Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection
2005; Elsevier BV; Volume: 46; Issue: 5 Linguagem: Inglês
10.1194/jlr.d400033-jlr200
ISSN1539-7262
AutoresDominic J. Harrington, Robin Soper, Christine Edwards, Geoffrey F. Savidge, Stephen Hodges, Martin J. Shearer,
Tópico(s)Hormonal Regulation and Hypertension
ResumoWe describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3′-3′-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5′-carboxy-3′-methyl-2′-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 ⩾ 0.999) and high sensitivity with an on-column detection limit of <3.5 fmol (<1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7′-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans.We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status. We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3′-3′-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5′-carboxy-3′-methyl-2′-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 ⩾ 0.999) and high sensitivity with an on-column detection limit of <3.5 fmol (<1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7′-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status. Compounds with vitamin K activity have a common 2-methyl-1,4-naphthoquinone nucleus and a variable alkyl substituent at the 3 position. The major naturally occurring K vitamins are the plant form phylloquinone (vitamin K1; abbreviated K1) and multiple forms of menaquinones (vitamins K2; abbreviated MK), predominately of bacterial origin. K1 has a 20 carbon phytyl side chain, whereas MKs have multiple prenyl side chains, their number being indicated by a suffix (i.e., MK-n). In a typical Western diet, K1 and MK-n account for ∼90% and 10% of vitamin K intake, respectively (1Schurgers L.J. Geleijnse J.M. Grobbee D.E. Pols H.A.P. Hofman A. Witteman J.C.M. Vermeer C.C. Nutritional intake of vitamin K-1 (phylloquinone) and K-2 (menaquinone) in The Netherlands.J. Nutr. Environ. Med. 1999; 9: 115-122Crossref Scopus (98) Google Scholar). Dietary MK-n mainly comprises MK-4, MK-7, MK-8, and MK-9 (2Schurgers L.J. Vermeer C. Determination of phylloquinone and menaquinones in food. Effect of food matrix on circulating vitamin K concentrations.Haemostasis. 2000; 30: 298-307Crossref PubMed Google Scholar), although MKs with longer side chains up to MK-13 are present in human liver (3Shearer M.J. Vitamin K metabolism and nutriture.Blood Rev. 1992; 6: 92-104Crossref PubMed Scopus (159) Google Scholar, 4Suttie J.W. The importance of menaquinones in human nutrition.Annu. Rev. Nutr. 1995; 15: 399-417Crossref PubMed Scopus (193) Google Scholar). Menadione (abbreviated K3) is a synthetic vitamin K homolog that lacks the side chain at the 3 position and that, despite toxicity concerns and restricted biological activity, is still available in some countries as a pharmaceutical vitamin K preparation in the form of menadiol sodium phosphate (5British National Formulary. 2004. British Medical Association and Royal Pharmaceutical Society of Great Britain. No. 48 (September).Google Scholar) or similar water-soluble salt. The biological activity of K3 in vivo depends entirely on its prenylation to MK-4 (6Martius C. Esser H.O. Über die Konstitution des im Tierkörper aus Methylnaphthochinon gebildeten K-Vitamines.Biochem. Z. 1958; 331: 1-9PubMed Google Scholar, 7Taggart W.V. Matschiner J.T. Metabolism of menadione-6,7-3H in the rat.Biochemistry. 1969; 8: 1141-1146Crossref PubMed Scopus (58) Google Scholar).At the cellular level, the cofactor role of vitamin K for the posttranslational conversion of specific peptide-bound glutamate to γ-carboxyglutamate (Gla) is well established, as is the intimately associated metabolic cycle whereby the vitamin K 2,3-epoxide metabolite generated during γ-glutamyl carboxylation is salvaged (8Olson R.E. Vitamin K.in: Shils M.E. Olson J.A. Shike M. Ross A.C. Modern Nutrition in Health & Disease. 9th ed. Williams & Wilkins, Baltimore, MD1999: 363-380Google Scholar, 9Suttie J.W. Vitamin K and human nutrition.J. Am. Diet. Assoc. 1992; 92: 585-590Abstract Full Text PDF PubMed Google Scholar).Other aspects of the intermediary metabolism of vitamin K, including the processes leading to vitamin K catabolism and excretion, are much less understood. During the 1970s, human studies using radiolabeled tracer and unlabeled pharmacological doses showed that K1 was rapidly and extensively catabolized via the urine and bile (10Shearer M.J. Barkhan P. Studies on the metabolites of phylloquinone (vitamin K1) in the urine of man.Biochim. Biophys. Acta. 1973; 297: 300-312Crossref PubMed Scopus (30) Google Scholar, 11Shearer M.J. McBurney A. Barkhan P. Studies on the absorption and metabolism of phylloquinone (vitamin K1) in man.Vitam. Horm. 1974; 32: 513-542Crossref PubMed Scopus (154) Google Scholar, 12McBurney A. Shearer M.J. Barkhan P. Preparative isolation and characterization of the urinary aglycones of vitamin K1 (phylloquinone) in man.Biochem. Med. 1980; 24: 250-267Crossref PubMed Scopus (24) Google Scholar). These studies established that the major aglycone metabolites of K1 were two side chain-shortened carboxylic acids with the structures 2-methyl-3-(3′-3′-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone; side chain length of 5 carbon atoms; structure IV in Fig. 1)and 2-methyl-3-(5′-carboxy-3′-methyl-2′-pentenyl)-1,4-naphthoquinone (7C-aglycone; side chain length of 7 carbon atoms; structure III in Fig. 1) (10Shearer M.J. Barkhan P. Studies on the metabolites of phylloquinone (vitamin K1) in the urine of man.Biochim. Biophys. Acta. 1973; 297: 300-312Crossref PubMed Scopus (30) Google Scholar, 11Shearer M.J. McBurney A. Barkhan P. Studies on the absorption and metabolism of phylloquinone (vitamin K1) in man.Vitam. Horm. 1974; 32: 513-542Crossref PubMed Scopus (154) Google Scholar, 12McBurney A. Shearer M.J. Barkhan P. Preparative isolation and characterization of the urinary aglycones of vitamin K1 (phylloquinone) in man.Biochem. Med. 1980; 24: 250-267Crossref PubMed Scopus (24) Google Scholar). Both 5C- and 7C-aglycones were excreted as water-soluble conjugates, mainly with glucuronic acid.At the time of isolation and characterization of these two metabolites, the role of vitamin K was thought to be limited to its classical coagulation function, and human deficiency at the population level was only considered a problem during the first 6 months of life (13Shearer M.J. Vitamin K.Lancet. 1995; 345: 229-234Abstract PubMed Scopus (393) Google Scholar). However, the subsequent discovery of many more vitamin K-dependent proteins (also called Gla proteins), with a widespread tissue distribution, has led to a reevaluation of the general physiological function and health roles of vitamin K. Putative roles of Gla proteins now extend to a diversity of functions, such as the regulation of bone turnover and calcification (14Vermeer C. Knapen M.H. Schurgers L.J. Vitamin K and metabolic bone disease.J. Clin. Pathol. 1998; 51: 424-426Crossref PubMed Scopus (42) Google Scholar, 15Shearer M.J. Role of vitamin K and Gla proteins in the pathophysiology of osteoporosis and vascular calcification.Curr. Opin. Clin. Nutr. Metab. Care. 2000; 3: 433-438Crossref PubMed Scopus (159) Google Scholar), inhibition of vascular calcification (16Shanahan C.M. Proudfoot D. Farzaneh-Far A. Weissberg P.L. The role of Gla proteins in vascular calcification.Crit. Rev. Eukaryot. Gene Expr. 1998; 8: 357-375Crossref PubMed Scopus (148) Google Scholar), and roles in vascular repair processes (17Benzakour O. Kanthou C. The anticoagulant factor, protein S, is produced by cultured human vascular smooth muscle cells and its expression is up-regulated by thrombin.Blood. 2000; 95: 2008-2014Crossref PubMed Google Scholar), cell cycle regulation, cell-cell adhesion, and signal transduction (18Tsaioun K.I. Vitamin K-dependent proteins in the developing and aging nervous system.Nutr. Rev. 1999; 57: 231-240Crossref PubMed Scopus (48) Google Scholar). Of particular note is the accumulating body of evidence that has linked suboptimal vitamin K reserves in bone to an increased risk of osteoporotic fracture (19Szulc P. Chapuy M.C. Meunier P.J. Delmas P.D. Serum undercarboxylated osteocalcin is a marker of the risk of hip fracture in elderly women.J. Clin. Invest. 1993; 91: 1769-1774Crossref PubMed Scopus (414) Google Scholar, 20Luukinen H. Käkönen S.M. Pettersson K. Koski K. Laippala P. Lövgren T. Kivelä S.L. Väänänen H.K.K. Strong prediction of fractures among older adults by the ratio of carboxylated to total serum osteocalcin.J. Bone Miner. Res. 2000; 15: 2473-2478Crossref PubMed Scopus (174) Google Scholar) or to reduced bone mineral density (21Szulc P. Arlot M. Chapuy M.C. Duboeuf F. Meunier P.J. Delmas P.D.D. Serum undercarboxylated osteocalcin correlates with hip bone mineral density in elderly women.J. Bone Miner. Res. 1994; 9: 1591-1595Crossref PubMed Scopus (255) Google Scholar). To address this issue and other questions, there has been a need to develop new biochemical measures to monitor vitamin K status in human populations (22Sokoll L.J. Sadowski J.A. Comparison of biochemical indexes for assessing vitamin K nutritional status in a healthy adult population.Am. J. Clin. Nutr. 1996; 63: 566-573Crossref PubMed Scopus (154) Google Scholar). Current measures include the direct measurement of circulating vitamin K (as an indicator of tissue stores) and functional assessments of the γ-carboxylation status of specific Gla proteins such as prothrombin and osteocalcin, representing hepatic and bone γ-carboxylation capacity, respectively. In addition, measurements of urinary free Gla offer an overall assessment of the γ-carboxylation status of Gla proteins. Each of these status assessments has a number of methodological and interpretational drawbacks (23Cham B.E. Smith J.L. Colquhoun D.M. Interdependence of serum concentrations of vitamin K1, vitamin E, lipids, apolipoprotein A1, and apolipoprotein B: importance in assessing vitamin status.Clin. Chim. Acta. 1999; 287: 45-57Crossref PubMed Scopus (25) Google Scholar, 24McKeown N.M. Jacques P.F. Gundberg C.M. Peterson J.W. Tucker K.L. Kiel D.P. Wilson P.W. Booth S.L.L. Dietary and nondietary determinants of vitamin K biochemical measures in men and women.J. Nutr. 2002; 132: 1329-1334Crossref PubMed Scopus (110) Google Scholar, 25Gundberg C.M. Nieman S.D. Abrams S. Rosen H. Vitamin K status and bone health: an analysis of methods for determination of undercarboxylated osteocalcin.J. Clin. Endocrinol. Metab. 1998; 83: 3258-3266Crossref PubMed Scopus (232) Google Scholar). Although useful in many situations, the measurement of circulating vitamin K to assess tissue stores has the major disadvantage that only K1 is commonly measured, to the detriment of MKs. MKs from the diet (1Schurgers L.J. Geleijnse J.M. Grobbee D.E. Pols H.A.P. Hofman A. Witteman J.C.M. Vermeer C.C. Nutritional intake of vitamin K-1 (phylloquinone) and K-2 (menaquinone) in The Netherlands.J. Nutr. Environ. Med. 1999; 9: 115-122Crossref Scopus (98) Google Scholar, 2Schurgers L.J. Vermeer C. Determination of phylloquinone and menaquinones in food. Effect of food matrix on circulating vitamin K concentrations.Haemostasis. 2000; 30: 298-307Crossref PubMed Google Scholar) and possibly from intestinal synthesis (3Shearer M.J. Vitamin K metabolism and nutriture.Blood Rev. 1992; 6: 92-104Crossref PubMed Scopus (159) Google Scholar, 4Suttie J.W. The importance of menaquinones in human nutrition.Annu. Rev. Nutr. 1995; 15: 399-417Crossref PubMed Scopus (193) Google Scholar) may make a significant contribution to total daily vitamin K intake (1Schurgers L.J. Geleijnse J.M. Grobbee D.E. Pols H.A.P. Hofman A. Witteman J.C.M. Vermeer C.C. Nutritional intake of vitamin K-1 (phylloquinone) and K-2 (menaquinone) in The Netherlands.J. Nutr. Environ. Med. 1999; 9: 115-122Crossref Scopus (98) Google Scholar), and the liver stores of vitamin K are predominately long-chain MKs (3Shearer M.J. Vitamin K metabolism and nutriture.Blood Rev. 1992; 6: 92-104Crossref PubMed Scopus (159) Google Scholar, 4Suttie J.W. The importance of menaquinones in human nutrition.Annu. Rev. Nutr. 1995; 15: 399-417Crossref PubMed Scopus (193) Google Scholar).Here, we describe the development of chromatographic techniques to quantify the two major urinary aglycones of vitamin K with high sensitivity, enabling their measurement at low physiological concentrations. We chose electrochemical detection (ECD) in the redox mode for the final analytical HPLC stage because this method is especially suitable for quinone compounds and has been used successfully for the determination of vitamin K in serum (26Hart J.P. Shearer M.J. McCarthy P.T. Rahim S. Voltammetric behaviour of phylloquinone (vitamin K1) at a glassy-carbon electrode and determination of the vitamin in plasma using high-performance liquid chromatography with electrochemical detection.Analyst. 1984; 109: 477-481Crossref PubMed Google Scholar, 27Hart J.P. Shearer M.J. McCarthy P.T. Enhanced sensitivity for the determination of endogenous phylloquinone (vitamin K1) in plasma using high-performance liquid chromatography with dual-electrode electrochemical detection.Analyst. 1985; 110: 1181-1184Crossref PubMed Google Scholar). To determine whether the 5C- and 7C-aglycones are common to all K vitamins, we obtained urine samples from adults before and after supplementation with different doses of K1, MK-4, MK-7, and K3. We also analyzed urine samples from newborn infants, who, as a group, are known to have precariously low vitamin K stores and are routinely given vitamin K prophylaxis at birth.MATERIALS AND METHODSReagents5C-aglycone, 7C-aglycone, and the compound [2-methyl-3-(7′-carboxy-heptyl)-1,4-naphthoquinone] used for the internal standard (IS) are not commercially available and were synthesized for this study (28Soper, R. J. 2004. The Synthesis and Biological Activities of Natural Quinone Metabolites. PhD Dissertation. University of Essex, Essex, UK.Google Scholar). The γ-lactone form of the 7C-aglycone [2-methyl-3-(5′-carboxy-3′-hydroxy-3′-methylpentyl)-1,4-naphthoquinone lactone], also known as vitamin K γ-lactone, was a gift to M.J.S. from Hoffmann-La Roche and Co. (Basel, Switzerland). Organic solvents were of HPLC grade (Rathburns Chemicals, Walkerburn, Scotland). Water for ECD was purified using a Purite Neptune water purification system (Jencons-PLS, Leighton Buzzard, UK). Potassium hydroxide (Ultra grade), 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), and ethylenediaminetetraacetic acid (disodium salt, dihydrate, Analar grade) were obtained from Sigma-Aldrich (Dorset, UK). Anhydrous sodium acetate, glacial acetic acid, and 3 M HCl, all AristaR grade, were obtained from BDH (Lutterworth, Leicstershire, UK). Nitrogen gas (oxygen free) for removal of solvents was obtained from BOC (Guildford, Surrey, UK).ApparatusSolid-phase extraction (SPE) procedures were performed using Isolute™ SPE C18 (100 mg, 1 ml reservoir) cartridges (Jones Chromatography, Mid Glamorgan, UK) and Sep-Pak™ silica plus cartridges (Waters Ltd., Elstree, Hertfordshire, UK) with a SPE vacuum manifold.For HPLC, we used a Gynkotek model 480 pump with pulse damper, a Waters™ 717 plus Autosampler, and an in-line DeJour X-Act HPLC degassing unit. For on-line detection, we used a DECADE electrochemical detector (Antec, Leyden, The Netherlands) equipped with an in-line cell (model 5011) containing dual porous graphite coulometric electrodes in series (ESA Analytical Ltd., Aylesbury, Buckinghamshire, UK) and an amperometric wall jet electrode situated immediately downstream (Antec model VT-03). Chromatographic data were captured using a data chromatography manager (Waters).Urine collection and human subject protocolsTwenty four hour and spot urine collections were made into plastic containers. Owing to the photosensitive nature of the metabolites, the urine containers were protected from sources of strong light. Urine collections were stored at room temperature for the duration of the collection period, and aliquots were frozen at −70°C until analysis. No significant metabolite loss occurred under these conditions.We investigated urinary 5C- and 7C-aglycone excretion in healthy adult volunteers. Measurements were made in spot or 24 h urine collections in both unsupplemented and vitamin K-supplemented subjects. For the supplementation studies, the subjects took different forms of vitamin K orally at doses previously used for the treatment or prevention of several pathologies. The vitamin K compounds used were K1 (2 mg and 50 mg) (29McCarthy P.T. Cox A.D. Harrington D.J. Evely R.S. Hampton E. al-Sabah A.I. Massey E. Jackson H. Ferguson T.T. Covert poisoning with difenacoum: clinical and toxicological observations.Hum. Exp. Toxicol. 1997; 16: 166-170Crossref PubMed Scopus (22) Google Scholar); two homologs representative of the MK series, MK-4 (45 mg) (30Shiraki M. Shiraki Y. Aoki C. Miura M. Vitamin K2 (menatetrenone) effectively prevents fractures and sustains lumbar bone mineral density in osteoporosis.J. Bone Miner. Res. 2000; 15: 515-521Crossref PubMed Scopus (355) Google Scholar) and MK-7 (1 mg) (31Tsukamoto Y. Ichise H. Kakuda H. Yamaguchi M. Intake of fermented soybean (natto) increases circulating vitamin K2 (menaquinone-7) and γ-carboxylated osteocalcin concentration in normal individuals.J. Bone Miner. Metab. 2001; 18: 216-222Crossref Scopus (89) Google Scholar); and K3 (20 mg) (5British National Formulary. 2004. British Medical Association and Royal Pharmaceutical Society of Great Britain. No. 48 (September).Google Scholar). The amounts of 5C- and 7C-aglycone excreted in the urine were measured before and after supplementation.A number of spot urine samples were also collected from newborn infants (two males, one female) before and after intramuscular K1 prophylaxis with 1 mg of Konakion Neonatal (Roche, Basel, Switzerland).All adult volunteers and guardians of newborns gave full written informed consent before participation in these investigations.Assay procedureA flow chart showing the extraction and analytical procedures is shown in Fig. 2.Fig. 2Flow chart illustrating the analytical stages involved in the determination of the urinary vitamin K aglycone metabolites. ECD, electrochemical detection; SPE, solid-phase extraction.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Extraction and hydrolysis of metabolitesA C18 SPE cartridge was prewashed with 1 ml of methanol followed by 1 ml of deionized water. Aliquots of 0.5 ml (unsupplemented subjects) or 0.05 ml (after vitamin K supplementation) of urine were loaded onto the SPE cartridge and allowed to elute to waste under gravity. To remove urinary salts, the SPE cartridge was washed with 1 ml of deionized water at a flow rate of ∼0.5 ml/min, and the eluent was discarded (32Pope S.A.S. Clayton P.T. Muller D.P.R. A new method for the analysis of urinary vitamin E metabolites and the tentative identification of a novel group of compounds.Arch. Biochem. Biophys. 2000; 381: 8-15Crossref PubMed Scopus (45) Google Scholar). The conjugated vitamin K metabolites were then eluted into a clean tapered polypropylene test tube (12 ml capacity) with 2 ml of a methanolic stock solution containing 0.15 μg of the IS, and the eluent was evaporated to dryness under a stream of N2 at 50°C.Conjugated urinary vitamin K metabolites were hydrolyzed overnight at room temperature (in the dark) with 1.1 ml of methanolic HCl [prepared by combining 1 volume of concentrated (35%) HCl with 4 volumes of methanol] (33Imada I. Watanabe M. Matsumoto N. Morimoto H. Metabolism of ubiquinone-7.Biochemistry. 1970; 9: 2870-2878Crossref PubMed Scopus (45) Google Scholar). The metabolites, now as their aglycones, were extracted into chloroform by the addition of 1.1 ml of chloroform and 1.0 ml of water to the sample tube (34Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Med. Sci. 1959; 37: 911-917Google Scholar). The upper methanolic-aqueous layer was removed and discarded. To prevent acid-catalyzed lactonization of the 7C-aglycone during subsequent drying procedures, the lower chloroform layer was washed with 10 ml of deionized water to remove any residual mineral acid. After brief shaking and time to allow the two phases to separate, the upper aqueous layer was discarded and the lower chloroform layer was evaporated to dryness under a stream of N2 at 50°C.Methylation of aglyconesThe carboxylic acid forms of the vitamin K aglycones were converted to their methyl ester derivatives by reaction with freshly prepared ethereal diazomethane generated on a small scale by a modification of a method described elsewhere (35Fales H.M. Jaouni T.M. Babashak J.F. Simple device for preparing etheral diazomethane without resorting to codistillation.Anal. Chem. 1973; 45: 2302-2303Crossref Scopus (252) Google Scholar). In brief, a mixture of 5 M KOH (∼20 ml) and diethyl ether (∼15 ml) was placed into a small, screw-capped bottle (100 ml capacity; Duran), and solid MNNG was gradually added until sufficient diazomethane was generated to turn the upper ether layer yellow (caution: only perform in a well-ventilated fume hood). A volume of 0.5 ml of this ethereal diazomethane was then added to each urine extract. Complete methylation of the carboxylic acid group of the aglycone metabolites was achieved at room temperature within 5 min, after which the diethyl ether was removed under a stream of N2 at 50°C.Normal-phase SPE of methylated aglyconesFurther fractionation of the urine extract was achieved by normal phase-SPE using Sep-Pak™ cartridges. Each cartridge was attached to a 10 ml glass syringe with a Luer end fitting and activated by drawing through 2 ml of n-hexane. Each extract was dissolved in 2 ml of n-hexane and then pipetted into the glass syringe attached to the activated cartridge. The n-hexane was drawn through the cartridge and the eluent was discarded. Another volume of 8 ml of n-hexane was added to the sample tube and the washings were drawn through the cartridge and discarded as before. The vitamin K metabolites and IS were then eluted from the cartridge with 10 ml of n-hexane-diethyl ether (85:15, v/v) and collected into a fresh stoppered, tapered test tube of polypropylene. The solvent was removed under a stream of nitrogen at 50°C.Reversed-phase HPLC-ECDThe final separation of the methylated aglycones was achieved by reversed-phase HPLC using a Thermo Hypersil-Keystone HyPURITY C18 column (3 μm particle size, dimensions 100 × 4.4 mm; Hichrom, Reading, Berkshire, UK) and a mobile phase consisting of 60:40 (v/v) methanol-0.05 M sodium acetate buffer (pH 3.0; containing 0.1% EDTA) (26Hart J.P. Shearer M.J. McCarthy P.T. Rahim S. Voltammetric behaviour of phylloquinone (vitamin K1) at a glassy-carbon electrode and determination of the vitamin in plasma using high-performance liquid chromatography with electrochemical detection.Analyst. 1984; 109: 477-481Crossref PubMed Google Scholar). The flow rate was 1.0 ml/min. Effluent from the column passed through the coulometric dual-electrode cell in which the twin, porous graphite electrodes were set at a negative potential (−1.2 V), thereby reducing the urinary vitamin K quinone metabolites and IS to their quinol states. The effluent then passed to the amperometric wall jet electrode set at +0.3 V, which resulted in the reoxidation of the quinol forms of metabolites and IS to their respective quinones. Chromatograms were generated by monitoring the current (nanoamperes) at the wall jet electrode.Quantification of 5C- and 7C-aglycone metabolitesFrom methanolic stock solutions of the 5C- and 7C-aglycones and the IS (methyl ester forms), we prepared a series of standards containing all three analytes with spectrophotometrically determined (EM mM = 18.9 at 248 nm) weight ratios of 5C-aglycone/IS and 7C-aglycone/IS ranging from 0.003 to 0.121. Each chromatographic run included the direct injection of 10 μl of each calibration standard. For routine analyses (i.e., for physiological urinary concentrations of metabolites), the SPE fractionated extracts were reconstituted in 40 μl of methanol and a volume of 10 μl was injected onto the reversed-phase HPLC system. For the determination of the much higher levels after vitamin K supplementation, the extract was reconstituted in 100 μl of methanol and 10 μl was injected. Metabolite concentrations were quantified by the method of peak height ratios. For each chromatogram generated, the peak heights of 5C- and 7C-aglycones were expressed as a ratio to the IS peak. A calibration plot of peak height ratios of the standards versus their weight ratios gave excellent linearity (typically, r2 ⩾ 0.999) and was used to calculate the equivalent weight ratios of aglycone/IS for each unknown. Multiplication of this weight ratio by the amount of IS originally added gave the amounts of aglycones in the volume of urine processed.RESULTSOptimization of extraction and conjugate hydrolysis proceduresThe initial SPE procedure using C18 cartridges was an effective strategy for desalting the urine and concentrating the urinary metabolites in their conjugated forms. True recovery experiments at this C18 SPE stage were not possible because there is limited information on the chemical nature and variety of conjugated metabolites in vivo and appropriate standards are unavailable. Nevertheless, recovery experiments using the synthetic 5C-aglycone, 7C-aglycone, and IS showed that there was no significant loss of vitamin K moieties at this stage. Recoveries were reduced if lower eluting volumes (<2 ml) of methanol were used or if the urine was acidified (with HCl) to promote protonization of the carboxylic acid metabolites. Trials with other SPE cartridge chemistries with side chain lengths < C18 also resulted in reduced recovery. Analyte recovery was found to vary inversely with the flow rates of both the first aqueous wash and the second methanolic elution steps. Omission of this initial SPE stage increased the baseline current in the final electrochemical reversed-phase HPLC analytical stage, resulting in a decrease in assay sensitivity.After the initial SPE stage, conjugated metabolites of vitamin K were hydrolyzed with methanolic HCl. At room temperature, hydrolysis of conjugated metabolites with methanolic HCl was complete within 16 h. After hydrolysis, the lipid-soluble aglycones and IS were efficiently extracted into chloroform after the addition of greater volumes of chloroform and water to form a two-phase system, as described for the final stage of the Bligh and Dyer (34Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Med. Sci. 1959; 37: 911-917Google Scholar) total lipid extraction technique. A second extraction of the methanolic digest with chloroform did not improve the recovery of aglycones.Methyl ester derivatization of carboxylic acid aglyconesThere were two main reasons for adopting a pre-HPLC methylation procedure to quantitatively convert the aglycone carboxylic acids to their respective methyl esters. First, exposure to methanol during the chromatographic procedures resulted in some inadvertent methylation, which was enhanced by the use of methanolic HCl to deconjugate the aglycones. Second, the reduced polarity of these methylated derivatives improved their retention in our reversed-phase HPLC separation system.SPE purification of methylated aglyconesAn additional SPE purification stage using silica cartridges was introduced to remove interfering compounds, which prevented baseline resolution of the 5C- and 7C-aglycones in the final HPLC-ECD analytical stage. Optimization of the composition of the eluting solvent was carried out using a standard mixture containing
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