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Analytica Chimica Acta 591 (2007) 148–154

Determination of vitamin K homologues by high-performance liquidchromatography with on-line photoreactor and peroxyoxalate

chemiluminescence detection

Sameh Ahmed, Naoya Kishikawa, Kenichiro Nakashima, Naotaka Kuroda ∗Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki University,

1-14 Bunkyo-machi, Nagasaki 852-8521, Japan

Received 11 January 2007; received in revised form 22 March 2007; accepted 29 March 2007Available online 4 April 2007

bstract

A sensitive and highly selective high-performance liquid chromatography (HPLC) method was developed for the determination of vitamin Komologues including phylloquinone (PK), menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in human plasma using post-column peroxyoxalatehemiluminescence (PO-CL) detection following on-line ultraviolet (UV) irradiation. The method was based on ultraviolet irradiation (254 nm,5 W) of vitamin K to produce hydrogen peroxide and a fluorescent product at the same time, which can be determined with PO-CL detection. Theeparation of vitamin K by HPLC was accomplished isocratically on an ODS column within 35 min. The method involves the use of 2-methyl-3-

entadecyl-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 andK-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%.

he sensitivity and selectivity of this method were sufficient for clinical and nutritional applications.2007 Elsevier B.V. All rights reserved.

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eywords: Vitamin K; Peroxyoxalate chemiluminescence detection; Ultraviole

. Introduction

There is growing interest in the role, biochemical functionnd metabolism of vitamin K in vivo. Vitamin K is a cofactoror an enzyme that converts specific glutamyl residues in severalroteins such as plasma clotting factors II (prothrombin), VII,X and X, protein C and protein S as well as non-coagulationroteins as osteocalcin to �-carboxyglutamyl residues. Theseitamin K-dependent proteins play crucial roles in hemostasisnd calcification. In addition, neonatal and infantile vitamin Keficiency causes melena neonatorum and intracranial hemor-hagic disorders [1,2].

There are two major forms of vitamin K in nature (Fig. 1).

itamin K1 (phylloquinone, PK) is produced by plants and algaehereas vitamin K2 series (menaquinones, MKs) is synthesizedy bacteria. The length of the isoprenoid side chain in MKs is

∗ Corresponding author. Tel.: +81 95 8192894; fax: +81 95 8192444.E-mail address: n-kuro@nagasaki-u.ac.jp (N. Kuroda).

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003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2007.03.061

iation; High-performance liquid chromatography; Hydrogen peroxide

efined by its carbon number, or the number of isoprenoid units.he major dietary form of vitamin K has been considered to beK, which is contained in green and leafy vegetables. In con-

rast, MKs are found in fermented foods such as natto (fermentedoyabeans) and in the colon, where they are synthesized by thentestinal microflora [3]. In Japan, MK-4 has been given to osteo-orotic patients and PK has been used as a therapeutic agent foritamin-K deficient syndromes such as hypoprothrombinemian newborn babies and in antibiotic-treated patients. However,nformation on the physiological and pharmacological roles ofitamin K homologues in vivo is still limited [4]. One reasonor this is that the detection and monitoring of vitamin K homo-ogues in plasma have been difficult on account of quite smalloncentrations of vitamin K homologues and many interferingubstances from plasma. For pharmacokinetic and epidemiolog-cal purposes, sensitive, highly selective and accurate analytical

ethods are required that allow assays at low (ng mL−1) plasmaevels.

Previously, several methods were reported for separationnd determination of vitamin K homologues by gas liquid

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Fig. 1. Structures of vitamin K homologues.

hromatography (GLC) [5] and HPLC with ultraviolet (UV)6–8], fluorescence [9–18], electrochemical [19–21] and masspectrometric detection [22,23]. HPLC with ultraviolet detec-ion was the first choice for measuring the individual forms ofitamin K [24]. This method offered better selectivity than theraditional chick bioassay commonly used to measure vitamin

status, however its sensitivity is still insufficient. Recently,method for the determination of vitamin K homologues in

uman plasma by liquid chromatography–atmospheric pressurehemical ionization/mass spectrometry (LC–APCI-MS) waseveloped [22]. Although this method has great advantage inigh sensitivity and accuracy, it is very expensive for routinessay. In contrast, HPLC with fluorescence detection using post-olumn chemical reduction is relatively sensitive, convenientnd stable; vitamin K homologues are reduced by the platinum-eduction column and converted into hydroquinones which areighly fluorescent [9]. However, there is still a problem in sep-ration of vitamin K homologues from interfering compoundsn plasma. Especially, it is difficult to determine vitamin Komologues accurately in a routine assay, because its basallasma concentration is markedly low and elute at the sameetention time of a number of interfering compounds in plasma.

Peroxyoxalate chemiluminescence is based on the reactionetween hydrogen peroxide and aryloxalate, which producestrong luminescence in the presence of fluorophore through thehemically initiated electron exchange luminescence (CIEEL)echanism [25]. The PO-CL is easily combined with HPLC and

pplied to highly sensitive determination of fluorescent com-ounds and hydrogen peroxide. Recently, we have reported theetermination of quinones by HPLC–PO-CL detection, with-ut addition of hydrogen peroxide and fluorophore, combinedith on-line photochemical reaction. This method was basedn conversion of quinones to hydrogen peroxide and a fluores-ent 3,6-dihydroxyphthalic acid by ultraviolet irradiation, andhen detected via PO-CL reaction by mixing with aryloxalate26].

In this work, we adopted this principle to the determinationf vitamin K homologues as they have a common 2-methyl-1,4-

aphthoquinone structure. We found that vitamin K homologuesenerated hydrogen peroxide and a fluorophore when subjectedo UV irradiation, which can be determined by mixing with ary-oxalate through PO-CL reaction. It was found that there is a

4bat

a Acta 591 (2007) 148–154 149

irect relation between CL intensity and vitamin K homologuesoncentration. As far as we know, it is the first time to couplePLC method with CL detection for simultaneous determina-

ion of vitamin K homologues in human plasma. Because theetermined samples in this CL method are limited to compoundshat generate hydrogen peroxide and a fluorescent material at theame time after UV irradiation, it is thought that this method canetect vitamin K homologues selectively without interferencerom plasma components. Moreover, when hydrogen peroxides used as a post column reagent in PO-CL system, backgroundoise is generated. On the other hand, because this CL methodoes not need the addition of hydrogen peroxide, the decreasef the background noise can be expected compared with theeneral PO-CL reaction.

. Experimental

.1. Chemicals and reagents

MK-4 and MK-7 were kindly provided by Eisai Pharmaceu-icals (Tokyo, Japan). bis[2-(3,6,9-Trioxadecyloxycarbonyl)--nitrophenyl] oxalate (TDPO) was obtained from Wakoure Chemical Industries (Osaka, Japan). bis(2,4,6-richlorophenyl) oxalate (TCPO), bis(2,4-dinitrophenyl)xalate (DNPO), bis(2,6-difluorophenyl) oxalate (DFPO)nd bis(pentafluorophenyl) oxalate (PFPO) were obtainedrom Tokyo Chemical Industry (Tokyo). Vitamin K1 and K32-methyl-1,4-naphthoquinone), silver nitrate, ammoniumersulfate and acetonitrile (LC/MS grade) were from Kantohemical (Tokyo). 2-Methyl-3-pentadecyl-1,4-naphthoquinones an internal standard was synthesized in our laboratory accord-ng to the previous reported method [27]. Stock solutions ofitamin K homologues and internal standard (100 �M) wererepared in ethanol and stored in the dark at −20 ◦C. Theseolutions were diluted appropriately with ethanol to prepare theorking solutions. Imidazole was obtained from Tokyo Chem-

cal Industry; imidazole was recrystallized from acetonitrileefore use. Distilled water was obtained using Simpli Lab-UVMillipore, Bedford, MA, USA) water device. Other chemicalsere of extra pure grade.

.2. HPLC–PO-CL system

The HPLC system (Fig. 2) consisted of two L-7100 liquidhromatographic pumps (Hitachi, Tokyo, Japan), a Rheodyne125 injector (Cotati, CA, USA) with a 10-�L sample loop, aevelosil ODS UG-5 (50 mm × 1.5 mm, i.d., Nomura Chem-

cals, Tokyo), a low-pressure mercury lamp (15 W, 254 nm,igemi Standard, Tokyo), a CLD-10A chemiluminescenceetector (Shimadzu) and SIC chromatorecorder (Tokyo, Japan).TFE tubing (6.0 m × 0.25 mm i.d., GL Sciences, Tokyo) coiledround a low-pressure mercury lamp was used as on-line pho-oreactor. Temperature of the photoreactor was maintained at

5 ◦C by an oven (Jasco, Tokyo). A mixture of imidazole–HNO3uffer (600 mM, pH 9.0) and acetonitrile (5:95, v/v) was useds a mobile phase and 0.6 mM TDPO in acetonitrile was used ashe post column CL reagent. The mobile phase and CL reagent

150 S. Ahmed et al. / Analytica Chimic

Fig. 2. HPLC–PO-CL system for the determination of vitamin K homo-logues. P1, pump 1; P2, pump 2; I, injector; Column, Develosil ODS UG-5(pt

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50 mm × 1.5 mm, i.d.); Cooling unit, oven equipped with cold insulator; L, low-ressure mercury lamp; RC, reaction coil (6.0 m × 0.25 mm, i.d.); M, mixingee; D, CL detector; Rec, recorder.

ere filtered through a 0.45-�m filter prior to use. The flow-ates of the mobile phase and the CL reagent were set at 0.25nd 0.80 mL min−1, respectively.

.3. HPLC-fluorescence system

HPLC-fluorescence system was used for identification ofhe fluorescence characteristics of photoproducts producedrom UV irradiation of vitamin K homologues. The HPLCystem consisted of L-7100 liquid chromatographic pumpHitachi), a Rheodyne 7125 injector (Cotati) with a 10-�Lample loop, a low-pressure mercury lamp (15 W, 254 nm,igemi Standard), PTFE tubing (6.0 m × 0.5 mm, i.d.) coiledround low-pressure mercury lamp was used as an on-linehotoreactor. A Capcellpak ODS UG 120 (250 × 4.6 mm,.d., Shiseido), which was set after UV-irradiation assembly,n agilent 1100 series multi-wavelength fluorescence detectorHewlett-Packard, Waldbronn, Germany) for multi-wavelengthuorescence detection. A mixture of imidazole–HNO3 buffer50 mM, pH 7.5) and acetonitrile (20:80, v/v) was used as aobile phase with a flow-rate 0.80 mL min−1.

.4. Sample preparation

The present experiments were approved by the Ethics Com-ittee of the School of Pharmaceutical Sciences, Nagasakiniversity, and performed in accordance with established guide-

ines. Blood samples were collected from a convenient forearmein into tubes containing EDTA. The samples were centrifugedt 3500 rpm for 10 min at room temperature then the plasmaas separated and stored at −30 ◦C in dark until analysis.xactly 1 mL of donated human plasma sample was placed in arown screw-capped tube. An aliquot of 1 mL of approximatelynM solution of internal standard (2-methyl-3-pentadecyl-1,4-aphthoquinone) was added. Extra ethanol (1 mL) was thendded to denature protein. The sample was extracted using 4 mL

f hexane with shaking for 5 min before centrifuging at 3000 rpmor 5 min. The supernatant was separated and applied to a Sep-ak silica cartridge (Waters, Milford, MA, USA), which wasashed with 10 mL of hexane. Vitamin K homologues were

efli

a Acta 591 (2007) 148–154

luted with 5 mL of hexane/diethyl ether (97:3,v/v). The eluateas evaporated under reduced pressure, and the residue wasissolved with 50-�L of ethanol then injected to the HPLCystem.

. Results and discussion

.1. Optimization of HPLC–PO-CL method

PK, MK-4 and MK-7 were detected in almost all plasma sam-les from healthy subjects tested here. In contrast, MK-5, MK-6,K-8, Mk-9 and MK-10 were not detected in all samples. Thus,

t was decided to measure PK, MK-4 and MK-7. A highly sen-itive and selective method is required for the determination ofitamin K homologues in biological samples because vitamin

homologues usually exists at low concentration. Therefore,he conditions for the determination of vitamin K homologuesith the proposed method were optimized in order to obtain theaximum sensitivity.

.1.1. Optimization of UV irradiation conditionsIt is thought that the conversion efficiency of vitamin K homo-

ogues by UV irradiation to hydrogen peroxide and a fluorescentompound depends on the exposure time to UV irradiation, typef UV sources and the temperature of photoreactor. Therefore,e studied effects of different UV irradiation lamps, the length of

he reaction coil wrapped around UV lamp and the temperaturef UV irradiation device.

Three types of UV lamps were evaluated as UV sources;oshiba GL-10 (10 W, 254 nm), Shigemi standard AL-15 H15 W, 254 nm) and National FL-10 BL-B (10 W, 350 nm). Weound that lamps, which emit at 254 nm wavelengths, gaveigher CL intensity and signal-to-noise (S/N) ratio than the lamphat emits at longer wavelength 350 nm. In addition, 15 W pow-red lamp gave CL intensity and S/N ratio about twice thatroduced by a 10 W powered lamp. As 15 W lamp that emitst 254 nm wavelengths is the highest energy lamp, it gave highonversion efficiency and CL intensity. Therefore, it was chosenor subsequent work.

The effect of coil length ranging from 3.0 to 8.0 m with.25 mm internal diameter corresponding to 36–96 s of UV irra-iation on CL intensity was examined. It was found that CLncreased as the length of the reaction coil increased due tohe increase in exposure time and then decreased. The reasonor this phenomenon is that the generation and decompositionf hydrogen peroxide have occurred simultaneously becausef the UV irradiation for a long time. Moreover, the detectionensitivity (S/N ratio) was measured against coil length for allamples. It was found that S/N ratio increase by increasing coilength till maximum then decrease because the background noisencreases as the length of the reaction coil increases. The opti-

um coil length was found to be 6.0 m corresponding to 72 s ofV irradiation.

The temperature of the photoreactor was studied due to its

ffect on generation efficiency of hydrogen peroxide and theuorophore. It was found that both CL intensity and S/N ratio

ncreased by the increase in temperature till 45 ◦C and then

S. Ahmed et al. / Analytica Chimica Acta 591 (2007) 148–154 151

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Fig. 3. Effects of pH of imidazole–HNO3 buffer on (A) CL intensity

ecreased. This may be due to the increase in generation effi-iency by the rise in temperature; the more rise in temperatureead to an increase in decomposition of hydrogen peroxide.herefore, 45 ◦C was selected.

.1.2. Optimization of CL conditionsImidazole has been widely used as a catalyst for the PO-

L reaction. The complex effects of imidazole on the reactioninetics have been attributed to a combination of nucleophilicnd general-base catalysis [25]. In this study, we selected aombination of imidazole–HNO3 and acetonitrile, accordingo previous report [28]. The usage of imidazole–HNO3 forO-CL reaction is intended for the elimination of quenchingffect of inorganic salts such as halides and also the solubil-ty in the organic constituent in the eluent [29]. The effect ofmidazole–HNO3 buffer solution on CL intensity was exam-ned. It was found that the best CL intensity and S/N ratio werebtained at 600 mM imidazole–HNO3 buffer solution. High con-entration of imidazole was required because of using smallercentage of imidazole buffer in the mobile phase (5%). Also,he influence of pH of the imidazole–HNO3 buffer solution onL was studied. It was found that both CL intensity and S/N ratio

ncreased with the increase in pH, which could be attributed toaster reaction kinetics at higher pH. Taking the durability ofhe ODS into consideration where develosil ODS-UG column istable at pH (1–10), pH value of 9.0 was selected for subsequentork (Fig. 3).The nature of the aryloxalate used in a PO-CL system is an

mportant factor in view of the produced CL intensity. There-

ore, several studies of aryloxalates have reported a general trendf increasing quantum yield and light intensity [30,31]. It wasound that aryloxalates with strongly electron-withdrawing sub-tituents provided the highest quantum yields, whereas those

p

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Fig. 4. Effects of different aryloxalates on CL intensity and S/N r

B) S/N ratio. Samples were 2 �M from each vitamin K homologue.

ith electron-donating or weak electron-withdrawing groupsesulted in either ineffective or poor production of chemilu-inescence. Among them, TCPO, TDPO, DNPO, DFPO andFPO are the most efficient aryloxalates. Therefore, we aimed

n this experiment at the study of the effect of these aryloxalatesn CL intensity and S/N ratio of vitamin K homologues. Amonghe investigated aryloxalates, TDPO gave the best CL intensitynd S/N ratio for all the studied vitamin K homologues (Fig. 4).oreover, TDPO provides improved solubility (1010 mM) and

hemical stability in acetonitrile relative to other aryloxalateshat further provides superior reproducibility [30]. Therefore,DPO was selected for subsequent work.

Moreover, the effect of different concentrations of TDPO wasnvestigated. It was found that CL intensity increases by increas-ng the concentration of TDPO whereas S/N ratio increases tilloncentration 0.6 mM and then decreased. The reason for this ishe increase in background noise. Therefore, the concentrationf 0.6 mM TDPO was used.

Because the emission intensity in PO-CL reaction is timeependent as the reaction proceeds and the excited intermediater product is formed, effect of flow rate of the TDPO solution onL intensity was also examined. It was found that CL intensity

ncreased by the increase in flow rate and reached maximumt 0.80 mL min−1 and then CL intensity decreased due to theecrease in reaction time between CL reagent, hydrogen perox-de and the fluorophore. Therefore, flow rate of 0.80 mL min−1

as selected.

.2. Fluorescence characteristics of the fluorescent

hotoproduct

Recently, we have reported the determination of quinonesy HPLC–PO-CL method combined with on-line photochem-

atio. Samples were 2 �M from each vitamin K homologue.

1 himica Acta 591 (2007) 148–154

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52 S. Ahmed et al. / Analytica C

cal reaction [26]. We have confirmed UV irradiation ofuinones produced hydrogen peroxide and a fluorescent 3,6-ihydroxyphthalic acid at the same time. Because vitamin Komologues have a common 1,4-naphthoquinone structure, itas assumed that vitamin K homologues produce the same

ction by UV irradiation. Vitamin K homologues have extremelyeak fluorescence and does not show efficient luminescence inO-CL system. However, it was found that the compound pro-uced by UV irradiation of vitamin K homologues had stronguorescence so it was detected via PO-CL reaction. The fluores-ence intensity of the photoproduct of vitamin K homologuesas measured using HPLC system with multi-wavelength fluo-

escence detection (as described in Section 2). The peak of theuorescent photoproduct newly appeared at 5.0 min after UV

rradiation. The excitation maximum and emission maximumavelengths, relative fluorescent intensities (RFI), and retention

imes were recorded for each vitamin K homologue. The excita-ion maximum wavelengths were 273 ± 2 nm whereas emission

aximum wavelengths were 330 ± 4 nm. The retention timesere 5.04 ± 0.04 min. In addition, the possibility that the gen-

rated compounds are the same was strongly suggested becausehe retention times and the fluorescence spectra of fluorescenthotoproducts were almost the same for all the studied vitamin

homologues. Moreover, the data obtained show correspon-ence with those obtained by authentic 3,6-dihydroxyphthaliccid with matching percentage of more than 98.5%. The flu-rescent product is assumed to be a degradation product foritamin K or its reduced form. Therefore, the generated fluo-ophore is suggested to be 3,6-dihydroxyphthalic acid althoughurther identification study is necessary in future.

.3. Validation of the proposed method

Calibration curves for standard vitamin K homologues wereonstructed by plotting the peak area ratio of vitamin Knd internal standard against concentration of vitamin K. Forach vitamin K calibration curve, retention time, calibrationange, correlation coefficient and detection limit were recordedTable 1). The correlation coefficient of 0.995 or more wasbtained in the concentration ranges of 0.01–10 �M for PK orK-4 and 0.02–10 �M for MK-7. Also, the detection limits

or quinones obtained with the proposed method (S/N = 3) were2, 38 and 85 fmol for PK, MK-4 and MK-7, respectively. Therder of CL intensity and detection sensitivity for all the stud-ed vitamin K homologues after UV irradiation were as follows;

eush

able 1etention times, calibration curves and detection limits for vitamin K homologues

ompound tR (min) Calibration curvea

Range (�M) Slopec (±S.E.)

K 10.7 0.01–10 0.69 ( ± 0.12)K-4 4.7 0.01–10 0.62 ( ± 0.08)K-7 31.5 0.02–10 0.53 ( ± 0.09)

a Peak area ratio of vitamin K and internal standard vs. concentration (�M).b Detection limit at a S/N ratio of 3.c Data presented as mean ± S.E. of three experiments.

ig. 5. Chromatogram of a mixture of standard vitamin K homologues obtainedsing the proposed method. Samples were 2 �M from each compound.

K > MK-4 > MK-7. The difference in the detection sensitivityas because of the difference in the generation efficiency ofydrogen peroxide and the fluorophore that were generated byV irradiation.A typical chromatogram of a standard mixture of vitaminhomologues and the internal standard using the proposed

ethod is shown in Fig. 5. Vitamin K homologues and thenternal standard were separated efficiently and all peaks wereluted within 35 min. The precision of the proposed methodithin- and between-day was examined using standard vita-in K homologues solutions at three different concentration

evels low (0.1 �M), middle (0.5 �M) and high (4 �M) con-entrations (Table 2). It was found that the relative standardeviations (R.S.D.) within-day (n = 5) and between-day (n = 3)ere 1.9–5.2% and 2.9–5.4%, respectively, so excellent repro-ucibility was obtained.

Recovery experiments were carried out by spiking plasmaamples with different concentrations of standard vitamin Komologues (low (1.0 nM plasma), middle (5.0 nM plasma) andigh (10.0 nM plasma) concentrations)). It was found that a per-entage recovery of 82.0–93.5% was obtained for all vitamin Komologues (Table 3). The extractive efficiency is important forccurate determination of plasma concentration. In our method,

xtractive efficiency from plasma can be completely adjustedsing 2-methyl-3-pentadecyl-1,4-naphthoquione as an internaltandard, which has the same chemical properties as vitamin Komologues. Therefore, the accuracy of determination of vita-

Detection limitb

fmol/injection (nM)Interceptc (±S.E.) r

0.57 ( ± 0.07) 0.995 32 (3.2)0.19 ( ± 0.03) 0.998 38 (3.8)0.26 ( ± 0.05) 0.997 85 (8.5)

S. Ahmed et al. / Analytica Chimica Acta 591 (2007) 148–154 153

Table 2Precision of the proposed method using standard vitamin K homologues

Compound Concentration(�M)

Precision(R.S.D.%)

Within-day (n = 5) Between-day (n = 3)

PK 0.1 3.9 4.90.5 1.9 4.04 2.1 3.6

MK-4

0.1 4.5 5.10.5 3.7 2.94 3.2 4.5

MK-7

0.1 5.2 5.4

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Table 4Circulating plasma vitamin K homologues concentrations in healthy humanvolunteers

Subject (sex) PK (nM) MK-4 (nM) MK-7 (nM)

1 (M) 2.48 1.35 3.022 (M) 2.13 0.94 1.813 (M) 2.95 1.84 2.264 (F) 3.05 2.13 2.055 (F) 2.70 1.37 2.936 (F) 2.83 1.24 1.88

Mean ± SD (nM) 2.69 ± 0.34 1.48 ± 0.43 2.32 ± 0.53

F, female; M, male.

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in K homologues was improved largely by using the internaltandard.

.4. Application for the determination of vitamin Komologues in human plasma

This method was applied successfully to the determination ofitamin K homologues in human plasma samples obtained fromealthy subjects (3 male and 3 females). Plasma levels of MK-4,K and MK-7 in healthy subjects were 1.48 ± 0.43 (mean ± S.D.nM)), 2.69 ± 0.34 and 2.32 ± 0.53, respectively (Table 4). How-ver, it is conceivable that the individual differences derived fromiet. A typical chromatogram of plasma extract from one healthyubject determined by the proposed HPLC–PO-CL method ishown in Fig. 6. It is clear from the chromatogram that PK, MK-4nd MK-7 can be determined without interference from plasmaomponents. The chromatogram indicates the high selectivity ofhe proposed method. The method was found to be superior inelectivity to most of the reported methods [9,19,22]. The resultsbtained from the determination of plasma samples were quiteimilar to those obtained by the reported methods [9,19,22].

hese results suggest that the accuracy of quantitative determi-ation of vitamin K homologues by this method was sufficientor clinical and nutritional applications.

able 3ecovery of PK, MK-4 and MK-7 from spiked plasma samples

ompound Spiked conc.(nM plasma)

Recovery (%)

Mean ± S.D. (%) (n = 3) R.S.D. (%)

K 1 85.4 ± 3.1 3.65 86.9 ± 3.4 3.9

10 90.4 ± 2.7 3.0

K-4 1 90.0 ± 4.9 5.45 86.8 ± 3.9 4.5

10 93.5 ± 4.5 4.8

K-7 1 82.0 ± 4.9 6.05 89.1 ± 3.9 4.4

10 91.7 ± 4.4 4.8

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ig. 6. A typical chromatogram of plasma extract from a healthy subject spikedith an internal standard.

. Conclusion

A sensitive and highly selective HPLC–PO-CL method forhe determination of vitamin K homologues in human plasmaas developed. Because hydrogen peroxide and the fluores-

ent material were not needed as post column reagents, theethod was found to be selective for the determination of

itamin K homologues in human plasma. It is thought thathe proposed method provides superior performance over theeported methods. One advantage of this method is the highelectivity compared to the reported methods where selectivitys an important issue when analyzing plasma samples. More-ver, it can detect accurately the low plasma concentrationsf vitamin K homologues that could be attributed to the highensitivity of the proposed method. However, the proposedethod is more sensitive than some of the reported methods

6,7,10], it still less sensitive than others [9,19,22]. These meth-ds require either platinum-reduction column which loses itsctivity by the time and provides insufficient reproducibility orighly expensive instruments as LC–MS methods. In contrast,he proposed method does not require expensive instrumentsnd has good reproducibility so it provides a convenient tool

or routine assay of plasma vitamin K homologues. In addition,he proposed method shows satisfactory precision and accuracyue to the use of vitamin K analog (2-methyl-3-pentadecyl-1,4-

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aphthoquione) as an internal standard. Therefore, proposedethod is suitable for clinical and nutritional studies as well

s routine assay for MK-4, PK and MK-7. Due to its high sensi-ivity and selectivity, the method may provide a useful tool forlucidation of importance of vitamin K in bone metabolism.

cknowledgments

We are grateful to Eisai Co. Ltd. for providing the standardsf vitamin K2 homologues menaquinone-4 and menaquinone-7.

eferences

[1] M.J. Shearer, Lancet 345 (1995) 229.[2] P.M. Gallop, J.B. Lian, P.V. Hauschka, N. Engl. J. Med. 302 (1980) 1460.[3] J.W. Suttie, J. Am. Diet Assoc. 92 (1992) 585.[4] M. Yamaguchi, H. Taguchi, Y.H. Gao, A. Igarashi, Y. Tsukamoto, J. Bone

Miner. Metab. 17 (1999) 23.[5] G.H. Dialameh, R.E. Olson, Anal. Biochem. 32 (1969) 263.[6] E. Sau Man Po, J. Ho, B. Yi Gong, J. Biochim. Biophys. Methods 34 (1997)

99.[7] P. Chatzimichalakis, V. Samanidou, I. Papadoyannis, J. Chromatogr. B 805

(2004) 289.[8] O.Y. Hu, C.Y. Wu, W.W. Chan, F.Y. Wu, J. Chromatogr. B 666 (1995) 299.[9] M. Kamao, Y. Suhara, N. Tsugawa, T. Okano, J. Chromatogr. B 816 (2005)

41.10] W.A. MacCrehan, E. Schonberger, J. Chromatogr. B 670 (1995) 209.11] E. Jacob, I. Elmadfa, Food Chem. 68 (2000) 219.12] L.Y. Wang, C.J. Bates, D.J. Harrington, M.J. Shearer, Ann prentice, Clin.

Chim. Acta 347 (2004) 199.

[

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a Acta 591 (2007) 148–154

13] Z.H. Gao, R.G. Ackman, Food Res. Int. 28 (1995) 61.14] T. Perez-Ruiz, C. Martinez-Lozano, J. Martin, M.D. Garcia, Anal. Bioanal.

Chem. 384 (2006) 280.15] K. Hirauchi, T. Sakano, T. Nagaoka, A. Morimoto, J. Chromatogr. 430

(1988) 21.16] Y. Haroon, D.S. Bacon, J.A. Sadowski, Clin. Chem. 32 (1986) 1925.17] J.P. Lagenberg, U.R. Tjaden, J. Chromatogr. 305 (1984) 61.18] T. Kojima, M. Asoh, N. Yamawaki, T. Kanno, H. Hasegawa, A. Yonekubo,

Acta Paediatr. 93 (2004) 457.19] H. Wakabayashi, K. Onodera, S. Yamato, K. Shimada, Nutrition 19 (2003)

661.20] H. Wakabayashi, K. Onodera, Nippon Yakurigaku Zasshi 166 (2000) 43.21] Z. Liu, T. Li, J. Li, E. Wang, Anal. Chim. Acta 338 (1997) 57.22] Y. Suhara, M. Kamao, N. Tsugawa, T. Okano, Anal. Chem. 77 (2005)

757.23] M. Gao, M. Yang, J. Hu, B. Shao, H. Zhang, H. Li, J. Chromatogr. A 1007

(2003) 31.24] M.F. Lefevere, A.P. De Leenheer, A.E. Claeys, J. Chromatogr. 186 (1979)

749.25] A.M. Garcia-Campna, W.R. Baeyens, Chemiluminescence in Analytical

Chemistry, M. Dekker, New York, 2001, Chapter 7, p.141.26] S. Ahmed, S. Fujii, N. Kishikawa, Y. Ohba, K. Nakashima, N. Kuroda, J.

Chromatogr. A 1133 (2006) 76.27] L. Benli, G. Lianzuan, Z. Jingling, Recl. Trav. Chim. Pays-Bas 110 (1991)

99.28] K. Nakashima, M. Wada, N. Kuroda, S. Akiyama, K. Imai, J. Liq. Chro-

matogr. 17 (1994) 2111.

29] K. Imai, A. Nishitani, Y. Tsukamoto, W. Wang, S. Kanda, K. Hayakawa,

M. Miyazaki, Biomed. Chromatogr. 4 (1990) 100.30] K. Imai, H. Nawa, M. Tanaka, H. Ogata, Analyst 111 (1986) 209.31] K. Nakashima, K. Maki, S. Akiyama, W. Wang, Y. Tsukamoto, K. Imai,

Analyst 114 (1989) 1413.