Determination of vitamins A, D, E, and K in human and bovine serum, and β-carotene and vitamin A palmitate in cosmetic and pharmaceutical products, by isocratic HPLC

Summary An HPLC method has been developed for the determination of fat-soluble vitamins. Ten fat-soluble vitamins were separated simultaneously on a 25 cm×4.6 mm i.d. Hypersil C₁₈ column with acetonitrile-dichloromethane-methanol, 60:20:20 (v/v) as mobile phase at 1.0 mL min⁻¹ with wavelength-programmed ultraviolet-visible-absorbance detection. Total analysis time was 12 min. The limits of detection were 0.03, 0.01, 0.55, 1.84, 0.02, 0.02, 0.01, 0.16, 0.33, 0.01, and 0.01 ng mL⁻¹ for retinol, retinyl acetate, retinyl palmitate, β-carotene, ergocalciferol, cholecalciferol, tocopherol, tocopherol acetate, phylloquinone, menatetrenone, and menadione, respectively. Analysis of human serum 2–7 days after ingestion of oral vitamins and Chinese herbs led to the conclusion that the concentration of vitamins was higher than for control serum.     

Simultaneous determination of vitamin K1, vitamin K1 2,3-epoxide and menaquinone-4 in human plasma by high-performance liquid chromatography with fluorimetric detection

A highly sensitive method for measuring endogenous vitamin K1, menaquinone-4 (which is one of the K2 vitamins) and vitamin K1 2,3-epoxide in human plasma was developed, based on high-performance liquid chromatography with coulometric reduction and fluorimetric detection, following extraction from plasma and purification on a Sep-Pak silica cartridge. The detection limits of vitamin K1, menaquinone-4 and vitamin K1 2,3-epoxide were 5, 5 and 8 pg per injection for the standard substances and 30, 30 and 50 pg/ml in human plasma, respectively.     

Quantitative determination of lansoprazole in human serum by HPLC

Summary Lansoprazole is a new inhibitor of gastric proton secretion. An HPLC method for the quantitative determination of lansoprazole in serum is described. The method consists of liquid-liquid extraction and enrichment of the analyte and subsequent reverse-phase liquid chromatography with UV detection. The method is specific, sensitive and practical. It has been applied to serum from healthy volunteers. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Automated method for the determination of vitamin D3 hydroxymetabolites in serum

An almost automated method for the determination of hydroxymetabolites of vitamin D₃ (cholecalciferol) in human serum is reported. The method consists of three steps: 1) a batch liquid–liquid extraction step with 2-propanol and hexane, and drying of the extract and reconstitution with phosphate buffer. 2) A cleanup and preconcentration step based on solid-phase extraction using Prospekt equipment, with CN group cartridges and elution with the chromatographic mobile phase. 3) A chromatographic step for individual separation of the target analytes starting with a 90:10 methanol–water mixture, then a linear gradient to obtain 100% methanol; followed by photometric detection. The method provides a linear range between 1.0 and 100 ng mL^–1 for 24,25-(OH)₂ vitamin D₃ and for 25-(OH)₂ vitamin D₃, and between 1.5 and 100 ng mL^–1 for 1,25-(OH) vitamin D₃, with correlation coefficients ranging between 0.993 and 0.987, repeatability between 1.9% and 4.8% and within-laboratory reproducibility between 2.8% and 8.8%.     

Flow-injection analysis of vitamin D3 and 25-hydroxy-vitamin D3 by amperometric detection

A simple, rapid and sensitive flow-injection method with amperometric detection for the determination of vitamin D₃ and 25-hydroxy-vitamin D₃ (25-OH-D₃) in pharmaceutical preparation is described. The method is based on the anodic electrochemical behaviour of these substances in methanol-water using a glassy carbon electrode. The optimum working potential was + 1.050 V (vs. Ag/AgCl). The influence of flow-rate, coil length and injection volume on sensitivity was established. Calibration graphs for both vitamin D₃ and 25-OH-D₃ show good linearity in the range 1.8×10^?7?1.0×10^?5 M. Detection limits of 7 ng (vitamin D₃) and 11 ng (25-OH-D₃) and relative standard deviations of 1.5% were obtained.     

High-performance liquid chromatography of fat-soluble vitamins: separation and identification of vitamins D2 and D3 and their isomers in food samples in the presence of vitamin A, vitamin E and carotene

Vitamins D2 and D3 and their corresponding previtamins and provitamins were resolved by reversed-phase high-performance liquid chromatography using a ternary solvent system (acetonitrile-methanol-water) pumped according to a gradient elution programme. The D vitamins were also resolved in the presence of other lipid-soluble vitamins (A, E and K1) and carotene. The peaks were monitored with a UV-visible variable-wavelength detector and were detected at their maximum absorbance, resulting in maximum sensitivity. Lipid-soluble vitamins and carotene were resolved in extracts obtained from oils and butter, thus permitting their identification in a single chromatographic run.     

Development and optimization of an LC-MS/MS-based method for simultaneous quantification of vitamin D2 , vitamin D3 , 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3

Simultaneous and accurate measurement of vitamin D and 25-hydroxyvitamin D in biological samples is a barrier limiting our ability to define "optimal" vitamin D status. Thus, our goal was to optimize conditions and evaluate an LC-MS method for simultaneous detection and quantification of vitamin D(2) , vitamin D(3) , 25-hydroxyvitamin D(2) and 25-hydroxyvitamin D(3) in serum. Extraction and separation of vitamin D forms were achieved using acetone liquid-liquid extraction and by a reversed phase C8 column, respectively. Detection was performed on a triple quadrupole tandem mass spectrometer (QQQ-MS/MS) equipped with atmospheric pressure photo ionization source. The LOQs for all analytes tested were 1 ng/mL for hydroxylated molecules and 2 ng/mL for the parent vitamin Ds. RSD at lower LOQ (2 ng/mL) and in medium (80 ng/mL) and high (200 ng/mL) quality control samples did not exceed 20 and 15% CV, respectively. Accuracy of the method for determination of hydroxylated molecules was also validated using National Institutes of Standards and Technology standard samples and found to be in the range of 90.9-111.2%. In summary, a sensitive and reproducible method is reported for simultaneous quantification of vitamin D(2) , vitamin D(3) , 25-hydroxyvitamin D(2) and 25-hydroxyvitamin D(3) molecules in biological samples.     

HPLC determination of carotenoids in human serum by gradient elution technique

Summary Polarities of the carotenoids in human serum are very different; many nonpolar carotenoid hydrocarbons (e.g. -carotene, lycopene) and highly polar hydroxycarotenoids (e. g. -cryptoxanthin, zeaxanthin, lutein) can be found among them.Gradient elution chromatography was used for the separation of -carotene, lycopene, -cryptoxanthin, lutein and zeaxanthin in serum samples applying amino and cyano packings (Chromsil-NH₂, Chromsil-CN). The effects of different stationary phases on the selectivity were compared. The method is particularly suitable for the direct determination of serum levels of carotenoid in serum extracts.     

High-performance liquid chromatography with electrochemical detection for the simultaneous determination of vitamin A, D3 and E in milk.

A high-performance liquid chromatographic electrochemical detection for the rapid and simultaneous determination of the vitamin A, D3 and E is described. The separation is carried out by using a C18 reversed-phase column and 0.1 M LiClO4 in methanol-water (99:1, v/v) as the mobile phase. The compounds are eluted with good resolution in the above order within about 15 min and are determined by amperometric detection with a glassy carbon electrode at +1050 mV (vs. Ag/AgCl). The method gave reproducible results and the detection limits were of the order of 0.07, 4 and 0.2 ng of vitamin A, D3 and E, respectively. The method was successfully applied to the determination of vitamin A, D3 and E in liquid cow milk and milk powder samples. After saponification, fat-soluble vitamins were extracted with hexane and a methanolic solution of the dried extract was injected directly into the chromatographic system, avoiding the clean-up step that is necessary for vitamin D3 when electrochemical detection is not used. Good recoveries were obtained.     

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 K in serum using HPLC with post-column reaction and fluorescence detection

A new, sensitive and selective detection method for vitamin K1 in human serum is reported. After the chromatographic separation, vitamin K is converted to its hydroquinone form in a wet-chemical post-column reduction reaction with tetramethylammonium octahydridotriborate. The reaction proceeds in an open tubular knitted reaction coil at elevated temperature. This system, when combined with a two-step multidimensional liquid chromatographic separation, has proven useful for the quantitative determination of serum concentrations of vitamin K1 in normal, healthy individuals.     

Fast and accurate determination of K, Ca, and Mg in human serum by sector field ICP-MS

Electrolytes in serum are important biomarkers for skeletal and cellular health. The levels of electrolytes are monitored by measuring the Ca, Mg, K, and Na in blood serum. Many reference methods have been developed for the determination of Ca, Mg, and K in clinical measurements; however, isotope dilution thermal ionization mass spectrometry (ID-TIMS) has traditionally been the primary reference method serving as an anchor for traceability and accuracy to these secondary reference methods. The sample matrix must be separated before ID-TIMS measurements, which is a slow and tedious process that hindered the adoption of the technique in routine clinical measurements. We have developed a fast and accurate method for the determination of Ca, Mg, and K in serum by taking advantage of the higher mass resolution capability of the modern sector field inductively coupled plasma mass spectrometry (SF-ICP-MS). Each serum sample was spiked with a mixture containing enriched ⁴⁴Ca, ²⁶Mg, and ⁴¹K, and the ⁴²Ca⁺:⁴⁴Ca⁺, ²⁴Mg⁺:²⁶Mg⁺, and ³⁹K⁺:⁴¹K⁺ ratios were measured. The Ca and Mg ratios were measured in medium resolution mode (m/Δm?≈?4 500), and the K ratio in high resolution mode (m/Δm?≈?10 000). Residual ⁴⁰Ar¹H⁺ interference was still observed but the deleterious effects of the interference were minimized by measuring the sample at K?>?100 ng g^?1. The interferences of Sr⁺⁺ at the two Ca isotopes were less than 0.25 % of the analyte signal, and they were corrected with the ⁸⁸Sr⁺ intensity by using the Sr⁺⁺:Sr⁺ ratio. The sample preparation involved only simple dilutions, and the measurement using this sample preparation approach is known as dilution-and-shoot (DNS). The DNS approach was validated with samples prepared via the traditional acid digestion approach followed by ID-SF-ICP-MS measurement. DNS and digested samples of SRM 956c were measured with ID-SF-ICP-MS for quality assurance, and the results (mean ± expanded uncertainty in mg dL^?1 unit) for Ca (DNS?=?10.14?±?0.13, digested?=?10.11?±?0.10), Mg (DNS?=?2.093?±?0.008, digested?=?2.098?±?0.007), and K (DNS?=?15.48?±?0.11, digested?=?15.50?±?0.28) were in good agreement with the certified values (Ca?=?10.17?±?0.06, Mg?=?2.084?±?0.023, K?=?15.55?±?0.13). Major sources of uncertainty are sample measurement, spike calibration, and instrument factor including mass discrimination of the spectrometer and the detector deadtime.     

Identification and determination of 1 alpha,25-dihydroxyvitamin D3 in rat skin by high-performance liquid chromatography and radioreceptor assay

Endogenous 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in normal rat skin was identified by using thermal isomerization to convert the metabolite into its pre-isomer, high-performance liquid chromatography (HPLC) and displacement potency with a chick intestinal cytosol receptor. When the metabolite in normal rat skin was determined by a radioreceptor assay after purification by Sep-Pak silica cartridge column chromatography and HPLC, the concentration was 71.0 +/- 6.6 pg/g of wet tissue (mean +/- S.D.). It is also shown that [3H]-1,25-(OH)2D3 and [3H]-25-hydroxyvitamin D3 administered intravenously in the mouse are located in the skin. These results suggest that the metabolite may play an important role in the skin.     

A stereocontrolled partial synthesis of 1α-hydroxy vitamin D3

A novel and efficient partial synthesis of 1α-hydroxy vitamin D₃ ($\text{6}$), starting from 7-dehydrocholesterol ($\text{1}$), is reported. The crucial step in the synthesis involves a selective Diels-Alder reaction of 4-phenyl-1,2,4-triazoline-3,5-dione with the 6,8-diene system of previtamin D₃ ($\text{3}$), generating an adduct $\text{7}$ suitable for the stereoselective introduction of the 1α-hydroxyl group. Cycloreversion of $\text{13}$ leads to the title compound.     

Determination of vitamins D2, D3, K1 and K3 and some hydroxy metabolites of vitamin D3 in plasma using a continuous clean-up-preconcentration procedure coupled on-line with liquid chromatography-UV detection

A semi-automatic procedure for the continuous clean-up and concentration of several fat-soluble vitamins prior to their separation by HPLC and UV detection is reported. The procedure is based on the use of a minicolumn packed with aminopropylsilica as sorbent located prior to the chromatographic detection system. The overall process was developed and applied to the main liposoluble vitamins (A, D2, D3, E, K1, K3) and several hydroxy metabolites of vitamin D3 [25-(OH)-D3,24,25-(OH)2-D3 and 1,25-(OH)2-D3]. All the analytes were monitored at a compromise wavelength of 270 nm. Calibration graphs were constructed between 0.01 and 100 ng ml-1 for vitamin D2 and D3 and their hydroxy metabolites, between 0.1 and 100 ng ml-1 for vitamin A, K1 and K3 and between 1 and 100 ng ml-1 for vitamin E, with excellent regression coefficients (> or = 0.9901) in all cases. The precision was established at two concentration levels with acceptable RSDs in all instances (between 3.6 and 8.7%). The method was appropriate for the determination of vitamin D2, D3, K1 and K3 and the 24,25-dihydroxy and 25-hydroxy metabolites of vitamin D3 in human plasma. The method was applied to plasma samples spiked with the target analytes and the recoveries ranged between 78 and 109%.