Identification and Structural Elucidation of Vitamin D3 Metabolites in Human Urine Using LC-MS-MS

An analytical method was developed for the identification of primary vitamin D₃ metabolites in human urine using liquid chromatography tandem mass spectrometry in positive mode. Urine samples were purified using C₁₈ solid-phase extraction cartridges and analytical separations were performed by reversed phase liquid chromatography in gradient mode using ammonium acetate (0.01 mol L^?1) and acetonitrile as the mobile phases. Identification and structural elucidation of the metabolites were carried out by comparison with mass spectral fragmentation behavior of vitamin D₃ and retention characteristics. Three primary urinary vitamin D₃ metabolites were identified as 25-hydroxyvitamin D₃, 1α,25-dihydroxyvitamin D₃ and vitamin D₃ sulphate, respectively.     

Determination of Vitamin D3 and 25-Hydroxyvitamin D3 Using Liquid Chromatography with Amperometric Detection

Abstract

The electrochemical behaviour of vitamin D₃ and 25-hydroxyvitamin D₃ (25-OH D₃) in a high performance liquid chromatography system using amperometric detection is described. Separation is carried out using a C18 reversed-phase column and the optimum mobile phase was a 0.1 M LiClO₄ solution in methanol-water (97:3, v/v) at a flow rate of 1.25 ml/min. 25-OH D₃ and vitamin D₃ were eluted with good resolution at retention times of 3 and 6 minutes respectively, and determined by amperometric detection with a glassy carbon electrode at + 1.050 V (vs Ag/AgCl). Calibration graphs for both substances showed good linearity when amounts of vitamin D₃ between 18 and 312 ng and 27 and 412 ng of 25-OH D₃ were injected. Detection limits of 8 ng (vitamin D₃) and 25 ng (25-OH D₃); relative standard deviations of 3.2% (vitamin D₃) and 5.8% (25-OH D₃) were obtained.     

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.     

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%.     

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.     

Quantification of physiological levels of vitamin D3 and 25‐hydroxyvitamin D3 in porcine fat and liver in subgram sample sizes

Most methods for the quantification of physiological levels of vitamin D₃ and 25‐hydroxyvitamin D₃ are developed for food analysis where the sample size is not usually a critical parameter. In contrast, in life science studies sample sizes are often limited. A very sensitive liquid chromatography with tandem mass spectrometry method was developed to quantify vitamin D₃ and 25‐hydroxyvitamin D₃ simultaneously in porcine tissues. A sample of 0.2–1 g was saponified followed by liquid–liquid extraction and normal‐phase solid‐phase extraction. The analytes were derivatized with 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione to improve the ionization efficiency by electrospray ionization. The method was validated in porcine liver and adipose tissue, and the accuracy was determined to be 72–97% for vitamin D₃ and 91–124% for 25‐hydroxyvitamin D₃. The limit of quantification was <0.1 ng/g, and the precision varied between 1.4 and 16% depending on the level of spiking. The small sample size required for the described method enables quantification of vitamin D₃ and 25‐hydroxyvitamin D₃ in tissues from studies where sample sizes are limited.     

Dilatometric constant for polymerization of α-methylstyrene

By measurement of the specific volume of solutions of poly-α-methylstyrene in α-methylstyrene monomer at 25°C, the dilatometric constant was found to be K_D = (0.002007 ± 0.000030)%^?1. Estimation of the temperature dependence resulted in the equation (K_D)_t = 1.81 × 10^?3 + 7.82 + 10^?6 t, where t denotes temperature in °C.     

The reaction of O(1D) with H2O and the reaction of OH with C3H6

N₂O was photolyzed at 2139 Å to produce O(¹D) atoms in the presence of H₂O and CO. The O(¹D) atoms react with H₂O to produce HO radicals, as measured by CO₂ production from the reaction of OH with CO. The relative importance of the various possible O(¹D )–H₂O reactions is The relative rate constant for O(¹D) removal by H₂O compared to that by N₂O is 2.1, in good agreement with that found earlier in our laboratory. In the presence Of C₃H₆, the OH can be removed by reaction with either CO or C₃H₆: From the CO₂ yield, k₃/k₂ = 75,0 at 100°C and 55.0 at 200°C to within ± 10%. When these values are combined with the value of k₂ = 7.0 × 10^?13exp (–1100/RT) cm³/sec, k₃ = 1.36 × 10^?11 exp (–100/RT) cm³/sec. At 25°C, k₃ extrapolates to 1.1 × 10^?11 cm³/sec.     

Rate Constants and Activation Energies for Gas‐Phase Reactions of Three Cyclic Volatile Methyl Siloxanes with the Hydroxyl Radical

Reaction with hydroxyl radicals (OH) is the major pathway for removal of cyclic volatile methyl siloxanes (cVMS) from air. We present new measurements of second‐order rate constants for reactions of the cVMS octamethylcyclotetrasiloxane (D₄), decamethylcyclopentasiloxane (D₅), and dodecamethylcyclohexasiloxane (D₆) with OH determined at temperatures between 313 and 353 K. Our measurements were made using the method of relative rates with cyclohexane as a reference substance and were conducted in a 140‐mL gas‐phase reaction chamber with online mass spectrometry analysis. When extrapolated to 298 K, our measured reaction rate constants of D₄ and D₅ with the OH radical are 1.9 × 10^?12 (95% confidence interval (CI): (1.7–2.2) × 10^?12) and 2.6 × 10^?12 (CI: (2.3–2.9) × 10^?12) cm³ molecule^?1 s^?1, respectively, which are 1.9× and 1.7× faster than previous measurements. Our measured rate constant for D₆ is 2.8 × 10^?12 (CI: (2.5–3.2) × 10^?12) cm³ molecule^?1 s^?1 and to our knowledge there are no comparable laboratory measurements in the literature. Reaction rates for D₅ were 33% higher than for D₄ (CI: 30–37%), whereas the rates for D₆ were only 8% higher than for D₅ (CI: 5–10%). The activation energies of the reactions of D₄, D₅, and D₆ with OH were not statistically different and had a value of 4300 ± 2800 J/mol.     

Determination of gold in geological samples by x-ray fluorescence spectrometry after extraction with tributyl phosphate

The behaviour of gold and elements impeding its x-ray fluorescence spectrometric (XRF) determination, namely zinc, lead and arsenic, was studied during their extraction from hydrochloric acid, nitric acid, and aqua regia solutions using tributyl phosphate as a solid extractant [SE(TBP)]. Extraction of gold from pulps after aqua regia leaching was found to be the most favourable approach for the quantitative and selective recovery of gold. The gold distribution ratio, D_Au, is ca. 10⁴ ml g^?1. For extraction from hydrochloric acid solutions the D_Au value also exceeds 10⁴ in the whole range of gold concentrations studied (10^?8?10^?4 M), but it decreases substantially with increasing extraction temperature, from 5 × 10⁵ ml g^?1 at 20°C to 9 × 10³ ml g^?1 at 70°C. An anomalously high distribution ratio of lead, D_Pb ≈ 10³ ml g^?1, was observed during extraction from hydrochloric solutions in the presence of chlorine. This could be explained by the formation of the chloro complexes of lead(IV). An XRF method for the determination of gold in natural samples was developed, which includes back-extraction of gold from SE(TBP) using a hot 0.025 M thiourea solution, providing a thin sample layer for secondary XRF. For 25 g of sample material the limit of determination is 10 ng g^?1 (10^?6%). The accuracy of the technique was checked using different reference materials. The results agreed within 10%.     

Lifetime and collisional depopulation of the metastable 5D 3/2-state of Yb+

The lifetime and collisional depopulation rates of the metastable 5D _3/2 state of Yb⁺ have been determined in a radiofrequency ion trap by observation of the fluorescence count rate after ion excitation by a short laser pulse. From measurements using He, N₂ and H₂ as buffer gases between 10^?8 and 10^?6 mbar pressure and linear extrapolation to zero pressure we obtain a lifetime of τ=52.15±1.00 ms and rate constants ofR(H₂)=(1.02±0.10)×10^?9 cm³/s andR(N₂)=(1.78±0.19)×10^?10 cm³/s. The lifetime is in fair agreement with a calculated value of 74 ms.     

Ultrasensitive Determination of Vitamin B12 Using Disposable Graphite Screen‐Printed Electrodes and Anodic Adsorptive Voltammetry

A facile, rapid and ultra‐sensitive method for the determination of vitamin B₁₂ (cyanocobalamin) at the sub‐nanomolar concentration range by using low‐cost, disposable graphite screen‐printed electrodes is described. The method is based on the cathodic preconcentration of square planar vitamin B_12s, as occurred due to the electro reduction of Co(III) center in vitamin B_12a to Co(I), at ?1.3 V versus Ag/AgCl/3 M KCl for 40 s. Then, an anodic square wave scan was applied and the height of the peak appeared at ca. ?0.73 V versus Ag/AgCl/3 M KCl, due to the oxidation of Co(I) to Co(II) in the adsorbed molecule, was related to the concentration of the vitamin B₁₂ in the sample. EDTA was found to serve as a key‐component of the electrolyte by eliminating the background signal caused by metal cations impurities contained in the electrolyte (0.1 M phosphate buffer in 0.1 M KCl, pH 3). It also blocks trace metals contained in real samples, thus eliminating their interference effect. The method was optimized to various working parameters and under the selected conditions the calibration curve was linear over the range 1×10^?10–8×10^?9 mol L^?1 vitamin B₁₂ (R²=0.994), while the limit of detection for a signal‐to‐noise ratio of 3 (7×10^?11 mol L^?1 vitamin B₁₂) is the lowest value of any reported in the literature for the electrochemical determination of vitamin B₁₂. The sensors were successfully applied to the determination of vitamin B₁₂ in pharmaceutical products.     

Photocurrent kinetics during electron emission from metal into electrolyte solution: Part III. H3O+ solutions

The photocurrent kinetics in acid solutions have been investigated. The diffusion coefficients of atoms H?((7±2)×10^?5cm²s^?1) and D?((4±1)×10^?5cm²s^?1) and OH^? and OD^? radicals ((1±0.3)×10^?5cm²s^?1) are found. The rate constants of capture of solvated electrons by H₃O⁺ and D₃O⁺ ions are identical and equal to (8±1)×10⁹M^?1s^?1. From the shape of the kinetic curves it follows that electrochemical desorption of atomic hydrogen occurs from the adsorbed state. The rate constant of this process has been measured. It is shown that the rate constant of electrochemical desorption depends only slightly on the potential.     

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 Albumin Using the Chemiluminescence of Tb3+/K2S2O8 System

A new chemiluminescence system of Tb³⁺/K₂S₂O₈ was developed for the determination of albumin. Some experimental conditions were examined and optimized. The linear ranges of the calibration curves are 5.0 × 10^?9–5.0 × 10^?6 mol/L for bovine serum albumin, 5.0 × 10^?8–1.0 × 10^?5 mol/L for human serum albumin and 2.5 × 10^?7–1.0 × 10^?5 mol/L for ovalbumin, and the corresponding detection limits are 1.9 × 10^?9 mol/L, 1.5 × 10^?8 mol/L, and 1.5 × 10^?7 mol/L, respectively. The method was applied to the determination of albumin in human serum samples, and the results were in agreement with those obtained by the bromcresol green method. The relative errors for the analytical results were from ?2.0% to 4.3%.     

Untersuchungen über (1:1)-Chelate des Curcumins mit Borsäure und Phenylborsäuren

The existence of 1∶1 boron-curcumin chelates in solution has been proved by the straight-line method of Asmus. The compounds with boric resp. phenylboronic acid propably exist as diacetato-resp. phenylacetato-boron chelates of curcumin. The dissociation constants of the 1∶1 complexes of curcumin and boron are K _D =4×10^?5 with boric acid in acetic acid and 3×10^?4 in dioxan, 1×10^?3 with phenylboronic acid in dioxan and 1×10^?4 when stabilized with phenol, 7×10^?5 with diphenylborinic acid in dioxan solution. All boron-curcumin chelates are formed from the quinonoid protonized form of curcumin, which can be stabilized by phenol.     

Analysis of urinary vitamin D3 metabolites by liquid chromatography/tandem mass spectrometry with ESI-enhancing and stable isotope-coded derivatization

The determination of the urinary vitamin D₃ metabolites might prove helpful in the assessment of the vitamin D status. We developed a method for the determination of trace vitamin D₃ metabolites, 25-hydroxyvitamin D₃ [25(OH)D₃] and 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], in urine using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using an ESI-enhancing reagent, 4-(4′-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), and its isotope-coded analogue, ²H₄-DAPTAD (d-DAPTAD). The urine samples were treated with β-glucuronidase, purified with an Oasis® hydrophilic–lipophilic balanced (HLB) cartridge, and then subjected to the derivatization. The DAPTAD derivatization enabled the highly sensitive detection (detection limit, 0.25 fmol on the column), and the use of d-DAPTAD significantly improved the assay precision [the intra- (n?=?5) and inter-assay (n?=?3) relative standard deviations did not exceed 9.5 %]. The method was successfully applied to urine sample analyses and detected the increases of the urinary 25(OH)D₃ and 24,25(OH)₂D₃ levels due to vitamin D₃ administration. Graphical Abstract

Scheme of procedure for urinary vitamin D₃ metabolite analysis based on LC/MS/MS with ESI-enhancing and isotope-coded derivatization.     

Simultaneous determination of dopamine, uric acid and ascorbic acid with LaFeO3 nanoparticles modified electrode

LaFeO₃ nanoparticles of approximately 22 nm in size were synthesized and characterized by XRD and TEM. A novel glassy carbon electrode modified with LaFeO₃ nanoparticles was constructed and characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The modified electrode exhibited strong promoting effect and high stability toward the electrochemical oxidation of dopamine (DA), which gave reversible redox peaks with a formal potential of 0.145 V (vs. Ag/AgCl) in pH 7.0 phosphate buffer solution. The anodic peak current (measured by constant potential amperometry) increased linearly with the concentration of dopamine in the range from 1.5?×?10^?7 to 8.0?×?10^?4 M. The detection limit was 3.0?×?10^?8 M. The relative standard deviation of eight successive scans was 3.47% for 1.0?×?10^?6 M DA. The interference by ascorbic acid was eliminated efficiently. The method was used to determine DA in dopamine hydrochloride injections and showed excellent sensitivity and recovery.     

Determination of trace amounts of nickel by differential pulse adsorptive cathodic stripping voltammetry using calconcarboxylic acid as a chelating agent

A method for the determination of nickel(II) by stripping voltammetry is described. The method is based on the adsorptive accumulation of nickel(II) calconcarboxylic acid complex onto a hanging mercury drop electrode (HMDE), followed by the reduction of the adsorbed complex using differential pulse voltammetry. The optimum operating conditions and parameters were found to be 0.05 M NH₃/NH₄Cl buffer (pH = 9.5) as the supporting electrolyte, a ligand concentration of 1 × 10^?6 M, accumulation potential of ?0.5 V (vs. Ag/AgCl) and accumulation time of 60 s. At the optimized conditions, the peak current is proportional to the concentration of nickel in the range of 1.7 × 10^?9 to 4.7 × 10^?7 M (0.1–28 ng ml^?1) with a detection limit of 0.05 ng ml^?1. The relative standard deviation (n = 10) at nickel concentrations of 2, 10 and 15 ng ml^?1 varies in the range 0.76 to 2.1%. Possible interferences by metal ions, which are of great significance in real matrices, have been studied. The method was successfully applied to the determination of nickel content in a chocolate sample.