Practices (cgmps) required for activities in manufacturing, packaging, labeling and holding Dietary Supplements to ensure the identity, purity, quality, strength and composition of these products. Depending on the business size determined by the number of employees, FDA set 3 different timelines by which the companies must comply with the new requirements. For businesses with 500 or more employees, the compliance date was June 25, For businesses with 20 to 499 employees, the compliance date was June 25, For businesses with 19 or fewer employees, the compliance date is June 25, The new regulation means more testing needs to be performed by companies that manufacture Dietary Supplements. The United States Pharmacopeia (USP) is an official authority that sets standards for all prescription and over the counter drugs, dietary supplements and food ingredients that are intended for sale in the United States. USP develops standards for the quality, purity, strength, and consistency of these products. In its annual book, the United States Pharmacopeia National Formulary (USP NF), USP publishes pharmacopeial standards along with validated analytical methods that have been the choice of many commercial laboratories. The pharmacopeial forum (USP PF) discusses proposed method revisions. Table 1 shows examples of USP techniques for the analysis of some dietary supplements.

2 Analysis of Vitamin B12 in Multivitamin Tablets Here we used an LC-UV to conduct analysis of Cyanocobalamin in multivitamin tablets from a popular brand. Sample preparation and analysis followed the USP32-NF27 procedure. 30 tablets corresponding to approximately 48 g were finely powdered. 6.1 g of the powder was transferred to a flask and 100 ml of water was added volumetrically. The mixture was carefully extracted for 2 minutes. 10 ml of the extract was filtered and the filtrate used for analysis. Analysis Results Detector Used SPD-20AV (UV-Vis) SPD-M20A (PDA) Concentration of Cyanocobalamin in HPLC sample mg/l mg/l Calculated amount of Cyanocobalamin in one tablet mg mg Labeled amount of Cyanocobalamin in one tablet mg Analytical Conditions Instrument : Prominence UFLC system + SPD-20A (UV-Vis) and SPD-M20A (PDA) detectors Column : Shim-pack VP-ODS (150 mm x 4.6 mm i.d., 5 μm particle size) Mobile Phase : Water/Methanol = 75/25 (v/v) Flow Rate : 0.5 ml/min Column Temperature : 40 C Detection : UV; 550nm Sample Volume : 50 μl

3 Analysis of Vitamin D in Multivitamin Tablets Here we used an LC-UV to conduct analysis of Cholecalciferol (Vitamin D3) and Ergocalciferol (Vitamin D2) in multivitamin tablets from a popular brand. Sample preparation and analysis followed the USP32-NF27 procedure. common procedure for Vitamins D, A and E 20 Tablets were finely powdered and a weighed portion of the powder, equivalent to 5 tablets, was transferred to a container with a POLYTEF-lined screw cap. 10 ml of dimethyl sulfoxide and 15 ml of n-hexane were added, and the mixture was shaken for 45 minutes on a wrist-action shaker in a water bath maintained at 60 C followed by centrifugation for 10 minutes at 3000 rpm. The hexane layer was transferred to a 100 ml volumetric flask. 15 ml of n- hexane was added to the dimethyl sulfoxide layer, shaken thoroughly for 5 minutes, and the hexane layer was transferred to the 100 ml volumetric flask. This extraction was repeated with 3 additional 15 ml portions of n-hexane. The extracts were brought to 100 ml volume with n-hexane. 10 ml of the resulting solution was diluted quantitatively with n-hexane to a final concentration of about 15 µg/ml of vitamin A. System Suitability Test USP acceptance criteria: %RSD must be no greater than 3% (with n 5, peak height) must be greater than 10% n Cholecalciferol Ergocalciferol R.T. Height R.T. Height , , , , , , , , , ,551 Average , ,496 %RSD % % % % R.T. Area Cholecalciferol Height R.T. Area Ergocalciferol Height Precursor of Vitamin D Vitamin D % %

4 Analysis of Vitamin D in Multivitamin Tablets Analysis Results Vitamin D (as Cholecalciferol) is calculated using the following formula (μg): Hu 1.09 C D Hs, 5 where 1.09 is a correction factor, C = 2.0 μg/ml, D = 50 ml, Hu is a peak height in the assay, and Hs is a peak height of the standard (Cholecalciferol). Weight of 1 tablet Calculated amount of Vitamin D in 1 tablet Labeled amount of Vitamin D in 1 tablet Analysis Time : g (based on the average of 20 tablets) : 428 IU (1 IU = μg) : 400 IU : 60 minutes Analytical Conditions Instrument : LC-2010 CHT Column : Luna NH 2 100A (150 mm x 4.6 mm i.d., 3 μm particle size) Mobile Phase : n-hexane/isopropyl Alcohol = 99/1 (v/v) Flow Rate : 1.0 ml/min Column Temperature : 40 C Detection : UV; 265nm Sample Volume : 100 μl

5 Analysis of Vitamin A in Multivitamin Tablets Here we used an LC-UV to conduct analysis of Retinyl Acetate and Retinyl Palmitate (Vitamin A) in multivitamin tablets from a popular brand. Sample preparation and analysis followed the USP32-NF27 procedure. common procedure for Vitamins D, A and E (see Vitamin D section above) n 1 Retinyl Palmitate System Suitability Test USP acceptance criteria: %RSD must be no greater than 3% must be greater than 10% Retinyl Acetate Standard R.T. Area Standard R.T. Area mg/l mg/l Average 10.8 % %RSD % % % % Analysis Results Concentration of Vitamin A in the LC sample : mg/l Calculated amount of Vitamin A per tablet : 2479 IU Labeled amount of Vitamin A per tablet : 1700 IU 5000 IU (66% as Beta-Carotene) Recovery : 94% performed by spiking with vitamin A standard Analytical Conditions Instrument : Prominence UFLC system + SPD-M20A (PDA) detector Column : Luna NH 2 100A (150 mm x 4.6 mm i.d., 3 μm particle size) Mobile Phase : n-hexane Flow Rate : 0.8 ml/min Column Temperature : 30 C Detection : UV; 325nm Reference Data from RF-10Axl; Excitation 340 nm, Emission 460 nm Sample Volume : 40 μl

6 Analysis of Vitamin E in Multivitamin Tablets Here we used an LC-UV to conduct analysis of Alpha Tocopheryl Acetate (Vitamin E) in multivitamin tablets from a popular brand. Sample preparation and analysis followed the USP32-NF27 procedure. common procedure for Vitamins D, A and E (see Vitamin D section above) Parameter USB Acceptance Criteria System Suitability Test Method Value %RSD 3 % % (RT, D) % (RT, E) % (P. Area D) % (P. Area E) > 12 % 17.3 % Relative R.T Tailing Factor (D) (E) n 1 Cholecalciferol (Vitamin D) α Tocopheryl Acetate (Vitamin E) Standard R.T. Area Standard R.T. Area mg/ml mg/ml %RSD % % % % Analysis Results and Analytical Conditions Concentration of Vitamin E in the LC sample : mg/ml Calculated amount of Vitamin E per tablet : 31 IU Labeled amount of Vitamin E per tablet : 75 IU Instrument : Prominence UFLC system + SPD-M20A (PDA) detector Column : Luna NH 2 100A (150 mm x 4.6 mm i.d., 3 μm particle size) Mobile Phase : n-hexane Flow Rate : 0.8 ml/min Column Temperature : 30 C Detection : UV; 325nm Reference Data from RF-10Axl; Excitation 340 nm, Emission 460 nm Sample Volume : 40 μl Shimadzu Scientific Instruments 7102 Riverwood Drive, Columbia, MD Phone: , Fax:

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