Journal of Chromatography B, 877 (2009) 351354

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Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Short communication

Determination of vitamin K1 in plasma by solid phase extraction and HPLC with uorescence detection
Rita Paroni a, , Elena Maria Faioni b , Cristina Razzari b , Gessica Fontana b , Marco Cattaneo b
a b

Analytical Chemistry and Biochemistry Unit, Department of Medicine, Surgery and Dentistry, University of Milan, H San Paolo, via di Rudini 8, 20142 Milan, Italy Hematology and Thrombosis Unit, Department of Medicine, Surgery and Dentistry, University of Milan, H San Paolo, via di Rudini 8, 20142 Milan, Italy

a r t i c l e

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a b s t r a c t
We describe a procedure for quantication of vitamin K1 in human plasma by HPLC. Samples, enriched with a vitamin K derivative as internal standard, were deproteinized, puried on polymeric RP-SPE cartridges and injected into HPLC equipped with a post-column on-line zinc metal reactor and a uorometric detector. Median level in blood donors (n = 87) was 1.967 nmol/L (0.934.01, 5th95th percentiles), with a signicant correlation between plasma levels and age (r = 0.276, p = 0.00958) and a lower (not signicant) value in women than in men. This method, easy-to-handle and with a high throughput, can be used to identify covert states of vitamin K intake deciency in patients thus at risk of alterations in blood clotting or bone mineralization. 2008 Elsevier B.V. All rights reserved.

Article history: Received 18 September 2008 Accepted 9 December 2008 Available online 25 December 2008 Keywords: Vitamin K1 HPLC Reference values Haemostasis alteration Bone mineralization

1. Introduction Vitamin K is a general term comprising phylloquinone (vitamin K1 , synthesized by green leaves, the main vitamin K form introduced with the diet) and menaquinones (vitamin K2 , synthesised by bacteria) [1]. All forms of vitamin K in humans serve as cofactor of an enzyme that catalyzes the post-translational carboxylation of glutamic acid residues to gamma-carboxyglutamic acid (Gla), resulting in the hepatic synthesis of biologically active coagulation factors [2] and of some proteins pivotal for the homeostasis of bone, cartilage, vessel wall and nervous system (matrix Gla protein, protein S, Gas6) [36]. Patients with severe cholestasis or fat malabsorption, and breast-fed newborns are at risk of developing vitamin K deciency [7]. In addition, a quantity of epidemiological studies have been carried out on the potential role of vitamin K in slowing down the rate of postmenopausal bone loss and maintaining bone health, but this role in this eld is still controversial [810]. Measurement of vitamin K in circulating blood is still considered an analytical challenge, due to its instability at UV-visible light and its very low levels nmol/L range) compared to those of interfering endogenous lipids, from which it must be isolated by means of several pre-analytical steps before assay. Many technical approaches have been used, most of which based on liquidliquid extraction followed by separation on adsorption chromatography and a second chromatographic stage involving reversed-phase conditions.

Detection modes include ultraviolet [11], electrochemical [12], uorescence [13], chemiluminescence [14] and mass spectrometry [15]. The technical problems associated with vitamin K measurement account for considerable discrepancies among the reported reference values, spanning from 1.64 2.15 to 4.8 2.9 nmol/L in studies on different populations, using different laboratory techniques [1519]. With the aim to get a simple and high throughput procedure for vitamin K1 measurement in human plasma starting from the procedure described by Kirk and Fell [20], we tested an improvement based on solid-phase extraction purication on a polymeric reversed-phase SPE sorbent with enhanced selectivity for polar and non polar compounds, followed by HPLC with post-column reduction e uorometric detection. 2. Experimental Analytical reagent grade chemicals, HPLC grade solvents and vitaminK1 standard with a purity of 99%, were from SigmaAldrich (Milano, Italy). After obtaining informed consent, blood from 87 healthy blood donors of the Blood Transfusion Centre of the San Paolo Hospital in Milan (mean age 44 yrs, range 2268 yrs, 62 men, and 25 women) was collected in EDTA, taking care to avoid light exposure; plasma aliquots (0.25 mL) were stored at 80 C in 2 mL polypropylene screw-cap cryotubes. When needed, plasma samples were thawed at 37 C, spiked with 5 L of the proprietary vitamin K derivative (Immundiagnostik AG, Wiesenstrasse, Bensheim, Germany cod. KC2400is) as suggested by Wang et al. [13] and added

Corresponding author. Tel.: +39 02 503 23272; fax: +39 02 503 23245. E-mail address: rita.paroni@unimi.it (R. Paroni). 1570-0232/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2008.12.044

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Fig. 1. A representative chromatogram from plasma samples (0.25 mL spiked with 5 L of the proprietary internal standard solution) extracted following the SPE procedure described in the present method: A from a blood donor and B after spiking with standard phylloquinone (K1 ). Calculated plasma concentration: A, 1.39 nmol/L and B, 9.5 nmol/L.

to 1 mL absolute ethanol to disrupt the -lipoprotein structure to which vitamin K is associated. After mixing for 30 sec, samples were centrifuged at 12,000 g in an Eppendorf MiniSpin for 5 min. The ethanol supernatant was loaded onto the StrataTM -X 33 m Polymeric Reversed Phase SPE 30 mg/1 mL extraction cartridges (Phenomenex, Anzola Emilia, Italy) connected to Visiprep Solid Phase Extraction Vacuum Manifolds (Supelco), and pre-activated with 1 mL methanol followed by 1 mL distilled water (1 mL/min). After washings with 1 mL distilled water, 1 mL water: methanol 50:50 (by volume) and 1 mL water: methanol 20:80 (by volume), the columns were dried by ushing air for 1 min. Vitamin K1 plus the internal standard were collected into 5 mL glass disposable tubes (rinsed with acetone) by applying 1 mL of acetonitrile: isopropanol: dichloromethane 70:10:20 (by volume), evaporated under nitrogen and stored at 20 C for no more than 15 days. Before analysis the residue was dissolved with 50150 L of the HPLC mobile phase, loaded onto the autosampler at 5 C (Waters 717plus) and 50 L automatically injected. Separation was accomplished with a Waters 515 pump connected to a Spherisorb Ultrasphere ODS Beckman column (Palo Alto, CA, USA) (125 mm 4.6 mm, 5 m). Isocratic elution at 0.6 mL/min and at room temperature was performed with 95% eluent A (994.5 mL methanol plus 5.5 mL of a solution 2 mol/L ZnCl2 , 1 mol/L CH3 COONa, 1 mol/L CH3 COOH) and 5% eluent B (ethanol). As an alternative, HyperClone 3 m BDS C18 130 column (Phenomenex, 150 3.2 mm) was also tested. Vitamin K1 was detected uorometrically with a Waters 474 detector (ecc 244em 430) after post-column on-line reduction to the hydroquinone form in a 4.6 mm 5 cm column freshly lled with 98% purity zinc dust (<10 m) (SigmaAldrich) at each analytical batch. 3. Results and discussion Under the described analytical conditions, the peaks of vitamin K1 and the internal standard eluted in about 16 min (Fig. 1), with a baseline separation of the two peaks. By using the alter-

native column HyperClone, the vitamin K1 and the IS peak eluted in 10 min with still an acceptable resolution (data not shown), so for high throughput routine conditions the second column may be recommended. The presence of late eluting interfering compounds or a back pressure increase along analytical batches were never observed, thus ruling out the need for gradient elution; in any case, as a preventive measure, we raised the component B to 80% for 10 min every 10 injections in order to eliminate nonpolar adsorbed impurities. We checked the linearity of the method by adding increasing amounts of the working vitamin K1 standard solution (up to 71.04 nmol/L) to the plasma samples before extraction. Calibration curves were prepared by plotting the peak area ratios of vitamin K1 standard to internal standard against the concentrations (nmol/L) of vitamin K1 . The method gave a linear response in the tested plasma range (a = 0.3847, b = 0.772, r2 = 0.999), with a recovery of 110 13%, 98 25% and 103 14% at 1.32, 2.64 and 5.28 nmol/L (n = 5). Within-run reproducibility ranged between 3.1% and 10.2% at 1.3 and 5.28 nmol/L (n = 10), while between-days reproducibility on a six month period was 13.6% and 12.5%, respectively (n = 6). The vitamin K1 detection limit for this assay was 20 fmol per injection, based on a signal-to-noise ratio of 5:1. Starting from a plasma volume of 0.25 mL, the quantitation limit was 0.2 nmol/L. While the plasma phylloquinone concentration in relation to the intake and age has been studied in depth in a nation-wide sample in Great Britain or in the Japanese area [18,19], a similar kind of investigation is still limited in the Italian region. Using the present method we found that the plasma levels of vitamin K1 in 87 Italian healthy blood donors were not normally distributed and were positively skewed, with a median (5th95th percentiles) value of 1.967 nmol/L (0.934.01,) (Fig. 2A). There was no statistically signicant difference between the observed values in men (1.991, 1.1294.013) and women (1.961, 0.883.636). There was a statistically signicant correlation between plasma vitamin K1 levels and age (r = 0.276, p = 0.00958) (Fig. 1B).

Fig. 2. A: Histogram distribution of vitamin K1 levels found in 88 healthy subjects. B: Correlation of vitamin K1 with age. r = 0.322 by Pearson product moment correlation (n = 87).

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Fig. 3. A: Regression plot of the new method vs. the HPLC method by Wang et al. [13] referred as reference method. B: Regression plot of the new method vs. the commercial Kit method.

Fig. 4. A: Bland and Altman differences plots for the new HPLC method versus the reference one [13]. The regression of the differences versus average values, 95% condence limits and limits of agreements (mean 2SD) are shown. B: Bland and Altman differences plots for the comparison in D.

The performance of our method was compared to the previously validated HPLC method by Wang et al. [13], which we considered the reference method, and to a commercially available HPLC kit for vitamin K1 determination in plasma and in serum (Immundiagnostik AG, Wiesenstrasse, Bensheim, Germany). Vitamin K1 plasma levels (n = 20) found by the three methods were compared by ANOVA for repeated measures followed by an all pairwise multiple comparison procedure (HolmSidak method) to isolate the group that differs from the others. The mean value found with the present method was 1.86 (0.91), not signicantly different both from 2.00 (0.72) nmol/L determined by the considered reference procedure [13], and 1.62 (1.00) nmol/L found by the commercially available kit. This last value was on the contrary signicantly lower (p = 0.027) than that obtained by the HPLC method by Wang et al. [13]. Linear regression analysis of the new method vs. the reference method yielded the following equation: Y = 0.922 (0.205) X + 0.012 (0.434, p = 0.978) (SE), r = 0.728 (p < 0.01), while a similar comparison vs. the commercial method produced: Y = 0.743 X (0.124) + 0.652 (0.235, p = 0.012) (SE), r = 0.816, p < 0.001 (Fig. 3A and B). The regression plot of differences in vitamin K1 concentrations detected by the reference method and by our method vs. the average plasma concentration (Bland and Altman method) was not signicantly different from 0 (p = 0.16), indicating the two methods are equivalent, although the curve tended to diverge from equality, especially at the highest vitamin K1 concentrations (Fig. 4A). Similar results were obtained when our method was compared to the commercial method (p = 0.5) (Fig. 4B). No sample was outside the limits of agreement (2SD) for both comparisons. 4. Conclusions The main advantage of our procedure compared to others, consists in the vitamin K1 purication from plasma, which requires only 3.5 mL organic solvent per sample and allows to reduce

the amount of organic solvent (hexane) generally used for the liquidliquid extraction followed by the silica solid phase chromatography (20 mL) [13]. By our method, one can freeze the plasma aliquots (0.5 or 0.25 L) in the centrifuge screw-cap polypropylene tubes, use the same tube for the addition of the internal standard and ethanol deproteinization, and recover the vitamin from the SPE column without any further transfer. This, bearing in mind vitamin K1 instability to light and alkali, could be an additional advantage. The whole extraction procedure takes no more than 30 min plus 10 min of drying under nitrogen for 12 samples. In addition, our assay does not require a skilled technical staff and allows high throughput. Acknowledgments We thank Prof. Monica Ferraroni, Statistic Unit, and Dr. Federico Lussana, Hematology and Thrombosis Unit, Department of Medicine, Surgery and Dentistry, University of Milan, H San Paolo, for their helpful advice in the statistical analysis of data. A special acknowledgment to Dr. Anna Lecchi and to all her staff at the Dipartimento di Medicina e Specialit Mediche, IRCCS Fondazione Ospedale Maggiore, Mangiagalli e Regina Elena, Universit di Milano, Italy, for the warm and kind hospitality given during the setting up of the method. This study was supported by a grant from Italian Ministry of Research. References
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