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, 2008. For businesses with 20 to 499 employees, the compliance date was June 25, 2009. For businesses with 19 or fewer employees, the compliance date is June 25, 2010. 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 overthecounter 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 (USPNF), USP publishes pharmacopeial standards along with validated analytical methods that have been the choice of many commercial laboratories. The pharmacopeial forum (USPPF) discusses proposed method revisions. Table 1 shows examples of USP techniques for the analysis of some dietary supplements.

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. [Sample Preparation] 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 Concentration of Cyanocobalamin in HPLC sample Calculated amount of Cyanocobalamin in one tablet Labeled amount of Cyanocobalamin in one tablet

SPD-20AV (UV-Vis) SPD-M20A (PDA) 1.111 mg/L 1.110 mg/L 0.0269 mg 0.0268 mg 0.025 mg

Analytical Conditions Instrument Column Mobile Phase Flow Rate Column Temperature Detection Sample Volume : : : : : : : Prominence UFLC system + SPD-20A (UV-Vis) and SPD-M20A (PDA) detectors Shim-pack VP-ODS (150 mm x 4.6 mm i.d., 5 m particle size) Water/Methanol = 75/25 (v/v) 0.5 mL/min 40 C UV; 550nm 50 L

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. [Sample Preparation]

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 nhexane 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 g/mL of vitamin A. System Suitability Test

USP acceptance criteria: %RSD must be no greater than 3% (with n 5, peak height) Resolution must be greater than 10% n
1 2 3 4 5 Average %RSD Cholecalciferol R.T. Peak Height 9.788 51,431 9.796 50,818 9.805 50,905 9.814 51,453 9.823 51,281 9.805 51,178 0.142 % 0.582 % Cholecalciferol R.T. Precursor of Vitamin D Vitamin D 6.341 9.707 Peak Area 21508 567552 Peak Height 2557 48980 Resolution -13.0 % R.T. 6.427 9.801 Ergocalciferol R.T. Peak Height 9.85 46,865 9.856 46,710 9.86 46,439 9.867 45,914 9.875 46,551 9.862 46,496 0.098 % 0.780 % Ergocalciferol Peak Area 20786 525931 Peak Height 2511 43896 Resolution -12.7 %

Analysis of Vitamin D in Multivitamin Tablets

Analysis Results

Vitamin D (as Cholecalciferol) is calculated using the following formula (g):

1.09 C D 5
where 1.09 is a correction factor,

Hu Hs

C = 2.0 g/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

: : : :

1.585 g (based on the average of 20 tablets) 428 IU (1 IU = 0.025 g)

400 IU

60 minutes

Analytical Conditions Instrument Column Mobile Phase Flow Rate Column Temperature Detection Sample Volume

: : : : : : :

LC-2010 CHT Luna NH2 100A (150 mm x 4.6 mm i.d., 3 m particle size) n-Hexane/Isopropyl Alcohol = 99/1 (v/v)
1.0 mL/min 40 C UV; 265nm 100 L

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. [Sample Preparation]

common procedure for Vitamins D, A and E (see Vitamin D section above)

System Suitability Test

USP acceptance criteria: %RSD must be no greater than 3% Resolution must be greater than 10% n
1 2 3 4 5 6 Average %RSD 0.056 % 0.203 % Analysis Results Concentration of Vitamin A in the LC sample Calculated amount of Vitamin A per tablet Labeled amount of Vitamin A per tablet Recovery 0.066 % 0.163 % 10.664 mg/L Retinyl Palmitate Standard R.T. 4.514 4.507 4.508 4.509 4.509 4.507 Peak Area 2632751 2630743 2621867 2625191 2620967 2620151 9.270 mg/L Standard Retinyl Acetate R.T. 7.548 7.541 7.544 7.545 7.544 7.533 Peak Area 3492673 3490868 3481802 3481740 3478654 3482120 Resolution 10.753 10.773 10.779 10.791 10.773 10.795 10.8 %

3.311 mg/L

2479 IU

: :

1700 IU 94%

5000 IU (66% as Beta-Carotene) performed by spiking with vitamin A standard

Analytical Conditions Instrument Column Mobile Phase Flow Rate Column Temperature Detection Sample Volume : : : : : : : Prominence UFLC system + SPD-M20A (PDA) detector Luna NH2 100A (150 mm x 4.6 mm i.d., 3 m particle size) n-Hexane 0.8 mL/min 30 C UV; 325nm 40 L

Reference Data from RF-10Axl; Excitation 340 nm, Emission 460 nm

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. [Sample Preparation]

common procedure for Vitamins D, A and E (see Vitamin D section above)

System Suitability Test Method Value

Parameter

%RSD

USB Acceptance Criteria 3%

Resolution Relative R.T. Tailing Factor

> 12 % 0.5 0.8 1.2

0.226 % (RT, D) 0.272 % (RT, E) 0.111 % (P. Area D) 0.068 % (P. Area E) 17.3 % 0.5 0.998 (D) 1.152 (E)

n
1 2 3 4 5 6 %RSD

Cholecalciferol (Vitamin D) Standard R.T. 18.325 18.321 0.0074 mg/mL 18.31 18.359 18.399 18.406 0.226 % Peak Area 706082 705621 705352 705579 705235 703800 0.111 %

Tocopheryl Acetate (Vitamin E) Standard R.T. 37.114 37.12 1.013 mg/mL 37.099 37.225 37.297 37.332 0.272 % Peak Area 2592333 2592311 2591430 2594490 2593342 2589309 0.068 %

Resolution 17.237 17.237 17.261 17.303 17.35 17.382

Analysis Results and Analytical Conditions Concentration of Vitamin E in the LC sample Calculated amount of Vitamin E per tablet Labeled amount of Vitamin E per tablet Instrument Column Mobile Phase Flow Rate Column Temperature Detection Sample Volume : : : : : : : : : : 2.000 mg/mL 31 IU 75 IU

Prominence UFLC system + SPD-M20A (PDA) detector Luna NH2 100A (150 mm x 4.6 mm i.d., 3 m particle size) n-Hexane 0.8 mL/min 30 C UV; 325nm 40 L

Reference Data from RF-10Axl; Excitation 340 nm, Emission 460 nm

Shimadzu Scientific Instruments 7102 Riverwood Drive, Columbia, MD 21046 Phone: 800-477-1227, Fax: 410-381-1222 www.ssi.shimadzu.com webmaster@shimadzu.com