Rapid simultaneous determination of 7 fat-soluble vitamins in human serum by ultra high performance liquid chromatography tandem mass spectrometry.

Accurate detection of vitamins is critically important for clinical diagnosis, metabolomics and epidemiological studies. However, the amounts of different vitamins vary dramatically in human serum. It is a challenge to achieve simultaneous detection of multiple vitamins rapidly. Herein, we developed and validated a sensitive and specific method using ultra high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for simultaneous quantification of 7 fat-soluble vitamins (FSVs) across their physiological concentrations in serum for the first time, which was subjected to protein precipitation, liquid-liquid extraction to an organic phase, evaporation to dryness and reconstitution with acetonitrile. In the present procedure, retinol (vitamin A), ergocalciferol (25-OH-D2), cholecalciferol (25-OH-D3), α-tocopherol (vitamin E), phylloquinone (vitamin K1), menatetrenone-4 (MK-4), and menaquinone-7 (MK-7) were detected in one analytical procedure for the first time within 5.0 min by triple quadrupole tandem mass spectrometry. The limit of quantification (LOQ) for vitamin A was 10.0 ng mL-1, LOQs for 25-OH-D2 and 25-OH-D3 were 1.0 ng mL-1, LOQ for vitamin E was 100.0 ng mL-1, and LOQs for vitamin K1, MK-4 and MK-7 were 0.10 ng mL-1, respectively, with a correlation (R2) of 0.995-0.999. Recoveries ranged from 80.5% to 118.5% and the intra-day and inter-day coefficients of variance (CVs) were 0.72-8.89% and 3.2-9.0% respectively. The method was validated according to the European Medicines Agency (EMA) and U.S. Food and Drug guidelines and C62-A on bioanalytical methods, and was used for clinical routine determination.

Determination of vitamin K in infant formulas by liquid chromatography-online electrochemical reduction based on porous Pt/Ti electrode-fluorescence detection.

A highly sensitive and selective HPLC method for the determination of vitamin K vitamers including phylloquinone (PK) and menaquinones (MK-4) in infant formulas is described. The K vitamers were quantified with a fluorescence detector after online post-column electrochemical reduction occurring in a laboratory-made electrochemical reactor (ECR) equipped with platinum plated porous titanium (Pt/Ti) electrodes. The morphology of the electrode showed that the grain size of Pt was homogeneous and well plated on the porous Ti substrate, resulting in largely improved electrochemical reduction efficiency due to the large specific surface area. In addition, the operation parameters such as mobile phase/supporting electrolyte and working potential were optimized. The detection limits of PK and MK-4 were 0.81 and 0.78 ng g-1. Infant formula varying in stages were detected, showing PK ranged from 26.4 to 71.2 µg/100 g, while MK-4 was not detected.

Vitamin K content of cheese, yoghurt and meat products in Australia.

Vitamin K is vital for normal blood coagulation, and may influence bone, neurological and vascular health. Data on the vitamin K content of Australian foods are limited, preventing estimation of vitamin K intakes in the Australian population. We measured phylloquinone (PK) and menaquinone (MK) -4 to -10 in cheese, yoghurt and meat products (48 composite samples from 288 primary samples) by liquid chromatography with electrospray ionisation-tandem mass spectrometry. At least one K vitamer was found in every sample. The greatest mean (± standard deviation for foods sampled in multiple cities) concentrations of PK (4.9 µg/100 g), MK-4 (58 ± 9 µg/100 g) and MK-9 (8 ± 2 µg/100 g) were found in lamb liver, chicken leg meat and Cheddar cheese, respectively. Cheddar cheese (1.1 ± 0.3 µg/100 g) and cream cheese (1.0 µg/100 g) contained MK-5. MK-8 was found in Cheddar cheese only (4 ± 2 µg/100 g). As the K vitamer profile and concentrations appear to vary considerably by geographical location, Australia needs a vitamin K food composition dataset that is representative of foods consumed in Australia.

Carotenoids, tocopherols and phylloquinone content of 26 green leafy vegetables commonly consumed in Southeast Asia.

Carotenoids, tocopherols and phylloquinone are highly valued in vegetables due to their potential health benefits. The profile of eight carotenoids, four tocopherols, and phylloquinone in 26 green leafy vegetables (GLV) commonly consumed in Southeast Asia were analysed by high-performance liquid chromatography with atmospheric pressure chemical ionisation and tandem mass spectrometry (HPLC-APCI-MS/MS). Lutein, ß-carotene and α-tocopherol were the predominant carotenoids and tocopherol in the GLV. Among 26 GLV, sweet leaf bush contained the highest amount of total carotenoids (494 ± 22 µg/g fresh weight (FW)), tocopherols (214 ± 60 µg/g FW) and phylloquinone (18 ± 2 µg/g FW). Other underutilised GLV, including wolfberry leaves, cassava leaves and moringa leaves, are also a rich source of fat-soluble micronutrients. Overall, this study enhanced the understanding of micronutrient composition in underutilised GLV in Southeast Asia. Data will be important for diet recommendations to promote the nutritional status of the population in the region.

Dietary Vitamin K Intake and the Risk of Pancreatic Cancer: A Prospective Study of 101,695 American Adults.

No epidemiologic studies have been conducted to assess the association of intake of dietary vitamin K with the risk of pancreatic cancer. We used prospective data from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial between 1993 and 2009 to fill this gap. A total of 101,695 subjects were identified. Dietary intakes of phylloquinone (vitamin K1), menaquinones (vitamin K2), and dihydrophylloquinone (dihydrovitamin K1) were assessed using a food frequency questionnaire. Cox regression was applied to calculate hazard ratios and 95% confidence intervals. During a mean follow-up of 8.86 years (900,744.57 person-years), 361 cases of pancreatic cancer were documented. In the fully adjusted model, dietary intakes of phylloquinone (for quartile 4 vs. quartile 1, hazard ratio (HR) = 0.57, 95% confidence interval (CI): 0.39, 0.83; P for trend = 0.002) and dihydrophylloquinone (for quartile 4 vs. quartile 1, HR = 0.59; 95% CI: 0.41, 0.85; P for trend = 0.006), but not menaquinones (for quartile 4 vs. quartile 1, HR = 0.93; 95% CI: 0.65, 1.33; P for trend = 0.816), were found to be inversely associated with the risk of pancreatic cancer in a nonlinear dose-response manner (all P values for nonlinearity < 0.05), and this was not modified by predefined stratification factors and remained in sensitivity analyses. In conclusion, dietary intakes of phylloquinone and dihydrophylloquinone, but not menaquinones, confer a lower risk of pancreatic cancer. Future studies should confirm our findings.

Characterization of nutritionally important lipophilic constituents from brown kelp Ecklonia radiata (C. Ag.) J. Agardh.

This research study presents information for the first time on the nutritionally relevant lipophilic compounds obtained from Ecklonia radiata, a poorly studied brown kelp. The major lipophilic compounds were analyzed utilizing liquid chromatography (LC)-tandem mass spectrometry (MS/MS) and gas chromatography (GC)-mass spectrometry (MS). The LC-MS/MS results revealed the presence of eight major lipophilic compounds, including sterols, carotenoids, vitamin E, and phylloquinone (vitamin K1). Quantitative analysis showed that fucosterol was the most predominant phytosterol in the fronds and stipes of E. radiata. The carotenoids (all-E)-fucoxanthin and (all-E)-ß-carotene were present in higher yield. In terms of vitamin E, α-tocopherol was identified as the main tocol. The coenzyme, phylloquinone, important for protein synthesis, was also identified in E. radiata. GC-MS identified 13 fatty acids with palmitic (C16:0) and oleic acid (C18:1n9c) present in the highest quantities. To our knowledge, this is the first report on E. radiata, and the valuable data presented herein can be used as a baseline for developing novel nutraceuticals.

Vitamin K (phylloquinone and menaquinones) in foods - Optimisation of extraction, clean-up and LC-ESI-MS/MS method for quantification.

Growing evidence of vitamin K's importance in human health beyond blood coagulation and bone health necessitates its further research. A method involving extraction, lipase treatment, clean-up, and detection and quantification by LC-ESI-MS/MS of phylloquinone (PK), menaquinone-4 (MK-4), menaquinone-7 (MK-7) and menaquinone-9 (MK-9) was developed, and single-laboratory validated. The matrices included in the validation were hazelnut, cheese, broccoli, and pork. The LC-method runtime was 9 min. The LOQ for PK, MK-4 and MK-7 was 0.5 µg/100 g food, while for MK-9 it was 2.5 µg/100 g food. The intra- and inter-day precision was <15% for endogenous and spiked levels, except for low content at 4 times the LOQ. Trueness was assessed to be in the range 94-125% for spiking at levels approximately 4 and 10 times LOQ. It is further shown that deuterium labelled MK-7 can be used as an internal standard for MK-9.

Development, Optimization, and Comparison of Different Sample Pre-Treatments for Simultaneous Determination of Vitamin E and Vitamin K in Vegetables.

The absence of vitamin E from the diet can lead to cardiovascular disease, cancer, cataracts, and premature aging. Vitamin K deficiency can lead to bleeding disorders. These fat-soluble vitamins are important nutritional factors that can be determined in different methods in vegetables. In this work, the simultaneous determination of α-tocopherol, α-tocopheryl acetate, phylloquinone, and menaquinone-4 by gas chromatography-mass spectrometry (GC-MS) has been optimized using both direct injection and solid phase microextraction (SPME). Three different sample pre-treatment approaches based on: (A) solid-liquid-liquid-liquid extraction (SLE-LLE), (B) SLE, and (C) SPME were then applied to extract the target analytes from vegetables samples using menaquinone as internal standard. All the procedures allowed the determination of the target analytes in onion, carrot, celery, and curly kale samples. Similar results were obtained with the three different approaches, even if the one based on SPME offers the best performance, together with a reduced use of solvent, time consumption, and experimental complexity, which makes it the preferable option for industrial applications.

[Determination of vitamin K1 and vitamin K2 in modulation milk powder by post-column reduction-high performance liquid chromatography].

A method for the determination of vitamin K1 and vitamin K2 in modulation milk powder was developed by high performance liquid chromatography (HPLC) coupled with post-column reduction. The samples were dissolved in water, lipase hydrolyzed, saponified with 2.5 mol/L sodium hydroxide solution and ethanol solution, extracted with n-hexane, and dissolved in methanol after concentration. The vitamin K were first separated on an Xbridge C18 column and then on a zinc powder reduction column, and detected using a fluorescence detector. The excitation and emission wavelengths were 326 nm and 432 nm, respectively. An external standard method was used for quantification. The results showed that the linearities of vitamin K1 and vitamin K2 was in the ranges of 0.0025-2.0 µg/mL and 0.01-2.0 µg/mL, respectively, with correlation coefficients both greater than 0.999. The spiked recoveries were 80.39%-94.39% and the precisions were 0.85%-3.98%. The limits of detection of vitamin K1 and vitamin K2 were 0.07 µg/100 g and 0.2 µg/100 g, respectively. The limits of quantification of vitamin K1 and vitamin K2 were 0.2 µg/100 g and 0.8 µg/100 g, respectively. The method has high sensitivity and good repeatability, and gives accurate results. It is suitable for the analysis and determination of the vitamin K1 and vitamin K2 in formula milk powder.

Dietary Vitamin K and Association with Hepatic Vitamin K Status in a Yup'ik Study Population from Southwestern Alaska.

SCOPE: The relationship between dietary vitamin K and plasma PIVKA-II concentration, a biomarker of hepatic vitamin K status, in a Yup'ik study population in southwestern Alaska is investigated. METHODS AND RESULTS: A total of 659 male and female, self-reported Yup'ik people, ≥14 years of age, were enrolled. Blood is collected for genotyping and plasma PIVKA-II biomarker analysis. A Yup'ik-specific dietary food frequency questionnaire is used to assess vitamin K intake. Among the participants, 22% report not consuming foods rich in vitamin K during the past year and 36% have a PIVKA-II concentration ≥ 2 ng mL-1 , indicating vitamin K insufficiency. The odds of an elevated PIVKA-II concentration are 33% lower in individuals reporting any versus no consumption of vitamin-K-rich foods. The association is significant after adjusting for CYP4F2*3 genotype. Tundra greens are high in vitamin K1 content, but an exploratory analysis suggests that subsistence meat sources have a greater effect on vitamin K status. CONCLUSIONS: A substantial proportion of the Yup'ik population exhibits vitamin K insufficiency, which is associated with low consumption of vitamin K rich foods and which might affect an individual's response to anticoagulant drugs such as warfarin that target the vitamin K cycle.

Introduction of a carbon paste electrode based on nickel carbide for investigation of interaction between warfarin and vitamin K1.

In this paper a novel electrochemical sensor based on nickel carbide (Ni3C) nanoparticles as a new modifier was constructed. Ni3C nanoparticle was synthesized and characterized by scanning electron microscopy, X-ray diffraction and first-principles study. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) studies confirmed the electrode modification. Afterwards, the new electrode for the first time was used for interaction study between vitamin K1 and warfarin as an anticoagulant drug by differential pulse voltammetry. The adduct formation between the drug and vitamin K1 was improved by decreasing in anodic peak current of warfarin in the presence of different amounts of vitamin K1. The binding constant between warfarin and vitamin K1 was obtained by voltammetric and UV-vis and fluorescence spectroscopic methods. The molecular modeling method was also performed to explore the structural features and binding mechanism of warfarin to vitamin K1. The different aspects of modeling of vitamin K1 and warfarin and their adduct structures confirmed the adduct formation by hydrogen bonding.

Phytonadione Content in Branded Intravenous Fat Emulsions.

BACKGROUND: Intravenous fat emulsions (IVFE) with different fatty acid compositions contain vitamin E as a by-product of vegetable and animal oil during the refining processes. Likewise, other lipid-soluble vitamins may be present in IVFE. No data, however, exist about phytonadione (vitamin K1) concentration in IVFE information leaflets. Therefore, our aim was to evaluate the phytonadione content in different IVFE. MATERIALS AND METHODS: Analyses were carried out in triplicate on 6 branded IVFE as follows: 30% soybean oil (100%), 20% olive-soybean oil (80%-20%), 20% soybean-medium-chain triglycerides (MCT) coconut oil (50%-50%), 20% soybean-olive-MCT-fish oil (30%-25%-30%-15%), 20% soybean-MCT-fish oil (40%-50%-10%), and 10% pure fish oil (100%). Phytonadione was analyzed and quantified by a quali-quantitative liquid chromatography-mass spectrometry (LC-MS) method after its extraction from the IVFE by an isopropyl alcohol-hexane mixture, reverse phase-liquid chromatography, and specific multiple-reaction monitoring for phytonadione and vitamin d3 (as internal standard). This method was validated through specificity, linearity, and accuracy. RESULTS: Average vitamin K1 content was 500, 100, 90, 100, 95, and 70 µg/L in soybean oil, olive-soybean oil, soybean-MCT coconut oil, soybean-olive-MCT-fish oil, soybean-MCT-fish oil, and pure fish oil intravenous lipid emulsions (ILEs), respectively. The analytical LC-MS method was extremely effective in terms of specificity, linearity ( r = 0.99), and accuracy (coefficient of variation <5%). CONCLUSIONS: Phytonadione is present in IVFE, and its intake varies according to IVFE type and the volume administered. It can contribute to daily requirements and become clinically relevant when simultaneously infused with multivitamins during long-term parenteral nutrition. LC-MS seems adequate in assessing vitamin K1 intake in IVFE.

Development of isotope dilution-liquid chromatography/tandem mass spectrometry for the accurate determination of trans- and cis-vitamin K1 isomers in infant formula.

A method based on isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC/MS/MS) using a C30 column has been developed for the separate and accurate determination of trans- and cis-vitamin K1 in infant formula. Vitamin K1 and the deuterium-labeled internal standard eluted at slightly different retention times experiencing different matrix effects, and this possibly resulted in biased measurement. The matrix effect profiles obtained from post-column infusion experiments showed that atmospheric pressure chemical ionization (APCI) was less susceptible to matrix effects near the retention time than electrospray ionization (ESI); therefore, APCI was used in this study. The developed method was validated by measuring fortified samples, and the results agreed with the gravimetric values. Its repeatability and reproducibly were within 2% relative standard deviation. The relative expanded uncertainty was approximately 5%, indicating that the method was of higher-order metrological quality as a reference method.

Phylloquinone (Vitamin K1): Occurrence, Biosynthesis and Functions.

BACKGROUND: Phylloquinone is a prenylated naphthoquinone that is synthesized exclusively by plants, green algae, and some species of cyanobacteria, where it serves as a vital electron carrier in photosystem I and as an electron acceptor for the formation of protein disulfide bonds. OBJECTIVE: In humans and other vertebrates, phylloquinone plays the role of a vitamin (vitamin K1) that is required for blood coagulation and bone and vascular metabolism. Phylloquinone from green leafy vegetables and vegetable oil represents the major dietary source of vitamin K for humans. METHOD: In recent years, reverse genetics and biochemical approaches using the model plant Arabidopsis thaliana have shown that phylloquinone biosynthesis in plants involves paralogous and multifunctional enzymes, a compartmentation of the corresponding pathway in plastids and peroxisomes, and trafficking of some biosynthetic intermediates within plastids themselves. Furthermore, phylloquinone biosynthetic intermediates create crucial metabolic branch-points with other plastid-synthesized metabolites such as chlorophylls, tocopherols and salicylate. RESULTS & CONCLUSION: This review presents an update on recent studies of the central role of plastids in the biosynthesis of phylloquinone, in particular on the discovery of novel enzymatic steps that are likely paradigms for phylloquinone and menaquinone (vitamin K2)-synthesizing organisms alike.

Stabilization and detection of hydrophylloquinone as di-O-methyl derivative.

Phylloquinone is a redox active naphthoquinone involved in electron transport in plants. The function of this reduced form remains unclear due to its instability, which has precluded detection. Herein, a simple method that permits the stabilization of the reduced form of phylloquinone by di-O-methylation and HPLC detection is described.

Rapid high performance liquid chromatography-high resolution mass spectrometry methodology for multiple prenol lipids analysis in zebrafish embryos.

The analysis of lipid molecules in living organism is an important step in deciphering metabolic pathways. Recently, the zebrafish has been adopted as a valuable animal model system to perform in vivo metabolomics studies, however limited methodologies and protocols are currently available to investigate zebrafish lipidome and even fewer to analyze specific classes of lipids. Here we present an HPLC-HRMS based method to rapidly measure multiple prenol lipid molecules from zebrafish tissues. In particular, we have optimized our method for concurrent detection of ubiquinones (Coenzyme Q6, Coenzyme Q9, Coenzyme Q10), cholesterol, vitamin E (α-tocopherol), vitamin K1 and vitamin K2. The purpose of this study was to compare different ionization modes, mobile phases and stationary phases in order to optimize lipid molecules separation. After HPLC-HRMS parameters selection, several extraction conditions from zebrafish embryos were evaluated. We assessed our methodology by quantitation of analytical recovery on zebrafish extracts from wild-type or zebrafish mutants (barolo) affected by impaired biosynthesis of ubiquinones.

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 .

Quantification of phylloquinone and menaquinones in feces, serum, and food by high-performance liquid chromatography-mass spectrometry.

Vitamin K, comprising phylloquinone (PK) and menaquinones (MKn), is a family of vitamers found in multiple biological and environmental matrices. Advancing emerging evidence for novel and distinct physiologic roles of these vitamers in human health and disease necessitates sensitive and selective methods for quantifying PK and MKn in these matrices. We developed a novel method employing high-performance liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS) for simultaneous quantification of 11 vitamin K vitamers that can be applied in feces, serum and food. Minimal detectable concentrations of vitamin K vitamers ranged from 1 pmol/g to 30 pmol/g. Limits of quantification ranged from 5 pmol/g to 90 pmol/g. Inter-assay and intra-assay variations were <17% and <8%, respectively, in food, and <12% and <8%, respectively, in feces. Recovery exceeded 80% for all vitamers in both food and feces. The method successfully quantified PK and MKn concentrations in rat chow, feces and serum. In summary, this LC-APCI-MS method provides a sensitive and selective tool for quantifying vitamin K vitamers in feces, serum and food. This method can be applied in human and animal studies examining the role of vitamin K vitamers derived from the diet and gut bacteria synthesis in health and disease.

Vitamin K2 in different bovine muscles and breeds.

Meat is a natural source of vitamin K, a vitamin associated with reduced bone loss and prevention of osteoporosis. Whether vitamin K content varies between breeds and muscles in cattle is not known. In the present study, contents of vitamin K1 (phylloquinone) and K2 (menaquinone, MK) were analysed in three different muscles from steers of two different breeds, Norwegian Red and Jersey, respectively. Results showed that MK4 was the most dominant of the vitamin K2 analogues, while only traces were found of MK6 and MK7. Both breeds had higher levels of MK4 in M. biceps femoris (BF) and M. longissimus dorsi (LD) compared to M. psoas major (PM). The results also showed significantly higher MK4 levels in muscles from Jersey compared to Norwegian Red. Furthermore, MK4 was not associated with intramuscular fat, suggesting a physiological role for MK4 in skeletal muscle cells. There were no association between vitamin K content and tenderness.

Dispersive liquid-liquid microextraction for the determination of vitamins D and K in foods by liquid chromatography with diode-array and atmospheric pressure chemical ionization-mass spectrometry detection.

A simple and rapid method was developed using reversed-phase liquid chromatography (LC) with both diode array (DAD) and atmospheric pressure chemical ionization mass spectrometric (APCI-MS) detection, for the simultaneous analysis of the vitamins ergocalciferol (D2), cholecalciferol (D3), phylloquinone (K1), menaquinone-4 (K2) and a synthetic form of vitamin K, menadione (K3). The Taguchi experimental method, an orthogonal array design (OAD), was used to optimize an efficient and clean preconcentration step based on dispersive liquid-liquid microextraction (DLLME). A factorial design was applied with six factors and three levels for each factor, namely, carbon tetrachloride volume, methanol volume, aqueous sample volume, pH of sample, sodium chloride concentration and time of the centrifugation step. The DLLME optimized procedure consisted of rapidly injecting 3 mL of acetonitrile (disperser solvent) containing 150 µL carbon tetrachloride (extraction solvent) into the aqueous sample, thereby forming a cloudy solution. Phase separation was performed by centrifugation, and the sedimented phase was evaporated with nitrogen, reconstituted with 50 µL of acetonitrile, and injected. The LC analyses were carried out using a mobile phase composed of acetonitrile, 2-propanol and water, under gradient elution. Quantification was carried out by the standard additions method. The APCI-MS spectra, in combination with UV spectra, permitted the correct identification of compounds in the food samples. The method was validated according to international guidelines and using a certified reference material. The validated method was applied for the analysis of vitamins D and K in infant foods and several green vegetables. There was little variability in the forms of vitamin K present in vegetables, with the most abundant vitamer in all the samples being phylloquinone, while menadione could not be detected. Conversely, cholecalciferol, which is present in food of animal origin, was the main form in infant foods, while ergocalciferol was not detected.