Determination of vitamin D3 in cod-liver oil (Lebertran) by high-pressure liquid chromatography

Summary An HPLC separation of vitamin D₃ in cod-liver oil (Lebertran) on a Partisil-10-pxs (Whatman) column with a mobile phase of chloroform-n-hexaneglacial acetic acid (70301) and detection at 268 nm is described. The vitamin D₃ peak in the sample solution was positively identified by measuring absorbance ratios at different wavelengths and recording a peak spectrum with a LC 55-S digital scanner between 250–350 nm. Analytical results of analysis of 11 samples are compared with those obtained by a chemical method. The coefficient of variation of the HPLC method was found to be ± 2.4%.

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Bestimmung von Vitamin D₃ in Lebertran durch Hochdruck-Flüssigkeits-Chromatographie

Zusammenfassung Ein Verfahren zur HPLC-Bestimmung von Vitamin D₃ in Lebertran mit Hilfe einer Partisil-10 pxs-Säule (Whatman) mit der mobilen Phase Chloroform-n-Hexan-Eisessig (70301) und UV-Detektion bei 268 nm wird beschrieben. Die Identität des Vitamin D₃-Peaks wurde zusätzlich durch eine Peakanalyse bei verschiedenen Wellenlängen sowie durch Aufnahme des Peakspektrums zwischen 250–350 nm mit einem LC-55-S Digital-Scanner sichergestellt. Die Ergebnisse von 11 Lebertranproben wurden den mit einer chemischen Methode erzielten Werten gegenübergestellt. Der Variationskoeffizient der HPLC-Methode beträgt ± 2,4%.

The author thanks Mrs. Petra Grötsch for the very efficient assistance in the experimental work. He is further indebted to Mr. R. Jöster, Perkin-Elmer Corporation, Offenbach (GFR), for recording the peak spectra with a digital scanner.     

Biological generation of tritiated vitamin D3 metabolites and their purification by high-performance liquid chromatography

Summary A simple, reproducible method for the biological synthesis of tritiated 24,25-dihydroxycholecalciferol, 25,26-dihydroxycholecalciferol and 1,25-dihydroxycholecalciferol is described. Kidney homogenates from both vitamin D deficient and replete chicks were usedin vitro to generate these dihydroxylated metabolites using 25 (23,24-³H) hydroxycholecalciferol as the substrate. Tritiated products were purified by Sephadex LH 20 chromatography followed by high-performance liquid chromatography; the identity of each metabolite was established by chromatography with authentic crystalline preparations.Presented at the Symposium organised by the Chromatography Discussion Group, held at Hatfield Lodge on 29 November 1979.     

Determination of vitamin B12 in multivitamin tablets by multimode high-performance liquid chromatography

Quantitative determination of vitamin B₁₂ in B-complex tablets was performed by using multimode high-performance liquid chromatography. The multivitamin tablets (B₁, B₆ and B₁₂) were sonicated for 30 min in methanol–water (50:50, v/v) and diluted to appropriated volume with the same solvent. The resulting solution was filtered and the filtrate was analysed on a phenylpropanolamine bonded silica column (15 cm×4.6 mm I.D., 5 μm). The optimized mobile phase was 30 mM phosphate buffer (pH 3.00) containing 6% (v/v) acetonitrile at a flow-rate of 1 ml min⁻¹ and the detection was measured at 361 nm. The calibration graph prepared using standards was linear from 0.05 to 0.25 μg. The determination limit was 25 ng, the relative standard deviation was 0.47% and recovery from tablet solution was 100%. An analysis was completed in 5 min. The new method is simple, rapid and precise.     

Determination of thiamine by high-performance liquid chromatography

High-performance liquid chromatographic methods for the determination of thiamine (vitamin B1) in foodstuffs or biological tissues and fluids are outlined and discussed. The methods are often similar and interchangeable, sample extraction and clean up procedures being the major difference. Most of the methods use either ultraviolet or fluorescence detection. Fluorescence detection requires either precolumn or postcolumn oxidation of thiamine to thiochrome. A number of methods are recommended and problems with standardization are emphasized.     

Determination of vitamin E acid succinate in biodegradable microspheres by reversed-phase high-performance liquid chromatography

A simple, rapid, and reproducible reversed-phase high-performance liquid chromatographic (HPLC) method is applied to the routine assay of vitamin E acid succinate in biodegradable microspheres. Vitamin E acid-succinate-containing poly-(D,L-lactic-co-glycolic acid) microspheres are prepared by the solvent evaporation method. The starting drug-polymer ratio is 1:10 (w/w) and the total amount of drug and polymer processed is always 440 mg. The content of vitamin E acid succinate in the microspheres is evaluated by HPLC. Chromatography is carried out isocratically at 25 degrees C +/- 0.5 degrees C on an Extrasil ODS-2 column with a mobile phase composed of methanol-water (97:3, v/v) (pH 5.6) at a flow rate of 2 mL/min and UV detection at 284 nm. Parameters such as linearity, limits of quantitation (LOQ) and detection (LOD), precision, accuracy, recovery, specificity, and ruggedness are studied as reported in the International Conference on Harmonization guidelines. The stability of vitamin E acid succinate is also studied with satisfactory results after 48 h at 25 degrees C. The method is selective and linear for drug concentrations in the range 15-210 micro g/mL. The LOQ and LOD are 15 and 3 micro g/mL, respectively. The results for accuracy studies are good. Values for coefficient of variation for intra- and interassay are 2.08% and 2.32%, respectively. The mean percentage of vitamin E acid succinate in the recovery studies is 99.52% +/- 0.81%. The mean loading efficiency for microspheres is 96.53% +/- 1.31%.     

Determination of vitamin A in pharmaceutical preparations by high-performance liquid chromatography with diode-array detection

Summary A very simple high-performance liquid chromatographic method for determination of Vitamin A in pharmaceutical preparations without the need for saponification was developed. A reversed-phase (Nova-Pack C₁₈, 4 m) column was used with a mobile phase of acetonitrile-tetrahydrofuran-water (55378) and a flowrate of 1.5 ml/min. Sample treatment only consisted of the extraction of retinol acetate content from capsules or tablets with methanol. Total extraction was achieved by shaking vigorously with the aid of magnetic stirring for three hours at room temperature. No change of solvent is necessary to introduce the sample in the chromatographic system. This method is suitable for routine quantification of Vitamin A.     

Determination of aprotinin by high-performance liquid chromatography

Summary A highly accurate and reproducible method for the determination of aprotinin (bovine pancreatic trypsin inhibitor) by HPLC is described. In the experiments, the relative standard deviation was 1.2% and detection limit 1 FIP-U cm^–3. Also, the method is quick and selective and active ingredients from difference source correlate well with enzymatic method. Analyses at different laboratories can be compared directly.     

Preparative isolation of vitamin D2 from previtamin D2 by recycle high-performance liquid chromatography.

A preparative high-performance liquid chromatographic method, based on recycle chromatography, to separate vitamin D2 (ergocalciferol) from previtamin D is described. The method provides efficient separation by means of a mixture of methanol, acetonitrile and hexane as eluent on a reversed-phase C18 column. Scale-up to a 2-in. diameter column resulted in the collection of 100% pure fractions based on UV detection at 265 nm. The total throughput and the economics of the purification were also optimized.     

Determination of 25-hydroxyvitamin D3 in human serum by fluorescence labelling and high-performance liquid chromatography.

A method is described for the determination of 25-hydroxyvitamin D3 in human blood serum. The problems of sensitivity and selectivity encountered with previous techniques were avoided by the formation of a highly fluorescent Diels-Alder adduct following solid-phase extraction of the vitamin. After excess of reagent had been eliminated, quantification was achieved by high-performance liquid chromatography. The recovery of the vitamin from serum was 76.4 +/- 1.76%. The precision of the method was determined, and the relative standard deviations were 8.38% at a concentration of 47.0 x 10(-9) mol dm-3, 6.74% at a concentration of 99.8 x 10(-9) mol dm-3 and 3.79% at a concentration of 146.8 x 10(-9) mol dm-3. The detection limit for the adduct was 2.93 x 10(-14) mol injected, for a signal-to-noise ratio of 3:1, and serum concentrations of 0.25 x 10(-9) mol dm-3 could easily be quantified. No interference from endogenous or exogenous substances was observed.     

Determination of vitamin D2 in emulsified nutritional supplements by solid-phase extraction and column-switching high-performance liquid chromatography with UV detection

This paper deals with a method for solid-phase extraction of trace amounts of vitamin D2 (VD2, 19 ng/g) from emulsified nutritional supplements, which contain 50 kinds of compounds, followed by column-switching high-performance liquid chromatography (HPLC) with UV detection at 265 nm. VD2 is present at 1000-20,000,000 times lower concentration than other components. Bond Elut C18 cartridge was chosen as for the emulsified nutritional supplements after comparison with eight other types. A sample solution was applied to the solid-phase extraction cartridge and VD2 was eluted by methanol followed by HPLC. The effects of sample pH, eluent composition and eluate volume on the retention and elution of VD2 on Bond Elut C18 cartridge were examined. The resulting method was simple, rapid (analysis time: approximately 20 min), sensitive (detection limit: approximately 0.1 ng per injection (200 microl) at a signal-to-noise ratio 3:1), and reproducible (relative standard deviation: approximately 6.2%, n=5). The calibration graph for VD2 was linear in the range of 0.1-3 ng per injection (200 microl). Recovery of VD2 was approximately 80% by the standard addition method.     

Determination of sulfur anions by high-performance liquid chromatography

Sulfite, sulfate, sulfide and thiosulfate ions are separated by high-performance liquid chromatography under acidic conditions on commercial low-capacity silica-based and resin-based anion-exchange columns with potassium hydrogenphthalate as the eluent. The ions are detected by using indirect ultraviolet absorption or conductivity detectors. The effects of concentration, pH and flow rate of the eluent on the retention times of sulfur anions are reported. The resin-based column is preferable to the silica-based column for separations of sulfur anions.