Simultaneous determination of vitamin K1, vitamin K1 2,3-epoxide and menaquinone-4 in human plasma by high-performance liquid chromatography with fluorimetric detection

A highly sensitive method for measuring endogenous vitamin K1, menaquinone-4 (which is one of the K2 vitamins) and vitamin K1 2,3-epoxide in human plasma was developed, based on high-performance liquid chromatography with coulometric reduction and fluorimetric detection, following extraction from plasma and purification on a Sep-Pak silica cartridge. The detection limits of vitamin K1, menaquinone-4 and vitamin K1 2,3-epoxide were 5, 5 and 8 pg per injection for the standard substances and 30, 30 and 50 pg/ml in human plasma, respectively.     

Analysis of isotope ratios in vitamin K1 (phylloquinone) from human plasma by gas chromatography/mass spectrometry

Vitamin K(1) is a fat-soluble vitamin required for the gamma-carboxylation of vitamin K-dependent proteins. Recent work has suggested an important role for vitamin K(1) in bone health beyond its more established function in the control and regulation of blood coagulation. However, current UK recommended intakes do not reflect this recent evidence. The use of stable isotopes provides a powerful tool to investigate vitamin K kinetics, turnover and absorption in man, although published methods have reported difficulties in the extraction and analysis of isotope ratios of vitamin K in human plasma. In this paper, we report a new methodology for the extraction and measurement of isotope ratios in vitamin K(1). Sample clean-up is achieved with liquid-liquid extraction, enzyme hydrolysis with lipase and cholesterol esterase, and solid-phase extraction. Isotopic analysis of the pentafluoropropionyl derivative of vitamin K(1) is performed by gas chromatography/mass spectrometry (GC/MS). The limit of quantitation is equivalent to at least 0.3 nmol/L and the method is demonstrated to be linear over a range of enrichments. This method provides a robust alternative to previous work requiring the use of semi-preparative high-performance liquid chromatography (HPLC).     

Accelerated solvent extraction of vitamin K1 in medical foods in conjunction with matrix solid-phase dispersion

An extraction technique is described for vitamin K1 in medical foods, using accelerated solvent extraction (ASE) in conjunction with matrix solid-phase dispersion (MSPD). The medical food sample is treated as it would be with MSPD extraction, followed by ASE for a hands-free automated extraction. The vitamin K1 in the ASE extract is then quantitated by reversed-phase liquid chromatography with fluorescence detection. The chromatography specifications are identical to those in previous work that used MSPD only, with a limit of detection of 6.6 pg and a limit of quantitation of 22 pg on column. Recoveries, which were determined for an analyte-fortified zero control reference material for medical foods, averaged 97.6% (n = 25) for vitamin K1. The method provides a rapid, automatic, specific, and easily controlled assay for vitamin K1 in fortified medical foods with minimal solvent usage.     

Determination of vitamin K1 in emulsified nutritional supplements by solid-phase extraction and high-performance liquid chromatography with postcolumn reduction on a platinum catalyst and fluorescence detection

Determination of small amounts of vitamin K1 (0.8 microg/g) in nutritional supplements with high fat content (20 mg/g) was performed by solid-phase extraction and high-performance liquid chromatography (HPLC) with fluorescence detection after reduction on a platinum oxide catalyst. The concentration ratio of plant oils to vitamin K1 (0.8 microg/g) was about 25,000:1. A sample solution was applied to a solid-phase extraction cartridge and vitamin K1 was eluted with ethanol, followed by HPLC. The proposed method was simple, rapid (analysis time: ca. 12 min), sensitive [detection limit: ca. 0.1 pg per injection (100 microl) at a signal-to-noise ratio of 3:1], highly selective and reproducible [relative standard deviation: ca. 1.3%. (n=5)]. The calibration graph of vitamin K1 was linear in the range of 0-2 pg per injection (100 microl). Recovery of vitamin K1 was over 90% by the standard addition method.     

Measurement of cis and trans isomers of vitamin K1 in rat tissues by liquid chromatography with a C30 column

The purpose of this study was to validate a method for measuring vitamin K isomers in rat tissues by liquid chromatography (LC) with fluorescence detection after simple solvent extraction. This method uses separation on a C30 column, followed by zinc reduction and fluorescence measurement (243 nm, excitation; 430 nm, emission) to detect and quantitate vitamin K isomers. We were able to separate cis- and trans-vitamin K1 in methylene chloride extracts of homogenized rat livers and in hexane extracts of rat plasma. Tissue extracts were evaporated and rediluted with tetrahydrofuran-methanol (1 + 1) or methanol before being injected under isocratic conditions onto the LC column. Liver tissue of Fischer 344 rats fed a vitamin K1-containing diet ad libitum contained approximately 20 and 60 ng/g cis- and trans-vitamin K1, respectively. Mean recoveries of vitamin K1 isomers from spiked liver were 92 +/- 11% for cis-vitamin K1 and 106 +/- 5% for trans-vitamin K1. We recovered 96 +/- 8% of trans-vitamin K1 added at 1, 3, and 6 ng/mL to plasma (containing an endogenous level of 4 ng/g) from the same rats; we recovered 112 +/- 5% when trans-vitamin K1 was added to human serum (National Institute of Standards and Technology Standard Reference Material 968C). This direct method shows significant potential for the selective measurement of vitamin K1 isomers in tissues.     

Liquid chromatographic analysis of vitamin K1 in milk-based infant formula with matrix solid-phase dispersion.

A liquid chromatographic method for vitamin K1 in milk-based infant formula is described. The vitamins are extracted from infant formula by matrix solid-phase dispersion and quantitated by reversed-phase chromatography with fluorescence detection. Vitamin K1 is converted to the fluorescent hydroquinone with a postcolumn zinc reductive reactor. The limit of detection is 12 pg, and the limit of quantitation is 38 pg on-column. Linear responses were obtained in the range 0.55-22.1 ng/ml (r2 = 0.9998). Recoveries of vitamin K1 from an analyte-fortified blank material for milk-based infant formula averaged 91.7% (n = 25). The method provides a rapid, specific, and easily controlled assay for vitamin K1 in fortified infant formula.     

Measurement of vitamin K in human liver by gradient elution high-performance liquid chromatography using platinum-black catalyst reduction and fluorimetric detection

A sensitive and precise method for measuring endogenous phylloquinone (K1) and menaquinone (MK-n) in human liver was developed, based on gradient elution high-performance liquid chromatography using platinum-black catalyst reduction and fluorimetric detection. Subnanogram levels of vitamin K compounds in 1 g of liver specimen were detectable. We measured vitamin K concentrations in 38 human resected livers. K1 and MK-4 to MK-13 were detected. The concentrations of MK-10 to MK-12 in livers with chronic hepatitis (n = 10) and cirrhosis (n = 22) were significantly lower than in normal livers (n = 6). It is suggested that the decreased concentrations indicate functional damage of the hepatocytes.     

Determination of Vitamins E,D3, and K1 in Plasma by Liquid Chromatography-Atmospheric Pressure Chemical Ionization-Mass Spectrometry Utilizing a Monolithic Column

This paper reports a rapid, simple, and sensitive method for determination of vitamin D3, vitamin E acetate, and vitamin K1 in plasma using atmospheric pressure chemical ionization –high performance liquid chromatography–mass spectrometry. Plasma samples were prepared using solid phase extraction. The separation of compounds was achieved using a C₁₈ monolithic column and a mobile phase composed of methanol and 0.1% formic acid in gradient elution mode at a flow rate of 1.0 mL min^?1. Analytes were ionized using atmospheric chemical ionization in positive mode. Mass spectra were recorded at m/z = 385.23, 473.47, and 451.41 for vitamin D3, vitamin E, and vitamin K1, respectively. Vitamin D2 was used as an internal standard and its mass spectra was recorded at 397.28 m/z. The method was validated using ICH guidelines. The system suitability responses were calculated for retention time, number of theoretical plates, capacity factor, resolution, and the selectivity factor. System validation was evaluated for precision, specificity, and linearity of all compounds. The limits of detection for vitamin D3, vitamin E, and vitamin K1 were determined to be 0.1, 1.36, and 0.052 ng mL^?1, respectively. The accuracy, evaluated as % of recovery, was in the range of 96.4 to 102.4% and precision determined as the coefficient of variation was between 1.24 and 3.6%. The validated method was applied to real plasma samples.     

柱后还原-高效液相色谱法同时测定调制乳粉中维生素K1和维生素K2

建立了一种同时检测调制乳粉中维生素K₁和维生素K₂的柱后还原-高效液相色谱-荧光检测方法。样品用水溶解，经脂肪酶酶解，2.5 mol/L氢氧化钠溶液和乙醇溶液皂化，正己烷萃取，氮吹浓缩后，用甲醇复溶。通过Xbridge C₁₈色谱柱分离，锌粉还原柱柱后还原，荧光检测器检测，激发波长为326 nm，发射波长为432 nm，外标法定量。结果表明，维生素K₁在0.0025~2.0 μg/mL、维生素K₂在0.01~2.0 μg/mL范围内线性关系良好，相关系数均大于0.999，维生素K₁和维生素K₂的检出限分别为0.07 μg/100 g和0.24 μg/100 g，定量限分别为0.2 μg/100 g和0.8 μg/100 g；方法的加标回收率为80.39%~94.39%，精密度为0.85%~3.98%。该方法灵敏度高，重复性好，结果准确，适用于调制乳粉中维生素K₁和维生素K₂的分析检测。     

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.     

A possible ethanol-catalyzed rearrangement of vitamin K(1) detected by gas chromatography/mass spectrometry

We studied vitamin K(1)(20), vitamin K(1)(25), and vitamin K(1) epoxide in n-hexane and ethanol solutions by gas chromatography/mass spectrometry (GC/MS) utilizing a DB-5 MS fused-silica capillary column. In ethanol solutions of K(1), we observed an extra peak eluting from the GC column with somewhat longer retention time than K(1)(20). A similar peak following K(1)(25) was also found. These peaks were not found in n-hexane solutions of K(1). A close examination of the mass spectra of these peaks indicated that they were vitamin K(1) variants containing a base peak at m/z 225 characteristic of the methylnaphthoquinone ring with a four-carbon side chain. In addition, they contained the molecular ions of K(1)(20) and K(1)(25), respectively. We conclude that K(1)(20) and K(1)(25), but not K(1) epoxide, might undergo rearrangements in ethanol involving an intramolecular proton transfer and a shift of the beta,gamma-double bond on the phytyl side chain toward the ring. The conjugation of the phytyl double bond with the quinone ring is probably the driving force of the rearrangement. We emphasize, however, that our conclusion is based only on mass spectral analysis and would require further investigation by other spectroscopic methods.     

Flow injection determination of vitamin K3 by a photoinduced chemiluminescent reaction

The determination of vitamin K3 using the coupling between a photochemical reaction and a chemiluminescent reaction in a flow system is described. The method is based on the photooxidation of ethanol sensitized by vitamin K3 to yield hydrogen peroxide, which is monitored through the chemiluminescent reaction with luminol catalysed by hematin. The new approach allows the determination of vitamin K3 in a wide concentration range (1 x 10(-7)-5 x 10(-4) mol l-1) with a throughput of 30 samples h-1. The applicability of the method was demonstrated by the determination of vitamin K3 in pharmaceutical preparations.     

Determination of Water- and Fat-Soluble Vitamins in Multivitamin Preparations by High-Performance Liquid Chromatography

An optimized method based on high-performance liquid chromatography with multiwavelength UV detection is proposed. The method provides the determination of vitamins B1 and B2, nicotinic acid, nicotinamide, pantothenic acid, folic acid, vitamins B6, B12, K3, H, D2, and D3, and vitamin A and E acetates in multivitamin preparations using two chromatographic procedures. Relative standard deviations for most of vitamins are 3–10%.     

Determination of vitamins D2, D3, K1 and K3 and some hydroxy metabolites of vitamin D3 in plasma using a continuous clean-up-preconcentration procedure coupled on-line with liquid chromatography-UV detection

A semi-automatic procedure for the continuous clean-up and concentration of several fat-soluble vitamins prior to their separation by HPLC and UV detection is reported. The procedure is based on the use of a minicolumn packed with aminopropylsilica as sorbent located prior to the chromatographic detection system. The overall process was developed and applied to the main liposoluble vitamins (A, D2, D3, E, K1, K3) and several hydroxy metabolites of vitamin D3 [25-(OH)-D3,24,25-(OH)2-D3 and 1,25-(OH)2-D3]. All the analytes were monitored at a compromise wavelength of 270 nm. Calibration graphs were constructed between 0.01 and 100 ng ml-1 for vitamin D2 and D3 and their hydroxy metabolites, between 0.1 and 100 ng ml-1 for vitamin A, K1 and K3 and between 1 and 100 ng ml-1 for vitamin E, with excellent regression coefficients (> or = 0.9901) in all cases. The precision was established at two concentration levels with acceptable RSDs in all instances (between 3.6 and 8.7%). The method was appropriate for the determination of vitamin D2, D3, K1 and K3 and the 24,25-dihydroxy and 25-hydroxy metabolites of vitamin D3 in human plasma. The method was applied to plasma samples spiked with the target analytes and the recoveries ranged between 78 and 109%.     

Determination of vitamin K homologues by high-performance liquid chromatography with on-line photoreactor and peroxyoxalate chemiluminescence detection

A sensitive and highly selective high-performance liquid chromatography (HPLC) method was developed for the determination of vitamin K homologues including phylloquinone (PK), menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in human plasma using post-column peroxyoxalate chemiluminescence (PO-CL) detection following on-line ultraviolet (UV) irradiation. The method was based on ultraviolet irradiation (254 nm, 15 W) of vitamin K to produce hydrogen peroxide and a fluorescent product at the same time, which can be determined with PO-CL detection. The separation of vitamin K by HPLC was accomplished isocratically on an ODS column within 35 min. The method involves the use of 2-methyl-3-pentadecyl-1,4-naphthoquinone as an internal standard. The detection limits (signal-to-noise ratio = 3) were 32, 38 and 85 fmol for PK, MK-4 and MK-7, respectively. The recoveries of PK, MK-4 and MK-7 were greater than 82% and the inter- and intra-assay R.S.D. values were 1.9-5.4%. The sensitivity and selectivity of this method were sufficient for clinical and nutritional applications.     

Flow-injection fluorimetric determination of vitamin K(1) based on a photochemical reaction

The sensitizing effect of vitamin K(1) on the photo-oxidation of glucose has been used for the determination of the vitamin. The hydrogen peroxide formed in the photochemical reaction reacts with Fe(II) to yield hydroxylradical and this radical is scavenged by benzoic acid to form the fluorescent hydroxybenzoic acids, which are analysed by fluorescence detection. This analytical scheme was adapted to a flow-injection system, which permits the determination of vitamin K(1) between 1x10(-6) and 1x10(-4) M with a throughput of 20 samples h(-1) and relative standard deviation between 0.2 and 1%. The applicability of the method was demonstrated by determining vitamin K(1) in pharmaceutical preparations and vegetables.     

高效液相色谱法测定化妆品中的维生素K1

建立了化妆品中维生素K1的高效液相色谱分析方法.将样品采用饱和氯化钠溶液-正己烷溶剂体系按不同的添加顺序进行分散、提取后,用高效液相色谱/二极管阵列检测器(HPLC/DAD)进行测定.其中,对油脂含量较高样品的提取液需经硅胶固相萃取柱进一步净化后测定.对不同的提取和净化方法作了系统研究,在优化实验条件下,方法的回收率为...     

Determination of vitamin K1 in medical foods by liquid chromatography with postcolumn reduction and fluorometric detection

A liquid chromatographic (LC) method is described for the determination of vitamin K1 in medical foods. The sample is enzymatically digested with lipase and alpha-amylase and extracted with 1% sodium bicarbonate solution-isopropanol (1 + 1). After C18 solid-phase extraction, vitamin K1 is separated by nonaqueous reversed-phase LC, converted to the hydroquinone by postcolumn zinc reduction, and quantitated by fluorescence detection. The limit of detection is 8 pg (3 sigma), and the limit of quantitation is 27 pg (10 sigma) on column. Linear response ranged from 0.1 to 1.0 ng vitamin K1 (r= 0.9999). The mean recovery (n = 38) for all spiking levels was 101.6 +/- 2.85%. Analysis of Standard Reference Material 1846, Infant Formula, gave a mean value of 0.95 +/- 0.088 mg vitamin K/kg (K or K1?) (n = 31) with a coefficient of variation of 9.26.     

NP-HPLC method for determining phytonadione and its impurities

A normal-phase high-performance liquid chromatography method has been developed to separate vitamin cis-phytonadione (cis-K1), trans-phytonadione (trans-K1), trans-epoxyphytonadione (trans-EK1), menadione (K3), cyclo-menadione (CK3), hydroperoxy-phytonadione (K1OOH), and other impurities on a Zorbax RX-SIL column, characterized by very pure porous silica, low acidity, and low metal content. A dual-mode gradient elution was applied involving simultaneous changes in flow rate and mobile-phase composition. The present method was sensitive enough to detect trans-EK1 (limit of detection, LOD: 1.8?ng), K3 (LOD: 0.6?ng), CK3 (LOD: 1.2?ng), and K1OOH (LOD: 2.4?ng). A linear relationship between peak area and concentration was found for four compounds with correlation coefficient r exceeding 0.9994. The impurities (trans-EK1, K3, and K1OOH) were measured accurately (recovery rate 80–120%) and precisely (relative standard deviation less than 2%). Preparing samples under yellow light (≥520?nm), the K1OOH in the three commercial batches of K1 was no higher than 0.10% (w/w) and was measured with a reproducibility of 3.1% and with an intermediate precision of 15.8%. After validation, the developed method was applied for analyzing the impurities occurring in the commercial samples of synthetic K1 and proved to be suitable for routine quality control.     

Determination of vitamin K1 isomers in foods by liquid chromatography with C30 bonded-phase column

Vitamin K1 was determined in a variety of foods by using reversed-phase liquid chromatography with a C30 column followed by post-column reduction to the fluorescent hydroquinone derivatives. Lipids were removed by lipase digestion, followed by single extraction into hydrocarbon, and the protocol was extended to selected natural and processed foods. Biologically active trans- and inactive ci-vitamin K1 isomers were measured individually to evaluate the true nutritional status of the products. Method performance parameters confirmed the validity of the technique. The use of the triacontyl-bonded C30 phase for selective phylloquinone isomer measurement extends previously validated AOAC Method 999.15 for vitamin K1 in milk and infant formula to a wider range of foods important in the human diet. The cis-vitamin K1 isomer contributes up to about 15% of total phylloquinone in certain foods.