Determination of phylloquinone (vitamin K1) by high performance liquid chromatography with UV detection and with particle beam-mass spectrometry

A reversed-phase PB LC-MS method was used for the determination and unequivocal identification of vitamin K(1) in some vegetable samples. The method was also applied to the identification of other constituents in the food analyzed. LC-MS experiments were carried out in both EI and negative-ion CI modes. Preliminary sensitivity studies on vitamin K(1) showed a detection limit of 2 ng (signal-to-noise ratio=3) in negative-ion operation and in single-ion monitoring at m/z 450. Quantitative results obtained with LC-MS for the extracts of four vegetables were compared with those of LC-UV analyses performed at 247 nm.     

LC-UV Column-Switching for Quantitation of Vitamin K1 in Infant Formula

This study was undertaken in order to develop an analytical method for vitamin K₁ in infant formula. The content of vitamin K₁ was investigated by using a column-switching LC-UV method. A Certified Reference Material sample of infant formula containing 0.94 ± 0.04 mg kg^?1 of vitamin K₁ was extracted with hexane followed by enzymatic digestion of fat and precipitation of the fatty acids. The linearity of this method was calculated using five consecutive standard curves, and the coefficient of determination (r ²) was found to be 0.9995. The limit of detection (LOD) and the limit of quantitation (LOQ) was 3.31 and 11.12 μg L^?1, respectively. The accuracy of intra- and inter-day measurements was in the range from 96.67 to 108.67%, and the precision of intra- and inter-day measurements was less than 5.13%. The recoveries were 109.27 ± 5.92%, and the recoveries of inter-laboratory results were in the range from 97.59 ± 1.29 to 109.27 ± 5.92%. The newly developed method uses the optimum conditions required to determine the content of vitamin K₁ in infant formula.     

Selective chemiluminescence method for monitoring of vitamin K homologues in rheumatoid arthritis patients

Vitamin K is a fat-soluble vitamin involved in blood coagulation and bone metabolism. The detection and monitoring of vitamin K homologues in rheumatoid arthritis (RA) patients is a challenging problem due to the smaller concentrations of vitamin K and the presence of several interfering medications. Therefore, this study aimed to develop a new highly sensitive and selective chemiluminescence (CL) method designated to quantify vitamin K homologues in plasma of RA patients including phylloquinone (PK, vitamin K₁), menaquinone-4 (MK-4, vitamin K₂) and menaquinone-7 (MK-7, vitamin K₂). The method was based on the unique photochemical properties of vitamin K homologues that were exploited for selective luminol CL reaction. The correlation coefficients of 0.998 or more were obtained in the concentration ranges of 0.1-100 ng mL⁻¹ vitamin K homologues. The detection limits were 0.03-0.1 ng mL⁻¹ in human plasma for vitamin K homologues. The developed HPLC-CL system was successfully applied for selective determination of vitamin K homologues in plasma of RA patients. The developed method may provide a useful tool for monitoring vitamin K homologues in different clinical studies such as RA, osteoporosis and hepatocellular carcinoma in which vitamin K is intervented.     

Determination of the diastereomers of vitamin K1

A method is described for the gas-chromatographic determination of the diastereomers of vitamin K₁ (phylloquinone) in the form of their dihydro dimethyl ethers. The reported method of derivatization and chromatographic analysis in conjunction with optical rotation measurements are useful approaches for characterization of vitamin K₁ samples with respect to their origin.     

Determination of vitamin k1 in plasma by differential pulse polarography and its possible clinical application

Differential pulse polarography, following solvent extraction, is used to monitor the clearance of vitamin K₁ (2-methyl-3-phytyl-1,4-naphthoquinone) from human plasma after a 20-mg intravenous injection. The average recovery of vitamin K₁ added to plasma (200–3000 ng ml^-1) was 72.2%. The coefficient of variation was 3.0% at a concentration of 2.75 μg ml^-1 of plasma. Measurements of vitamin K₁ in plasma from patients given an intravenous injection of the vitamin, support the idea that a metabolic cycle involving vitamin K₁ underlies calcification of bone.     

An LC-MS/MS Method for Analysis of Vitamin D Metabolites and C3 Epimers in Mice Serum: Oral Supplementation Compared to UV Irradiation

Introduction: The most common forms of vitamin D in human and mouse serum are vitamin D3 and vitamin D2 and their metabolites. The aim of this study is to determine whether diet and sunlight directly affect the circulating concentrations of vitamin D metabolites in a mouse model. We investigated the serum concentrations of eight vitamin D metabolites—vitamin D (vitamin D3 + vitamin D2), 25OHD (25OHD3 + 25OHD2), 1α25(OH)₂D (1α25(OH)₂D2, and 1α25(OH)₂D3)—including their epimer, 3-epi-25OHD (3-epi-25OHD3 and 3-epi-25OHD2), and a bile acid precursor 7alpha-hydroxy-4-cholesten-3-one (7αC4), which is known to cause interference in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Method: The LC-MS/MS method was validated according to FDA-US guidelines. The validated method was used for the analysis of mouse serum samples. Forty blood samples from mice were collected and divided into three groups. The first group, the DDD mice, were fed a vitamin D-deficient diet (25 IU VD3/kg of diet) and kept in the dark; the second group, the SDD mice, were maintained on a standard-vitamin D diet (1000 IU VD3) and kept in the dark; and the third group, SDL, were fed a standard-vitamin D diet (1000 IU VD3) but kept on a normal light/dark cycle. LC-MS/MS was used for the efficient separation and quantitation of all the analytes. Results: The validated method showed good linearity and specificity. The intraday and interday precision were both <16%, and the accuracy across the assay range was within 100 ± 15%. The recoveries ranged between 75 and 95%. The stability results showed that vitamin D metabolites are not very stable when exposed to continuous freeze–thaw cycles; the variations in concentrations of vitamin D metabolites ranged between 15 and 60%. The overlapping peaks of vitamin D, its epimers, and its isobar (7αC4) were resolved using chromatographic separation. There were significant differences in the concentrations of all metabolites of vitamin D between the DDD and SDL mice. Between the groups SDD (control) and SDL, a significant difference in the concentrations of 3-epi-25OHD was noted, where C3 epimer was about 30% higher in SDL group while no significant differences were noted in the concentrations of vitamin D, 25OHD, 1α25(OH)₂D, and 7αC4 between SDD and SDL group. Conclusions: A validated method, combined with a simple extraction technique, for the sensitive LC-MS/MS determination of vitamin D metabolites is described here. The method can eliminate the interferences in LC-MS/MS analysis caused by the overlapping epimer and isobar due to them having the same molecular weights as 25OHD. The validated method was applied to mouse serum samples. It was concluded that a standard-vitamin D diet causes an increase in the proportion of all the vitamin D metabolites and C3 epimers and isobar, while UV light has no pronounced effect on the concentrations of the majority of the vitamin D metabolites except 3-epi-25OHD. Further studies are required to confirm this observation in humans and to investigate the biochemical pathways related to vitamin D’s metabolites and their epimers.     

THE USE OF CARBOXYMETHYL ETHOXYLATE SURFACTANT TO DISSOLVE VITAMIN K1 AND TO OBTAIN LOW INTERFACIAL TENSION AND STABLE EMULSION SYSTEMS

Abstract

The presence of vitamin K₁ in human body is important for preventing the hemorrhagic disease. Due to its very long side chain, vitamin K₁ is highly insoluble in water. We have successfully dissolve a substantial amount of vitamin K₁ in solutions of a commercial surfactant containing carboxymethyl ethoxylates (Hüls B433) and obtained low interfacial tension (IFT) and stable emulsion systems. This paper will present the details of these experiments. The solubilization of vitamin K₁ was estimated from UV absorption. The IFT values were measured by using a spinning drop apparatus and all particle sizes were determined by using laser light scattering. By using the Hüls B433 surfactant and an optimum amount of CaCl₂, we can dissolve vitamin K₁ in water and obtain low IFT systems in the order of 10^?2 dyne/cm. The emulsions obtained in these systems are stable and contain droplet sizes below 65 nm. The dissolution of vitamin K₁ and the IFT behavior in these systems follow the rules for crude oil and prefer larger surfactant micelles.     

Determination of vitamin K<Subscript>1</Subscript> in blood serum by high-performance liquid chromatography

A method is proposed for the determination of vitamin K₁ concentration in blood serum using HPLC on a Gemini C18 (250 mm × 4.6 mm ID, 5 μm) column and elution with a methanol–acetonitrile–dichloromethane (45 : 50 : 5, v/v) mobile phase at a flow rate of 1 mL/min and detection at 248 nm. The limit of detection for the vitamin is 0.5 ng/mL and relative error is 19%. The volume of sample in the column is 100 μL. The method has been applied to the evaluation of vitamin K₁ deficit in a human body in medical practice.     

Development and validation of a gas chromatographic—mass spectrometric procedure for the identification and quantification of residues of chloramphenicol

A method for the identification and quantification of residues of the antibiotic chloramphenicol was developed and validated. The method is based on combined gas chromatography-mass spectrometry with negative-ion chemical ionization and the use of [³⁷Cl₂]chloramphenicol as an internal standard. A set of identification criteria, in accordance with guidelines of the European Community, is described. For urine, muscle and eggs limits of detection and quantification of 0.1 μg kg^?1 are obtained. The method shows good repeatability and reproducibility. Results for urine were compared with those obtained with a radioimmunochemical procedure and an enzyme immunoassay (Quik-Card). Screening with an immunochemical procedure followed by confirmation with gas chromatography-mass spectrometry was found to be an effective strategy for monitoring residues of chloramphenicol in biological matrices.     

Sequential determination of fat- and water-soluble vitamins in Rhodiola imbricata root from trans-Himalaya with rapid resolution liquid chromatography/tandem mass spectrometry

A rapid method was developed to determine both types of vitamins in Rhodiola imbricata root for the accurate quantification of free vitamin forms. Rapid resolution liquid chromatography/tandem mass spectrometry (RRLC–MS/MS) with electrospray ionization (ESI) source operating in multiple reactions monitoring (MRM) mode was optimized for the sequential analysis of nine water-soluble vitamins (B₁, B₂, two B₃ vitamins, B₅, B₆, B₇, B₉, and B₁₂) and six fat-soluble vitamins (A, E, D₂, D₃, K₁, and K₂). Both types of vitamins were separated by ion-suppression reversed-phase liquid chromatography with gradient elution within 30 min and detected in positive ion mode. Deviations in the intra- and inter-day precision were always below 0.6% and 0.3% for recoveries and retention time. Intra- and inter-day relative standard deviation (RSD) values of retention time for water- and fat-soluble vitamin were ranged between 0.02–0.20% and 0.01–0.15%, respectively. The mean recoveries were ranged between 88.95 and 107.07%. Sensitivity and specificity of this method allowed the limits of detection (LOD) and limits of quantitation (LOQ) of the analytes at ppb levels. The linear range was achieved for fat- and water-soluble vitamins at 100–1000 ppb and 10–100 ppb. Vitamin B-complex and vitamin E were detected as the principle vitamins in the root of this adaptogen which would be of great interest to develop novel foods from the Indian trans-Himalaya.     

Characterization and traceability of two generations of standard reference material for the measurement of vitamin K1(phylloquinone) at endogenous concentrations in human plasma and serum

Vitamin K is an essential micronutrient required for blood coagulation, regulation of vascular calcification and bone mineralization. Plasma and serum measurements of vitamin K₁(phylloquinone, K₁) made using high-performance liquid chromatography with fluorescence detection, or tandem mass spectrometry are used clinically and in population studies to assess vitamin K status. Standard reference materials provide a validation tool for laboratories, helping assure clinical diagnosis and the comparability of data from different populations. We manufactured two K₁ standard reference materials, in 2009 (KEQAS SRM-001) and in 2019 (KEQAS SRM-002). The target concentrations of K₁ were assigned to each SRM using the All Laboratory Trimmed Mean of results reported by selected laboratories enrolled in the Vitamin K External Quality Assurance Scheme (KEQAS). The assigned concentrations of K₁ for KEQAS SRM-001 and SRM-002 were 0.25 and 0.36 μg/L respectively. In 2019 KEQAS SRM-001 was re-analysed simultaneously with KEQAS SRM-002 to provide traceability between the two standards, therefore aiding comparability of analysis performed using these materials. Both standards were stored as aliquots at −80°C in the dark; annual re-analysis of the materials indicated that K₁ is stable for at least 12 years in these conditions.     

The reaction of superoxide ion with vitamin K1 and its related compounds

The reaction of potassium superoxide with vitamin K₁ and its related compounds in the persence of crown ether gave the corresponding 2,3-oxide and phthalic acid. The oxidative cleavage of oxirane ring by superoxide ion has been observed with vitamin K₁ 2,3-oxide and certain epoxides.     

Electrochemical Studies of Vitamin K1 Microdroplets: Electrocatalytic Hydrogen Evolution

The voltammetry of a basal-plane pyrolytic graphite electrode modified with a random ensemble of unsupported microdroplets of vitamin K₁ is investigated when the electrode is immersed in aqueous electrolytes. It is shown that in dilute acidic solutions, electroreduction occurs in a single two-electron two-proton process to yield the corresponding hydroquinone at the electrode|vitamin K₁ microdroplet|aqueous-electrolyte three-phase boundary. On addition of ionic alkali-metal salts to the aqueous acidic phase, the electrochemical reduction of vitamin K₁ to the quinol is accompanied by catalytic hydrogen evolution within and alkali-metal-cation insertion into the organic microdroplets. In strongly alkaline solutions, electrochemical reduction of vitamin K₁ at the triple-phase junction is proposed as being a single two-electron process with concomitant uptake of alkali-metal cations in order to maintain electroneutrality within the oil phase. Surprisingly, the relative ease of cation insertion into the oil phase is demonstrated to be governed by the degree of ion-pair formation rather than by the Gibbs transfer energy of the cation across the liquid|liquid interface.     

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.     

Electrosorption of vitamin K1 at mercury and its determination at submicrogram levels by differential pulse voltammetry at a hanging mercury electrode

The electrochemical behaviour of vitamin K₁ has been studied in ethanolic and methanolic acetate buffers by cyclic voltammetry and differential pulse voltammetry (d.p.v.) at the h.m.d.e. and differential pulse polarography at the d.m.e. Increased adsorption occurs at the mercury electrode as the percentage of water is increased. The most sensitive signal was obtained by d.p.v. with acetate-buffered 60% methanolic solutions; vitamin K₁ could then be measured down to 10 ng ml^-1.     

高效液相色谱-质谱分离鉴定荧光试剂标记的脂肪酸

以2-(2-(10-蒽基)-萘[2,3-d]咪唑)-乙基-对甲苯磺酸酯(ANITS)作为柱前衍生化试剂,在Eclipse XDB-C8色谱柱上,梯度洗脱实现了20种游离脂肪酸(FFA)衍生物的完全基线分离。90℃下在DMF溶剂中以K2CO3作催化剂,选取衍生试剂摩尔数为脂肪酸的7倍,衍生反应40min可获得稳定的荧光产物。激发和发射波长分别为250nm和512nm。采用大气压化学电离源(APCI)正离子模式,实现了油菜蜂花粉中游离脂肪酸的质谱鉴定。所有脂肪酸的线性相关系数均大于0.9999,检出限为24.76~98.79fmol。     

Vitamin K1 distribution following intravenous vitamin K1–fat emulsion administration in rats

This study investigated vitamin K₁ (VK₁) distribution following intravenous vitamin K₁–fat emulsion (VK₁–FE) administration and compared it with that after VK₁ injection. Rats were intravenously injected with VK₁–FE or VK₁. The organ and tissue VK₁ concentrations were determined using high‐performance liquid chromatography method at 0.5, 2 and 4 h to determine distribution, equilibrium and elimination phases, respectively. In the VK₁–FE group, the plasma, heart and spleen VK₁ concentrations decreased over time. However, other organs like liver, lung, kidney, muscle and testis, reached peak VK₁ concentrations at 2 h. In the VK₁ injection group, the liver VK₁ concentrations were significantly higher than those in other organs at the three time points. However, VK₁ concentrations in the other organs peaked at 2 h. In addition, in VK₁–FE group, the heart, spleen and lung VK₁ concentrations were significantly higher than those in the VK₁ injection group at the three time points, and the liver VK₁ concentration was significantly higher than that in the VK₁ injection group at 4 h. The VK₁ amount was greatest in the liver compared with the other organs. Thus, the liver is the primary organ for VK₁ distribution. The distribution of VK₁ is more rapid when injected as VK₁–FE than as VK₁. Copyright © 2015 John Wiley & Sons, Ltd.     

Nuclear magnetic resonance and liquid chromatography-mass spectrometry combined with an incompleted separation strategy for identifying the natural products in crude extract

NMR and LC-MS combined with an incompleted separation strategy were proposed to the simultaneous structure identification of natural products in crude extracts, and a novel method termed as NMR/LC-MS parallel dynamic spectroscopy (NMR/LC-MS PDS) was developed to discover the intrinsic correlation between retention time (Rt), mass/charge (m/z) and chemical shift (δ) data of the same constituent from mixture spectra by the co-analysis of parallelly visualized multispectroscopic datasets from LC-MS and ¹H NMR. The extracted ion chromatogram (XIC) and ¹H NMR signals deriving from the same individual constituent were correlated through fraction ranges and intensity changing profiles in NMR/LC-MS PDS spectrum due to the signal amplitude co-variation resulted from the concentration variation of constituents in a series of incompletely separated fractions. NMR/LC-MS PDS was applied to identify 12 constituents in an active herbal extract including flavonol glycosides, which was separated into a series of fractions by flash column chromatography. The complementary spectral information of the same individual constituent in the crude extract was discovered simultaneously from mixture spectra. Especially, two groups of co-eluted isomers were identified successfully. The results demonstrated that NMR/LC-MS PDS combined with the incompleted separation strategy achieved the similar function of on-line LC-NMR-MS analysis in off-line mode and had the potential for simplifying and accelerating the analytical routes for structure identification of constituents in herbs or their active extracts.     

Analysis of triacylglycerol hydroperoxides in human lipoproteins by Orbitrap mass spectrometer

Herein, we represent a simple method for the detection and characterization of molecular species of triacylglycerol monohydroperoxides (TGOOH) in biological samples by use of reversed-phase liquid chromatography with a LTQ Orbitrap XL mass spectrometer (LC/LTQ Orbitrap) via an electrospray ionization source. Data were acquired using high-resolution, high-mass accuracy in Fourier-transform mode. Platform performance, related to the identification of TGOOH in human lipoproteins and plasma, was estimated using extracted ion chromatograms with mass tolerance windows of 5 ppm. Native low-density lipoproteins (nLDL) and native high-density lipoproteins (nHDL) from a healthy donor were oxidized by CuSO₄ to generate oxidized LDL (oxLDL) and oxidized HDL (oxHDL). No TGOOH molecular species were detected in the nLDL and nHDL, whereas 11 species of TGOOH molecules were detected in the oxLDL and oxHDL. In positive-ion mode, TGOOH was found as [M + NH₄]⁺. In negative-ion mode, TGOOH was observed as [M + CH₃COO]^–. TGOOH was more easily ionized in positive-ion mode than in negative-ion mode. The LC/LTQ Orbitrap method was applied to human plasma and three molecular species of TGOOH were detected. The limit of detection is 0.1 pmol (S/N?=?10:1) for each synthesized TGOOH.

Preparation of 2-methyl-1,4-naphthoquinone (vitamin K3) by catalytic oxidation of 2-methyl-1-naphthol in the presence of iron phthalocyanine supported catalyst

Iron tetrasulfophthalocyanine (FePcS) supported catalyst was prepared by covalent grafting onto amino-modified silica by a novel practical one-pot method using activation of sulfonate groups of FePcS by triphenylphosphine triflate. FePcS–SiO₂ in combination with ^tBuOOH behaved as an efficient catalyst for the oxidation of 2-methyl-1-naphthol to 2-methyl-1,4-naphthoquinone, vitamin K₃. To optimise the catalytic system, the influence of different reaction parameters on the efficiency of this oxidation has been studied. Vitamin K₃ was obtained with 59% selectivity at 96% conversion of 2-methyl-1-naphthol using only 0.5 mol% of catalyst. ¹⁸O labelling experiments indicate a non-radical mechanism for this oxidation.