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Abstract
A very simple nonaqueous reverse phase HPLC was developed for determining retinol acetate, retinol palmitate, cholecalciferol, alpha-tocopherol acetate, alphacalcidol, and phylloquinone in capsules without the need for saponification. A reverse phase (Lichrospher C8, 4.6 mm, id.) column was used with a mobile phase of acetonitrile–methanol (95:5, v/v) and flow rate of 1 mL/min. Sample treatment consisted only of the dilution of the capsule contents with n-hexane and methanol. The range of the assay method has been set at 2.5–150% of finished product label claim. The limit of detection of each vitamin was between 0.259 μg/mL (K1) and 2.379 μg/mL (alphacalcidol). The minimum average amount recovered was 98.1% (D3). The precision (relative standard deviation of label claim among six sample preparation as repeatability was not more than 0.82% (A palmitate) and, as intermediate (N = 24), was not more than 1.76% (E acetate). The Standard Preparation (at −15°C) and Sample Preparation (at 4°C) were stable for up to 48 and 24 h, respectively. This method is suitable for routine quantification in industrial quality assurance laboratories. The principal aspects of method validation are examined and discussed, and the validation studies indicate that the method is specific, accurate, precise, and robust over the concentration range investigated.
ACKNOWLEDGMENT
The work was supported by grant no. KBN 32/1271/T09/1999-2000.