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Shimadzu Analysis Vitamins 2007
Shimadzu Analysis Vitamins 2007
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
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 %
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
: : : :
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
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%
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
Parameter
%RSD
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 %
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
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