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.     

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.     

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%.     

Facile Construction of the 7,8‐Olefin Linkage in Vitamin D3: A Practical Synthesis Benefiting the Vitamin D3 Analog Study

Abstract

A facile procedure for construction of the 7,8‐olefin linkage in vitamin D₃ is described. Treatment of a mixture of A‐ring phosphine oxide and CD‐ring ketone in THF with lithium hexamethyldisilazide (LHMDS) at ?20°C followed by gradual heating to 50°C gives the key intermediate of vitamin D₃ analogs in excellent yield. This simplified procedure makes possible small‐scale synthesis benefiting the vitamin D₃ analog study.     

Liquid-Solid Extraction of Vitamin D3 Metabolites from Plasma for Analysis by HPLC,GC/MS and Protein Binding Techniques

Abstract

Liquid-solid extraction of vitamin D₃ metabolites from human plasma using octadecylsilane bonded silica has been studied. Steroid-protein interactions were minimized by diluting the plasma (or serum) with two volumes of saline and passing the solution through the sorbent at 64°C. Highly purified secosteroid fractions were obtained by washing with aqueous methanol, drying the sorbent in situ with a stream of nitrogen for one minute and eluting with mixtures of hexane/chloroform. Recoveries of vitamin D₃ metabolites were essentially quantitative. Applications to the rapid analysis of 25-hydroxy- and 1α, 25-dihydroxy-vitamin D in plasma by high-performance liquid chromatography, gas chromatography-mass spectrometry or by a receptor protein assay are reported.     

A gas chromatographic flow method for preconcentration and determination of vitamins D2 and D3 in pharmaceutical preparations

Summary A continuous-flow method that combines on-line preconcentration and isolation with gas chromatography for the direct determination of vitamins D₂ and D₃ in oily solutions is reported. A silica gel column permits preconcentration and isolation of analytes from the vitamin D matrix, although some triglyceride (ca. 25%) is also retained. To overcome problems associated with the low volatility of triglycerides, their retained fraction is further transesterified with potassium methylate to fatty acid methyl esters after elution. The proposed method was applied to the determination of vitamin D in pharmaceutical preparations.     

Metabolic profiling of major vitamin D metabolites using Diels–Alder derivatization and ultra-performance liquid chromatography–tandem mass spectrometry

Biologically active forms of vitamin D are important analytical targets in both research and clinical practice. The current technology is such that each of the vitamin D metabolites is usually analyzed by individual assay. However, current LC-MS technologies allow the simultaneous metabolic profiling of entire biochemical pathways. The impediment to the metabolic profiling of vitamin D metabolites is the low level of 1α,25-dihydroxyvitamin D₃ in human serum (15–60 pg/mL). Here, we demonstrate that liquid–liquid or solid-phase extraction of vitamin D metabolites in combination with Diels–Alder derivatization with the commercially available reagent 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) followed by ultra-performance liquid chromatography (UPLC)–electrospray/tandem mass spectrometry analysis provides rapid and simultaneous quantification of 1α,25-dihydroxyvitamin D₃, 1α,25-dihydroxyvitamin D₂, 24R,25-dihydroxyvitamin D₃, 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂ in 0.5 mL human serum at a lower limit of quantification of 25 pg/mL. Precision ranged from 1.6–4.8 % and 5–16 % for 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃, respectively, using solid-phase extraction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

利用薯蓣皂甙元完整骨架形式合成1α,25-二羟基维生素D3

首次利用薯蓣皂甙元的完整骨架经16步反应以7.6%的总收率合成了骨化三醇(1α,25-二羟基维生素D₃)的光化反应前体. 3-苄基保护的薯蓣皂甙元经还原开E/F环产生3,16,26-胆甾三醇-3-苄醚(5). 除去化合物5 C-16羟基后, 其C-26羟基经消除和羟基化反应转移到C-25位. 目标分子A/B环结构单元通过薯蓣皂甙元A/B环的官能团转化被构筑. 按照已知的光化反应, (1S,3R)-胆甾-5,7-二烯-1,3,25-三醇能被转化成为1α,25-二羟基维生素D₃.     

Zur Stereochemie der Vitamin D3-Epoxide Röntgenstrukturanalyse einer 5,6-7,8-Diepoxidverbindung

Epoxidation of vitamin D₃ with benzonitrile ¦H₂O₂¦ KHCO₃ (Payne's reagent) yields exclusively the 5,6-mono-oxirane2. Further epoxidation with the same reagent gives the 5,6-7,8-bisepoxide3 a. Its stereochemistry and therefore the stereochemistry of2 was established by single crystal X-ray analysis of thep-Brbenzoate3b. Thereby for the first time a directive effect of a homoallylic hydroxyl group during thePayne-epoxidation was established.

Herrn Prof.Mihailovic (Universität Belgrad) mit den besten Wünschen zum 60. Geburtstag gewidmet.     

Quantitative Determination of Octamethylcyclotetrasiloxane (D4) in Extracts of Biological Matrices by Gas Chromatography-Mass Spectrometry

Abstract

A method was developed and validated to measure octamethylcyclotetrasiloxane (D₄)^? quantitatively by gas chromatography-mass spectrometry (GC-MS) at low level in extracts of several biological matrices that include plasma, liver, lung, feces and fat from rats. The key to the successful determination lay in the use of extracts dried with anhydrous magnesium sulfate. This was necessary in view of the propensity of the methyl siloxane based GC-stationary phase to generate D₄ by its reaction with water present in the extracts. To enable quantiiation of D₄ at parts per billion (μg/L) levels, the base ion m/z 281 resulting from the loss of a methyl group from the parent molecule was selected for monitoring by SIM mode in GC-MS. The recovery of D₄ from any of the biological matrices was determined to be greater than 90% in three extractions. The D₄ response for the standards in GC-MS was linear (R² > 0.9900) and reproducible at concentrations ranging from 1—16,000 ng D₄/g solvent. Precision was less than 5%.     

Amperometric Immunosensor Based on Gold Nanoparticles/Fe3O4-FCNTs-CS Composite Film Functionalized Interface for Carbofuran Detection

In this paper, a novel amperometric immunosensor for the determination of carbofuran based on gold nanoparticles (GNPs), magnetic Fe₃O₄ nanoparticles-functionalized multiwalled carbon nanotubes-chitosan (Fe₃O₄-FCNTs-CS), and bovine serum albumin (BSA) composite film was proposed. First, GNPs were immobilized onto the glassy carbon electrode (GCE) surface, and then the magnetic Fe₃O₄ nanoparticles mixed with chitosan-functionalized multiwall carbon nanotubes (CS-FCNTs) homogeneous composite (CS-FCNTs-Fe₃O₄) was immobilized onto the GNPs layer by electrostatic interactions between amino groups of CS and GNPs. Because chitosan (CS) contains many amino groups, it can absorb more antibodies. FCNTs have high surface area, high electrical conductivity, and it can enhance the electron transfer rate; Magnetite (Fe₃O₄) nanoparticles can provide a favorable microenvironment for biomolecules immobilization due to their good biocompatibility, strong superparamagnetic property, and low toxicity; and GNPs possess high surface-to-volume reaction, stability, and high conductivity. Gold Nanoparticles/Fe₃O₄-FCNTs-CS composite film was constructed onto the GCE surface, which had significant synergistic effects toward immunoreaction signal amplification. The stepwise assembly process was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), respectively. Under the optimal conditions, the current response was proportional to the concentration of carbofuran ranging from 1.0 ng/mL to 100.0 ng/mL and from 100.0 ng/mL to 200 µg/mL with the detection limit 0.032 ng/mL. The proposed immunosensor exhibited good accuracy, high sensitivity, and stability, and it can be used for detection of carbofuran pesticide.     

A specific LC/ESI-MS/MS method for determination of 25-hydroxyvitamin D3 in neonatal dried blood spots containing a potential interfering metabolite, 3-epi-25-hydroxyvitamin D3

Vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes in later life. A method for the quantification of 25‐hydroxyvitamin D₃ [25(OH)D₃, the best‐established indicator of vitamin D status] in neonatal dried blood spots (DBSs) using LC/ESI‐MS/MS has been developed and validated. The method employed two steps of derivatization, a Diels–Alder reaction with 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione followed by acetylation, to enhance the detectability of 25(OH)D₃ in ESI‐MS/MS and to separate 25(OH)D₃ from 3‐epi‐25‐hydroxyvitamin D₃ [3‐epi‐25(OH)D₃], a potent interfering metabolite. 25(OH)D₃ was extracted from two DBS punches (3 mm in diameter, equivalent to 5.3 μL of whole blood), purified using an Oasis HLB^® cartridge, and subjected to derivatization prior to analysis with LC/ESI‐MS/MS. 25‐Hydroxyvitamin D₄ was used as the internal standard. This method was reproducible (intra‐ and inter‐assay RSDs, <6.9%) and accurate (analytical recovery, 95.2–102.7%), and the LOQ was 3.0 ng/mL. The developed method enabled specific quantification of 25(OH)D₃ in neonatal DBSs and detection of vitamin D deficiency without interference from 3‐epi‐25(OH)D₃.     

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.     

Determination of 25‐hydroxyvitamin D3 in human plasma using HPLC with UV detection based on SPE sample preparation

Interest in the metabolism and physiological action of vitamin D is increased exponentially. The most important metabolites of vitamin D are 25‐hydroxyvitamin and 1,25‐dihydroxyvitamin D₃. The aim of the study was to develop a rapid and simple HPLC method for the measurement of 25‐hydroxyvitamin D₃ in human plasma. A method for the measurement of 25‐hydroxyvitamin D₃ using HPLC with UV detection and investigation into the extraction techniques with regard to stability and recovery are described. For the separation, RP column LiChroCart 125‐4, Purospher RP‐18e, 5 μm, was used. The mixture of methanol and deionized water (95:5 v/v) was used as mobile phase. The analytical performance of this method is satisfactory: the intra‐ and inter‐assay coefficients of variation were below 10%. Quantitative recoveries from spiked plasma samples were between 92.0–103.2%. The LOD was 10 nmol/L. The preliminary reference range of 25‐hydroxyvitamin D₃ in a group of blood donors is 62 ± 26 nmol/L.     

Zur Epoxidation der Δ 7-Doppelbindung in Vitamin D3 Röntgenstrukturanalyse des 4-Phenyl-1,2,4-triazolin-3,5-dion-Addukts — eine Strukturrevision

Epoxidation of vitamin D₃ withMCPBA yields exclusively the 7,8-monooxirane. Its 7R, 8R stereochemistry was established by X-ray analysis of thePTAD-adduct-derivative. This result corrects the stereochemical assignment in a previous publication⁷.

     

Tetracyanonickelate compounds as sorptive materials and the substitution of their H2O content by D2O

The compounds NiNi(CN)₄·3,5H₂O and Ni(NH₃)₂Ni(CN)₄·H₂O have been studied to examine the possibility of substituting their H₂O or NH₃ content by D₂O. Contact with D₂O was performed after heating the compounds to several temperatures. Depending on the degree of decomposition of the original compounds different ranges of substitution were possible. In such manner the compounds NiNi(CN)₄·3,5D₂O, NiNi(CN)₄·5D₂O, Ni(NH₃)₂Ni(CN)₄·D₂O, and Ni(D₂O)₂Ni(CN)₄·D₂O were prepared and thermally they were less stable than the original ones. The substitution by D₂O is in agreement with the sorptive properties of the original tetracyanonickelate against different organic compounds using GC, since these could substitute the guest component and sometimes also the ligands during their decomposition.     

Rh(PPh3)3Cl/Tetrakis(Dialkylamino)Phosphonium Salts as Thermoregulated and Recyclable Catalytic System for Hydrosilylation Reaction

Abstract

Eleven tetrakis(dialkylamino)phosphonium salts have been prepared and were used as “soft” catalyst supports for the hydrosilylation reaction of styrene with triethoxysilane catalyzed by Rh(PPh₃)₃Cl. Among the Rh(PPh₃)₃Cl/tetrakis(dialkylamino)phosphonium salts tested, the best catalytic activity and selectivity in favor of the β-adduct were obtained when {[(C₄H₉)₂N]₃[(C₈H₁₇)₂N]P}PF₆ was used as the support, and Rh(PPh₃)₃Cl/ {[(C₄H₉)₂N]₃[(C₈H₁₇)₂N]P}PF₆ catalyst system can be reused more than 10 times without noticeable loss of catalytic activity and selectivity.

GRAPHICAL ABSTRACT      

Enhanced sensitivity by laser-induced fluorescence for the determination of calcitriol and other vitamin D3 metabolites in plasma

Summary A sensitive method for the determination of hydroxymetabolites of vitamin D₃, parficularly calcitriol (1,25-(OH)₂-D₃), in human plasma is reported. The method is based on the use of laser-induced fluorescence detection as an alternative to conventional fluorimetry in an integrated cleanup/preconcentration, HPLC separation and post-column derivatization system. The derivatization step is based on a dehydration reaction which takes place in secosteroid structures at high temperature in a strong-acid medium. A LOD of 0.01 pg mL⁻¹ (SNR=3) was obtained for each analyte with a linear dynamic range over 4 order of magnitude with excellent regression coefficients (≥0.9922) in all cases. The precision was studied at two concentration levels and the RSDs values (for n=5) were acceptable (between 2.6 and 4.7%). The method was also checked by applying it to human plasma spiked with the target analytes and excellent recoveries were obtained. This is the first time that these species have been determined at the sub-pg mL⁻¹ level.     

Liquid chromatography–tandem mass spectrometric method for the determination of salivary 25-hydroxyvitamin D<Subscript>3</Subscript>: a noninvasive tool for the assessment of vitamin D status

A sensitive liquid chromatography–electrospray ionization–tandem mass spectrometric (LC–ESI–MS/MS) method for the determination of 25-hydroxyvitamin D₃ [25(OH)D₃] in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with a Cookson-type reagent, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and subjected to LC–MS/MS. The PTAD derivative was much more easily ionized in positive-ESI–MS and efficiently produced a characteristic product ion during MS/MS, compared to the intact 25(OH)D₃. Methylamine was used as the mobile phase additive, and also effectively enhanced the assay sensitivity. Quantification was based on selected reaction monitoring, and 25-hydroxyvitamin D₄ was used as the internal standard. This method allowed the reproducible and accurate quantification of salivary 25(OH)D₃ using a 1.0-ml sample, and the limit of quantitation for 25(OH)D₃ was 2.0 pg/ml. The applicability of the developed method for clinical studies was then examined. There was a positive linear relationship (r ² = 0.830) between the serum 25(OH)D₃ level, which is conventionally used as a means of assessing the vitamin D status, and the salivary 25(OH)D₃ level measured using the proposed method. The method also enabled the detection of the increase in the salivary 25(OH)D₃ level after the supplementation of vitamin D₃.     

Komplexe des zweiwertigen Molybdäns,Derivate des Oktahalodimolybdat(II)-Ions, 4. Mitt.: Darstellungen und Kristallstrukturen von zwei neutralen Komplexen mit 4-Methylpyridin

Mo₂Cl₄ Pic ₄·CHCl₃ (A) (Pic=4-methylpyridine) and Mo₂Br₄ Pic ₄ (B) crystallize in the monoclinic space group.A inC2/c (No. 15) witha=15.175 (4),b=10.847 (2),c=19.946 (6) and =104.52 (2)°;D _o=1.71 (2),D _c =1.72 gcm^–3 forZ=4.B inP2_l/n (No. 14) witha=9.270 (3),b=16.614 (5),c=9.305 (3) and =91.96 (5)°;D _o=2.03 (3),D _c =2.05 gcm^–3 forZ=2.Two halogens and 4-methylpyridines of the MoX ₂ Pic ₂ group are in the trans position. Mo–Mo bond lengths are 2.153 96) forA and 2.150 92) forB. Both molecules are situated on the inversion center resulting in the eclipsed configuration of the ligands around the molybdenum pair. The structure ofB has been refined to the conventionalR factors of 0.08 and 0.098. Disorder on the part of 4-methylpyridines and chloroform molecules stopped the refinement ofA at the endR value of 0.175.Mean Mo–X and Mo–N bonding distances are 2.40 (2), 2.25 (5) forA and 2.53 (3), 2.25 (1) forB.