Chemische Vitamin D3-Bestimmung in Lebertran

The different aspects of Vitamin D₃ determination and the data reported in the literature are discussed. The spectrophotometric determination of Vitamin D₃ in cod-liver oil is carried out after the alkaline saponification, extraction of unsaponified parts, precipitation of accompanying sterols and the column- and thin-layer chromatographic purification and separation of vitamin D₃ on 40 cm plates from other vitamins. The dyestuff α-naphtholbenzein is suited well as standard substance for the better location and identification of vitamin D₃ zone on the thin-layer plate. The results obtained from the chemical method were checked through the simultaneous biological determination.     

Voltammetric determination of B<Subscript>1</Subscript> and B<Subscript>6</Subscript> vitamins using a pencil graphite electrode

Due to the importance of B₁ and B₆ vitamins for human health it is useful to develop new cheap and rapid methods for their determination. Voltammetric behavior of these vitamins on a pencil graphite electrode was investigated using cyclic voltammetry in different media. Direct quantitative determination of the two vitamins, one in the presence of the other, was done by differential pulse voltammetry. Vitamin B₁ was electroactive only in a NaOH solution generating two irreversible oxidation peaks; the first peak obtained at 250 mV is well-defined and was used in quantitative determinations. In case of vitamin B₆, a well-defined oxidation peak was observed in all investigated supporting electrolytes except for HCl. The linear concentration ranges were 10^?5–10^?3 M for vitamin B₁ in a NaOH solution and 5 × 10^?6–10^?3 M for vitamin B₆ in an acetate buffer solution. The obtained detection limits were 5.34 × 10^?6 M and 2.81 × 10^?6 M for vitamin B₁ and vitamin B₆, respectively. The developed method is simple and rapid and it was successfully applied in the determination of the two vitamins in pharmaceuticals.     

High Performance Liquid Chromatogrphy of Fat-Soluble Vitamins. III. Determination of Vitamin D3 in Pharmaceutical Preparations and in Blood

Abstract

A reversed phase high-performance liquid chromatographic method (HPLC) is described for separation and determination of colecalciferol (Vitamin D₃) in Vitamin preparations and in biological materials. Vitamin D₃ is extracted from the formulations and from the blood in a fully automated electronically controlled extraction apparatus. For HPLC a column of lichrosorb RP18 and methanol as eluent are used. The extraction, separation and determination of vitamin D₃ needs about 10–20 minutes. The described extraction and HPLC methods allow the detection of 1–2 ng per injection and are well reproduced with a maximum coefficient of variation of < 3,5%. Vitamin A-acetate is used as internal standard.     

Highly Sensitive Electrochemical Sensor for Determination of Vitamin D in Mixtures of Water‐Ethanol

This paper describes the electrochemical determination of vitamin D₂ (ergocalciferol) and D₃ (cholecalciferol) in mixed organic/water solvent, using a glassy carbon electrode (GCE). The mixing ratio of organic/water solvent has an important influence on the electrocatalytic response of D vitamins on the surface of the glassy carbon electrode. Well‐defined peaks for Vitamin D₂ and D₃ were observed in a 40 % ethanol/60 % water solution with lithium perchlorate as the support electrolyte. This study demonstrated that the glassy carbon electrode is highly sensitive for the determination of vitamin D₂ and D₃, with a limit of detection of 0.13 and 0.118 µmol L^?1, respectively. No significant interference was seen for vitamins A, E and K in the detection of vitamin D.     

The Measurement of Nanogram Quantities of Vitamin D3

Abstract

An isotope dilution method is described for the measurement of nanogram quantities of vitamin D₃ (cholecalciferol). Use is made of the Diels -Alder reaction between vitamin D₃ and tetracyanoethylene.

Increasing quantities of exogeneous vitamin D₃ added to a standard reaction mixture of ¹⁴C-labelled vitamin D₃ and tetracyanoethylene produced a decrease in the ratio of reacted to unreacted vitamin D₃. The ratio (y) was measured by radio-scanning of an eluted thin-layer chromatogram, and quantitation of added vitamin D₃ was thereby achieved.     

Determination of Vitamin D3 Metabolites Using High‐Performance Liquid Chromatography or Immunoaffinity Chromatography

Our investigation of the analysis of vitamin D₃ metabolites has been reviewed. The development of high‐performance liquid chromatographic methods for the quantitative determination of 25‐hydroxyvitamin D₃ 3‐sulfate and 25‐hydroxyvitamin D₃, which are the major circulating metabolites of vitamin D₃ in human serum/plasma, has been described. The developed methods were applied to the determination of the correlation between the concentration of the sulfate and its genin in healthy subjects and patients with chronic renal failure. The development of immunoaffinity chromatography immobilizing the highly specific anti‐1,25‐dihydroxyvitamin D₃ antibody for the pretreatment of radioreceptor assay of 1,25‐dihydroxyvitamin D₃, which is the active metabolite of vitamin D₃, is also described.     

Voltammetric determination of vitamin B<Subscript>6</Subscript> (pyridoxine) at a graphite screen-printed electrode modified with graphene oxide/Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>@SiO<Subscript>2</Subscript> nanocomposite

The preparation and study of electrochemical properties of a graphite screen-printed electrode (SPE) modified with the GO/Fe₃O₄@SiO₂ (GO is graphene oxide) nanocomposites are described. The morphologies of the GO/Fe₃O₄@SiO₂ nanocomposites were examined by scanning electron microscopy. The electrochemical oxidation of vitamin B₆ (pyridoxine) on SPE modified with the GO/Fe₃O₄@SiO₂ nanocomposite was investigated by cyclic voltammetry, differential pulse voltammetry, and chronoamperometry. Under optimum conditions (pH 7.0), the vitamin B₆ oxidation at the surface of the modified SPE occurs at a potential about 190 mV less positive than that at the unmodified SPE. A linear voltammetric response for vitamin B₆ was obtained in the concentration range 1.0?10 ⁶—9.0?10 ⁴ mol L^–1 with a detection limit of 5.2?10 ⁷ mol L^–1 using differential pulse voltammetry. The developed sensor was also successfully applied for determination of trace level of vitamin B₆ in both the standard vitamin B₆ sample and biological samples (urine).     

Determination of vitamin B<Subscript>1</Subscript> in seawater and microalgal fermentation media by high-performance liquid chromatography with fluorescence detection

A highly sensitive high-performance liquid chromatographic method with fluorescence detection has been developed for determination of vitamin B₁. Vitamin B₁ was converted into a fluorescent compound by treatment with hydrogen peroxide–horseradish peroxidase and the derivative was subsequently analyzed by HPLC on a Waters Spherisorb ODS2 column (250 mm×4.6 mm ID, 5 μm) with 40:60 methanol–pH 8.5 acetate buffer solution as mobile phase and fluorescence detection at 440 nm (with excitation at 375 nm). The calibration graph was linear from 5.00×10⁻¹⁰ mol L⁻¹ to 5.00×10⁻⁷ mol L⁻¹ for vitamin B₁ with a correlation coefficient of 0.9991 (n=9). The detection limit was 1.0×10⁻¹⁰ mol L⁻¹. The method was successfully used for determination of vitamin B₁ at pg mL⁻¹ levels in microalgal fermentation media and seawater after solid-phase extraction. Recovery was from 89 to 110% and the relative standard deviation was in the range 1.1 to 4.3%.     

Determination of Cobalt in the Form of an Ion Associate in Vitamin B<Subscript>12</Subscript>

The possibility of application of the ion-associated complex formed between the anionic chelate cobalt(II)-4-(2-thiazolylazo) resorcinol (TAR) with the cation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) for extraction-spectrophotometric determination of cobalt in the form of an ion associate in Vitamin B₁₂ was studied. The liquid–liquid extraction system Co(II)-TAR-MTT-H₂O-CHCl₃ was applied. This system was chosen by our previous research of the ion associates of the cobalt by spectrophotometric investigation of fourteen different liquid–liquid extraction systems, containing azo derivatives of resorcinol (TAR or 4-(2-pyridylazo) resorcinol (PAR)) and mono or ditetrazolium salts. Based on the obtained results, a sensitive, relatively simple, convenient and inexpensive method for determination of cobalt in the form of an ion associate in Vitamin B₁₂ was developed. The proposed method can be implemented for biological, medical and pharmaceutical samples containing cobalamin (Vitamin B₁₂).     

Vitamin D analysis in plasma by high performance liquid chromatography (HPLC) with C(30) reversed phase column and UV detection--easy and acetonitrile-free

Two physiologically important forms of vitamin D exist: vitamin D₂ and vitamin D₃, which by liver based hydroxylase enzymes are converted to 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃, respectively. These hydroxylated metabolites of vitamin D are measured in plasma to assess the vitamin D status of animals and humans. Therefore cheap and reliable analytical methods are very much in demand in nutritional and physiological research. After saponification and extraction of plasma or serum samples the current method uses reverse phase high performance liquid chromatography on a C₃₀ column and with UV detection at 265 nm for quantifying vitamin D₂, vitamin D₃, 25-hydroxyvitamin D₂, and 25-hydroxyvitamin D₃. The method proved versatile with respect to plasma lipid content, sample amount, and plasma concentration of the vitamin D metabolites as it was tested using plasma from six different species: cattle, pigs, poultry, mink, horses, and humans. In cattle plasma recoveries were between 86.6 and 101.0%, within day error between 0.9 and 5.9%, and between day error between 0.2 and 1.7%. However, depending on species and sample amount error percentages varied. When running the method on standard reference material^® 972 “Vitamin D in human serum” from the National Institute of Standards and Technology (NIST) (Gaithersburg, USA) the results for 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ concentrations were within the boundaries provided by NIST, reflected by Z-scores between 0.1 and 0.9.     

Electrochemical Determination of Vitamin D3 in Pharmaceutical Products by Using Boron Doped Diamond Electrode

A sensitive square‐wave voltammetry method was developed to determine cholecalciferol (vitamin D₃) in pharmaceutical products at boron‐doped diamond electrode as a working electrode. Vitamin D₃ provided a well‐defined voltammetric peak at around +1.00 V (vs. Ag/AgCl, 3.5 mol dm^?3) in 0.02 mol dm^?3 Britton‐Robinson buffer pH 5.0 prepared in 50 % ethanol. The influence of various factors such as type and pH of the supporting electrolyte, scan rate and square‐wave parameters were studied and optimized. Under optimum conditions, the oxidation peak current increased linearly with the concentration of vitamin D₃ over the range of 2 to 200 μmol dm^?3. The calculated limit of detection and limit of quantitation were 0.17 μmol dm^?3 and 0.51 μmol dm^?3, respectively. The boron‐doped diamond electrode exhibited specific recognition capability for cholecalciferol amongst possible interferences, and the determination of vitamin D₃ was possible in samples such as commercial pharmaceutical products without complicated sample pretreatments.     

Radioimmunologische Bestimmung von 25-Hydroxycholecalciferol

Zusammenfassung Der gegen Vitamin D₃ als Hapten gerichtete Antikörper bewährte sich bei der Bestimmung von 25-OH-Cholecalciferol (25-OH-CC). Der Versuch, auch Vitamin D₃ mit Hilfe dieses radioimmunologischen Verfahrens direkt zu bestimmen, scheiterte an der schlechten Löslichkeit des Vitamins in wäßrigen Systemen. Auf Grund der Kopplung an das C-Atom-3 wären der D-Ring und die C-17-Seitenkette als immundeterminante Stellen zu erwarten. Die Tatsache, daß der Antikörper zur Bestimmung des 25-OH-CC herangezogen werden kann, gleichzeitig aber keine Kreuzreaktion mit Cholesterin ergibt, weist auf die Bedeutung des offenen B-Ringes dieser Vitamine hin. Die Überprüfung der Methode ergab gute Reproduzierbarkeit und Genauigkeit. Der günstigste Auswertebereich liegt bei 0,1–2 ng/250l Ansatz. Das beschriebene Verfahren ermöglicht die Bestimmung von 25-Hydroxy-Vitamin-D₃ ohne vorherige Reinigung des Probenextraktes.

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Radioimmunological determination of 25-hydroxycholecalciferol

Summary The antibody to vitamin D₃ as hapten proved useful in determination of 25-hydroxycalciferol (25-OH-CC). The attempt also to determine vitamin D₃ directly with the aid of this radioimmunological procedure failed because of the poor solubility of the vitamin in aqueous systems. Because of the linkage to the C3 the D-ring and C17 side chain would be expected to be the immunodeterminant sites. Although the antibody can be used to determine 25-OH-CC, there is no cross-reaction with cholesterol. This indicates the importance of the open B-ring of this vitamin. Scrutiny of the method showed good reproducibility and accuracy. The most favorable evaluation region is 0.1–2 ng/250l sample. The procedure described enables 25hydroxy vitamin D₃ to be determined without previous purification of the sample extract.

     

The Spectrophotometric Determination of Vitamin a Using Iodine

Abstract

Methods are described for the spectrophotometric determination of vitamin A (0.15–12.5 μg) using iodine as the chromogenic reagent. The reaction is most sensitive in chloroform. Vitamin D₂ and ß-carotene do not interfere if the analysis is carried out in 1, 2 -dichloroethane.     

Determination of vitamin B<Subscript>12</Subscript> using a chemiluminescence flow system

A method to determine vitamin B₁₂ by measuring the chemiluminescence (CL) intensities using a flow injection (FI) system has been proposed. It is based on the catalytic effect of cobalt(II) in vitamin B₁₂ on the CL reaction between luminol and hydrogen peroxide in a basic medium. The increment of the CL intensity is proportional to the concentration of vitamin B₁₂ in the range 8.68–86.9 ng/mL (r ² = 0.9984) with a detection limit (3σ) of 0.89 ng/mL. The CL response is obtained in 10 s at a flow rate of 3.0 mL/min with a relative standard deviation (RSD) of less than 2.5% (n = 6). The method has been successfully applied to the determination of vitamin B₁₂ in pharmaceutical injections. The text was submitted by the authors in English.     

Simultaneous quantitative analysis of eight vitamin D analogues in milk using liquid chromatography–tandem mass spectrometry

Milk is an important source of nutrients for various risk populations, including infants. The accurate measurement of vitamin D in milk is necessary to provide adequate supplementation advice for risk groups and to monitor regulatory compliance. Currently used liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods are capable of measuring only four analogues of vitamin D in unfortified milk. We report here an accurate quantitative analytical method for eight analogues of vitamin D: Vitamin D₂ and D₃ (D₂ and D₃), 25-hydroxy D₂ and D₃, 24,25-dihydroxy D₂ and D₃, and 1,25-dihydroxyD₂ and D₃. In this study, we compared saponification and protein precipitation for the extraction of vitamin D from milk and found the latter to be more effective. We also optimised the pre-column derivatisation using 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD), to achieve the highest sensitivity and accuracy for all major vitamin D forms in milk. Chromatography was optimised to reduce matrix effects such as ion-suppression, and the matrix effects were eliminated using co-eluting stable isotope labelled internal standards for the calibration of each analogue. The analogues, 25-hydroxyD₃ (25(OH)D₃) and its epimer (3-epi-25(OH)D₃) were chromatographically resolved, to prevent over-estimation of 25(OH)D₃. The method was validated and subsequently applied for the measurement of total vitamin D levels in human, cow, mare, goat and sheep milk samples. The detection limits, repeatability standard deviations, and recovery ranges were from 0.2 to 0.4 femtomols, 6.30–13.5%, and 88.2–105%, respectively.     

Synthesis of C-11 linked active ester derivatives of vitamin D3 and their conjugations to 42-residue helix-loop-helix peptides

Derivatives of vitamin D₃ carrying an 8-carbon linker at C-11 terminating in an active ester were synthesized from commercial vitamin D₃ using a disassembly-reassembly strategy. Vitamin D₃ was cleaved at the C6-C7 double bond and the ‘upper’ fragment was converted, via a series of reactions, to derivatives substituted at C-11 with an 8-carbon linker terminating in an ethyl ester. Reassembly with modified ‘lower’ fragments using Horner-Wittig olefination followed by linker ester hydrolysis and re-esterification with p-nitrophenol gave C-11 substituted p-nitrophenyl esters. These vitamin D derivatives were conjugated to 42-amino acid helix-loop-helix peptides by reaction of their p-nitrophenyl esters with lysyl side-chain amino groups on the peptides. The vitamin D—peptide conjugates, being potential specific binder candidates for vitamin D-binding protein, were characterized by mass spectroscopy and CD measurements.     

Synthesis of a 1α-C-methyl analogue of 25-hydroxyvitamin D3: interaction with a mutant vitamin D receptor Arg274Leu

Vitamin D₃ analogues have been developed for a mutant vitamin D receptor (VDR), Arg274Leu. The mutant VDR has a mutation at Arg274, which forms an important hydrogen bond with 1α-OH of 1α,25-dihydroxyvitamin D₃ to anchor the ligand tightly in the VDR ligand binding pocket. Stereoselective synthesis of the A-ring part of the novel vitamin D analogue, 2α-(3-hydroxypropyl)-1α-methyl-25-hydroxyvitamin D₃ (4), from d-galactose was accomplished with the key steps of the introduction of the methyl and allyl groups to the chiral building blocks. The new analogue 4 is ca. 7.3-fold more active than the natural hormone 1α,25-dihydroxyvitamin D₃ (1).     

Quantitative determination of vitamin D metabolites in plasma using UHPLC-MS/MS

Vitamin D is an important determinant of bone health at all ages. The plasma concentrations of 25-hydroxy vitamin D (25-OH D) and other metabolites are used as biomarkers for vitamin sufficiency and function. To allow for the simultaneous determination of five vitamin D metabolites, 25-OH D₃, 25-OH D₂, 24,25-(OH)₂ D₃, 1,25-(OH)₂ D₃, and 1,25-(OH)₂ D₂, in low volumes of human plasma, an assay using ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) was established. Plasma samples were spiked with isotope-labeled internal standards and pretreated using protein precipitation, solid-phase extraction (SPE) and a Diels–Alder derivatization step with 4-phenyl-1,2,4-triazoline-3,5-dione. The SPE recovery rates ranged from 55% to 85%, depending on the vitamin D metabolite; the total sample run time was <5 min. Mass spectrometry was conducted using positive ion electrospray ionization in the multiple reaction monitoring mode on a quadrupole–quadrupole-linear ion trap instrument after pre-column addition of methylamine to increase the ionization efficiency. The intra- and inter-day relative standard deviations were 1.6–4.1% and 3.7–6.8%, respectively. The limit of quantitation for these compounds was determined to be between 10 and 20 pg/mL. The 25-OH D results were compared with values obtained for reference materials (DEQAS). In addition, plasma samples were analyzed with two additional Diasorin antibody assays. All comparisons with conventional methods showed excellent correlations (r ² = 0.9738) for DEQAS samples, demonstrating the high degree of comparability of the new UHPLC-MS/MS technique to existing methods.     

Spectrophotometric determination of vitamins D2 and K1 in presence of rutin

Summary Methods are given for determination of vitamin D₂ and K₁ in presence of rutin added as stabilizer, and applied to assessment of the rate of breakdown of these vitamins when exposed to ultraviolet light. Because the absorption maxima of these vitamins, rutin and some of the decomposition products are very near to each other a preliminary separation is necessary. Thin layer chromatography with chloroform on silica gel H is suitable.

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Spektropbotometrische Bestimmung von Vitamin D₂ und K₁ in Gegenwart von Rutin

Zusammenfassung Für die Bestimmung von Vitamin D₂ und Vitamin K₁ in Gegenwart von Rutin, das als Stabilisator zugegeben wird, wurden Methoden angegeben und zur Bestimmung der bei UV-Bestrahlung eintretenden Zersetzung dieser Vitamine angewendet. Da deren Absorptionsmaxima alle sehr nahe beieinander liegen, ist eine vorherige Trennung nötig. Dazu hat sich die Dünnschichtchromatographie mit Chloroform auf Silicagel H als geeignet erwiesen.

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Presented at the 8th International Microchemical Symposiums Graz, August 25–30, 1980.     

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.