Abstract & Introduction

Vitamin K is a group of fat-soluble vitamins divided into vitamin K1 (onecompound, phylloquinone) and K2 (a group of compounds, menaquinones).Among the K2 family, MK4 and MK7 are the most nutritionally recognized. WhileK1 plays an important role in controlling blood clotting, studies have shown thatMK4 and MK7 have distinct biological function in the regulation of bonemetabolism and vascular calcification. As an interest for their biological action inextra-hepatic tissues is increasing, an accurate and simple measurement ofvitamin K status remains a critical issue for both clinical research anddiagnostics. There is a high degree of analytical challenges in vitamin K analysis,as it is the most lipophilic and least abundant of the fat-soluble vitamins. In thisstudy, a simple and fast plasma sample preparation procedure was developedusing phospholipid removal in combination with a chromatographic analysisusing a Raptor Biphenyl column. The method provides a novel solution towardsvitamin K research and high-throughput clinical diagnosis.

Calibration Standards and Quality Control Samples

SeraFlx BIOMATRIX (BioreclamationIVT) was fortified with 3 analytes to preparecalibration standards and QC samples. The linearity ranges were from 0.10-10ng/mL. Three QC levels were prepared at 0.25, 0.75, and 8.00 ng/mL for all 3analytes. The fortified standard and QC samples were subjected to samplepreparation procedure as described.

Table 1: Analytical Conditions for Waters Xevo TQ-S with Acquity UPLC

Table 2: Analyte Transitions

Analyte Precursor Ion Product Ion Quantifier Product Ion QualifierMK4 445.47 187.15 81.09K1 451.47 187.15 128.17

MK7 649.73 187.19 81.09MK4-d7 452.47 194.16 -K1-d7 458.53 194.17 -

MK7-d7 656.67 194.16 -

Figure 2: Analysis of Blank and Fortified SeraFlx BIOMATRIX

Methods

A Novel Solution for Vitamin K1 and K2 Analysis in Human Plasma by LC-MS/MS Shun-Hsin Liang, Ravali Alagandula, Frances Carroll, Ty Kahler, Sue Steinike, Paul Connolly; Restek Corporation

Analytical ColumnRaptor Biphenyl 2.7µm, 50 mm x 2.1 mm (Restek Part No. 9309A52)

Mobile Phase A Water, 0.1% formic acid, 5mM ammonium formate

Mobile Phase B Methanol, 0.1% formic acid

Gradient Time (min) %B

0.00 90

1.00 100

3.00 100

3.01 90

4.00 90

Flow Rate 0.4 mL/min

Injection Volume 5 µL

Column Temp. 40°C

Ion Mode positive ESI

Chromatograms

Figure 1: Structure of Vitamin K1 and K2

K1

Sample Preparation

A 500 µL aliquot of sample was mixed with 5 µL of internal standard solution(K1-d7, MK4-d7, and MK7-d7 at 100 ng/mL in methanol) and 1.5 mL ofacetonitrile followed by vortexing for 20 seconds at 3000 rpm. Aftercentrifugation at 4300 rpm for 10 minutes, the supernatant was loaded onto aBiotage ISOLUTE PLD+ 96-well plate (50 mg) and vacuum was applied to collectthe eluate. The eluate was then evaporated to dryness at 50°C under a gentlestream of nitrogen. The dried extract was reconstituted with 100 μL of 15:85water:methanol and 5 μL of sample was injected for analysis.

Figure 3: Analysis of Endogenous Vitamin K in Human Plasma

Results and Discussion

Linearity: Using 1/x weighted linear regression, all 3 analytes showedacceptable linearity with r2 values of 0.995 or greater, and deviations <15%(from nominal concentration). The established limit of quantitation was 0.10ng/mL.

Figure 4: Standard Curves

Accuracy & Precision: Precision and accuracy analyses were performed on threedifferent days. The method accuracy was demonstrated with recovery valueswithin 10% of the nominal concentration for all QC levels. The %RSD was 0.207-7.83% and 3.39-5.75% for intra-day and inter-day, respectively, indicatingacceptable method precision (Table 3).

Table 3: Accuracy and Precision of QC Samples

Conclusions

A method was developed for fast, accurate, and precise measurement ofvitamin K1, MK4, and MK7 in plasma. With fast and simple phospholipid removalsample preparation and 4 minutes of chromatographic analysis using a RaptorBiphenyl column and MS/MS analysis, the established method provides areliable and high-throughput assay for the clinical research and assessment ofvitamin K1 and K2.

Chromatographic Performance: A fast 4-minute chromatographic analysis wasachieved with injection of reconstituted solution obtained from a simplephospholipid removal procedure. No chromatographic interferences wereobserved from the analysis of blank plasma samples (Figure 2).

Analyte

QC-1 (0.250 ng/mL)

QC-2(0.750 ng/mL)

QC-3(8.00 ng/mL)

Avg. Conc.

(ng/mL)

Avg.Accuracy %RSD

Avg. Conc.

(ng/mL)

Avg.Accuracy %RSD

Avg. Conc.

(ng/mL)

Avg.Accuracy %RSD

K1 0.246 98.2 5.75 0.763 102 3.39 8.23 103 4.36

MK4 0.249 100 4.55 0.729 97.2 3.83 8.11 101 5.17

MK7 0.245 98.0 3.88 0.715 95.3 4.98 8.23 103 4.76

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MK4

MK7

Blank 0.50 ng/mL

MK4

K1

MK7

Human Plasma Analysis

Multiple human plasma samples (BioreclamationIVT) were analyzed forendogenous vitamin K with established method. These included pooled plasma(from 20 persons), plasma of an individual taking vitamin K2 medication, andplasma from an individual of a Japanese descent .

Pooled Plasma Japanese Descent Plasma

Plasma from IndividualUsing Vitamin K2

Medication

MK4

K1

MK7

MK4

K1

K1

MK7

Human Plasma Analysis: Distinct endogenous vitamin K profiles were revealedby analyzing multiple plasma samples (Figure 3). The analysis of pooled humanplasma is a good indication of the average vitamin K level of a collectedpopulation. The result showed clear detection of K1 and MK4 in pooled plasmawith no detectable MK7, reflecting that the collected population has very lowconsumption of the MK7-containing food. Conversely, a plasma sample from anindividual of Japanese descent showed higher MK7 and no detectable MK4. Theplasma sample from an individual indicated as under vitamin K2 medicationshowed strong K1, MK4, and MK7 signals. Overall, these results demonstratedthat our established method can measure the variation of vitamin K1 and K2

concentrations in various individuals and is thus applicable for clinical analyses.

K1

y = 1.15 x + 0.0207 (r2 = 0.997)

MK4

y = 0.980 x – 0.0185 (r2 = 0.999)

MK7

y = 1.29 x + 0.0212 (r2 = 0.998)