Routine Supercritical Fluid ChromatographyTandem Mass Spectrometry Method forDetermination of Vitamin K1 Extracted fromSerum with a 96-Well Solid-Phase ExtractionMethod

Trude Athammer Sandvik,1,2* Asgeir Husa,1 Marie Buchmann,1 and Elsa Lundanes2

Background: The concentration of vitamin K1 in serum or plasma is themost common index for assessing vitaminK status. The aim of this studywas to develop and validate a rapid and reliable routinemethod for quantifying vitaminK1 above 0.1 ng/mL. Semi-automation of a simple sample preparation with fast analysis by supercritical fluidchromatography–tandemmass spectrometry (SFC-MS/MS) was exploited.Methods: Vitamin K1 was extracted from 250-μL serum samples by the use of protein precipitation and reversed-phase solid-phaseextraction (SPE) in96-well plates andquantifiedbySFCona2.1×100mmTorus1-Aminoanthracene(1-AA) column in 3.8 min with electrospray ionization—tandemmass spectrometry (MS/MS) detection.Results: This method shows good linearity in the concentration range of 0.1–50 ng/mL with a correlation coefficientof R2 >0.999. Imprecision was satisfactory, with repeatability and reproducibility <10% CV. The lower limit of themeasuring interval was 0.1 ng/mL, and no systematic bias was observed for themethod, which used vitamin K1-d7 asinternal standard. Recovery of vitamin K1 in external quality controls was satisfactory compared to other laboratoriesparticipating in theexternalqualityassurancescheme.Themethod iscurrently inroutineuseforanalysisofserumsamples.Conclusions: Themethod allows high-throughput reliable determination of vitamin K1 in serum in the range 0.1–50ng/mL.

IMPACT STATEMENTWith the developed method, measurement of vitamin K1 level in patient serum samples can be performed

rapidly and reliably. The method includes a semi-automated solid-phase extraction sample preparation method

without theneedofanevaporationstepbeforequantificationbysupercriticalfluidchromatography–tandemmass

spectrometry (SFC-MS/MS). The method provides a faster analysis than that of LC-MS/MS, and in a period of 3

months, the method has been applied to analysis of >5000 samples. The method is suitable for use in high-

throughput medical laboratories.

1Fürst Medical Laboratory, Oslo, Norway; 2University of Oslo, Oslo, Norway.*Address correspondence to this author at: Fürst Medical Laboratory, Søren Bulls vei 25, 1051 Oslo, Norway. Fax +47-22909606; e-mailtasandvik@furst.no.DOI: 10.1373/jalm.2016.021717© 2017 American Association for Clinical Chemistry

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Copyright 2017 by American Association for Clinical Chemistry.

Fat-soluble vitamin K is required for the synthe-sis of proteins involved in blood coagulation (1).Recent publications suggest that low vitamin K in-take is associated with increased fracture risk andlow bone mineral density (2, 3), and vitamin K mayhave a protective role in cardiovascular health (4,5). Vitamin K can be classified into 2 groups: vita-min K1 (phylloquinone) and vitamin K2 (group ofmenaquinones) (6, 7). The highest amounts of vita-min K1 are found in green leafy vegetables (7),while K2 vitamin is found in fermented food and tosome extent is produced by intestinal bacteria (3,8). The predominant circulatory form of vitamin Kin blood and the only vitamin routinely measuredis vitamin K1. Serum concentration of vitamin K1 isan indicator of vitamin K status, which directly re-flects storage and transport of vitamin K (5). Sev-eral methods for determining vitamin K1 havebeen published, mostly with extensive samplepreparation using liquid chromatography (LC)3

coupled with fluorescence detection or massspectrometry (9–21).Supercritical fluid chromatography (SFC) was in-

troduced in the early 1960s and has since experi-enced wavering popularity. Because of thesupercritical mobile phase properties, where lowviscosity and high diffusion rates allow for highspeed and efficient separations, SFC has advan-tages compared to LC. Previous generations of SFCequipment were seldom used for quantitativeanalyses because of poor performance regardingprecision, accuracy, and robustness. With the in-troduction of new redesigned instrumentation,most of these issues have been resolved, resultingin a recent revival of interest in SFC (22–25).Our in-house LC-MS/MS method for determina-

tion of vitamin A, vitamin E, and 25-OH-vitamin Dincludes evaporation of heptane in the sample

preparation step. The residue is reconstituted in amore polar solvent compatible with the reversed-phase liquid chromatography method. This evap-oration step is considered a “bottleneck” in thework flow. By using SFC, the work flow could besimplified, since organic solvents such as heptanehave similarmobile phase strength as CO2, makingdirect injection of heptane extract possible (22, 25).The higher efficiency of SFC compared to LC wasthe reason for developing a routine method fordetermination of vitamin K1 in serum using super-critical fluid chromatography–tandem mass spec-trometry (SFC-MS/MS).

MATERIALS AND METHODS

Chemicals and materials

Vitamin K1 was obtained from Sigma-Aldrich[Supelco analytical standard purity 99.9% and Cer-illiant certified referencematerial (CRM)] andChro-madex (primary grade, purity 99.3%). VitaminK1-d7 (5,6,7,8-d4, 2-methyl-d3) was purchased fromSigma-Aldrich (isotopic purity 99 atom % D). Car-bon dioxide 5.0 (99.999%) was from Yara PraxairAS. Methanol LC-MS grade and ethanol HPLCgrade were from Merck Millipore, and formic acidFluka LC-MS grade was from Sigma-Aldrich. Hep-tane LC-MS grade was from Rathburn Chemicals,2-propanol LC-MS gradewas fromVWRChemicals,and type 1 water was produced with a Milli-Q Sys-tem (Merck Millipore). Collision gas for the MS/MSwas argon 5.0 (Yara Praxair), and nitrogenwas pro-duced with a nitrogen generator (Atlas Copco).Polypropylene 96-deep-well plates (DWPs) andthermal sealing foil were from Porvair SciencesLimited. The solid-phase extraction (SPE) sorbentwas Oasis PRiME HLB μElution in 96-well plate for-mat (Waters Corporation).

3Nonstandard abbreviations: LC, liquid chromatography; SFC, supercritical fluid chromatography; SFC-MS/MS, supercritical fluid chromatogra-phy–tandem mass spectrometry; CRM, certified reference material; DWP, deep-well plate; SPE, solid-phase extraction; IS, internal standard; BS,bovine serum; KEQAS, external quality assurance scheme for phylloquinone; PPT, protein precipitation; RLH, robotic liquid handler; UPC2, UltraPerformance Convergence Chromatography; ESI, electrospray ionization; 1-AA, 1-Aminoanthracene; ABPR, automatic back pressure regulator;MRM, multiple reaction monitoring; LLMI, lower limit of the measuring interval; LLE, liquid-liquid extraction.

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Stock solutions and working solutions

Stock solutions for vitamin K1 were prepared byweighing appropriate amounts of vitamin K1 dis-solved in ethanol. Gravimetric values were ad-justed to compensate for impurity. Stock solutionof the internal standard (IS) was prepared by add-ing ethanol to the purchased vial. The concentra-tions of the stock solutions are presented in Table1. Working solutions in ethanol were made by ap-propriate dilutions of the stock solutions.

Validation solutions, calibrators, and QCmaterial

The matrix used to prepare the validation solu-tions, calibrators, and QC material was bovine se-rum (BS) fromSigma-Aldrich. TheBSwas irradiatedbyUV-light (Biosan PCR cabinet) for 30 h to removeendogenous vitamin K1. Validation solutions wereprepared at 12 concentration levels in the range of0.05–100 ng/mL. Five calibrators were preparedcovering the concentration range 0.1–10 ng/mL.Low and high QC samples were prepared at con-centrations of 0.3 and 1.75 ng/mL, respectively. Toensure an independent source of vitamin K1 whenassessing trueness, vitamin K1 stock solution fromanother vendor was used when preparing two ofthe validation solutions and the QC samples. A de-tailed overview of all the solutions is presented inTable 1 in the Data Supplement that accompaniesthe online version of this article at http://www.jalm.org/content/vol1/issue6.

Serum samples and external qualityassurance samples

Serum samples were obtained from Fürst Med-ical Laboratory. The method was also monitoredby participating in the external quality assurancescheme for phylloquinone (KEQAS) (26).

Sample preparation

Vitamin K1 was extracted from the samples byprotein precipitation (PPT) followed by SPE. Thesample preparation was semiautomated on a Mi-crolab STAR (Hamilton Robotics) robotic liquidhandler (RLH) equipped with 1000 μL 8-channelpipettes and a vacuum station. All pipetting andSPE steps were performed by using the RLH. Ali-quots of 250 μL serumwere pipetted from sampletubes into a 96-DWP, and 800 μL cold precipitationsolution consisting of ethanol/methanol (50/50,v/v) with IS (0.65 ng/mL) was added to ensure pro-tein denaturation. The 96-DWP was sealed withthermal sealing foil (MiniSeal heat sealer, Porvair)and vigorously shaken for 5 min at 2000 rpm (Mix-Mate vortex-type mixer, Eppendorf AG). The platewas left for 15 min before centrifugation at 1811gfor 15 min (Eppendorf 5810 Centrifuge). Afterpiercing (in-house–made piercer), the 96-DWPwasplaced in the RLH, and 750 μL of supernatants wastransferred from the 96-DWP to an Oasis HLBPRiME 96-well SPE plate, which was placed in thevacuum station (no pretreatment of the SPE platewasnecessary). The supernatantwas left to immerse

Table 1. Stock solutions of vitamin K1 and vitamin K1-d7.

Solution Concentration in ethanol Used for preparingVitamin K1 stock solution 1 (Supelco) 5mg/mL Working solutions for making validation

solutions and calibratorsVitamin K1 stock solution 2 (Chromadex) 0.5mg/mL Working solution for making validation

solutions and QC samplesVitamin K1 (Cerilliant) 10 μg/mL Working solution for spiking analysis in

human serumIS (Vitamin K1-d7) stock solution 0.98mg/mL Working solution for making the protein

precipitation solution containing IS

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into the SPE sorbent for 2 min and then was slowlypassed through the SPE sorbent by vacuum. Toeach of the wells of the SPE plate, 200 μL washsolution, consisting of methanol/water (80/20, v/v),was added andpassed through the SPE sorbent byvacuum. A drying step of 2 min (high vacuum) wasapplied before a 96-DWP collection plate wasplaced inside the vacuum station, and 50 μL hep-tane was aliquoted into each SPE well, slowly elut-ing the analytes into the collection plate assistedby vacuum. The elution step was repeated twice,hence a total elution volume of 150 μL for eachsample. The plate was sealed before transfer tothe SFC-MS/MS instrument. A schematic overviewof the sample preparation is presented in Fig. 1.The SPE procedure efficiently removed phospho-lipids (see Fig. 2 in the online Data Supplement).

SFC-MS/MS

Two Waters ACQUITY Ultra Performance Con-vergence Chromatography (UPC2) instrumentswithWaters Xevo TQ-S tandemquadrupoleMS/MSequipped with multimode ionization source [elec-trospray ionization (ESI)/atmospheric pressurechemical ionization] and isocratic solventmanagerwere used. The interface connecting the UPC2 andthe MS/MS was a pre–automatic back pressureregulator (pre-ABPR) splitter.The 2.1 × 100 mm Acquity UPC2 Torus 1-Amino-

anthracene (1-AA) (1.7 μm) column with precol-umn (2.1 × 5 mm) was from Waters. The mobilephasewas CO2with a linearmodifier gradient from0.1% to 15% methanol in 1.3 min at a flow rate of1.3 mL/min. Washout was performed by using a40% modifier (flow gradient from 1.3 to 1.0 mL/min) in 0.6 min with re-equilibration by 0.1%meth-anol (flow gradient from 0.9 to 1.3 mL/min) in 0.7min. The autosampler cycle was 1.2 min, giving atotal cycle time of 3.8 min. The column tempera-ture was 45 °C, the ABPR pressure was 1650 psi,and injection volume was 5 μL (heptane). To im-prove ionization, methanol with 0.3% formic acid(flow rate 0.3 mL/min) was used as solvent. Wash-

ing of the chromatographic systemwas performeddaily by column wash with 50% methanol in 7 minand 100% CO2 in 3 min (flow rate 0.7 mL/min), anda 30-min wash-out of the tubing (bypassing thecolumn)using100%methanol (flowrate1.0mL/min)for 30 min.The MS/MS was operated in ESI positive mode

with a capillary voltage of 3 kV, desolvation gas flowof 800 L/min, desolvation temperature of 600 °C,and source temperature of 120 °C. The measure-ments were carried out by multiple reaction

Fig. 1. Schematic presentation of the samplepreparation procedure.Sample preparation steps using automated liquid han-dling are marked with dark background. MeOH, metha-nol; EtOH, ethanol.

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monitoring (MRM) (27). All m/z transitions in theMRM method are described in Table 2.

Quantification

To quantify vitamin K1 in samples, a calibrationcurve was constructed using linear regression withweighting 1/x in MassLynx by plotting the ratio ofanalyte peak area/IS peak area as a function of theconcentration of the calibrators. Them/z transitionwith best signal was chosen as quantifier for bothvitamin K1 and IS. An additional m/z transition forvitamin K1 was chosen as a qualifier. Integration ofpeak area was performed in MassLynx, and theextracted ion chromatograms were smoothed be-fore integration.

Recovery

Recovery was evaluated by spiking vitamin K1and IS in blankmatrix (UV-depleted BS) before andafter extraction, solution B and solution C, respec-tively, in Fig. 3. The solutions were prepared in 7concentration levels of vitamin K1 while keepingthe IS concentration constant. The solutions wereanalyzed with single injections by the SFC-MS/MSmethod. The data were evaluated by linear regres-sion, calculating the slope of both the peak area ofvitamin K and the analyte/IS peak area ratio. Recov-ery was determined by dividing slopes for solutionB/solution C.

Matrix effects

Matrix effects were investigated by spiking vita-min K1 and IS to the injectionmatrix (heptane) andblank matrix after extraction, solution A and

solution C, respectively, in Fig. 3. Matrix effectswere calculated by dividing slopes for solution C bysolution A.In addition, a qualitative evaluation of matrix ef-

fects was performed by a postcolumn infusionexperiment. Ten serum samples (vitamin K1 con-centration level ≤0.1 ng/mL) were extracted andinjected with postcolumn infusion of vitamin K1and IS.

Linearity and lower limit of the measuringinterval

To evaluate linearity, 10 validation solutionsin duplicates, over the concentration interval of0.1–98 ng/mL, were analyzed. Peak area ratio ofcalibrator to IS was plotted against concentrationof calibrator in MassLynx.The aim was to achieve a lower limit of the mea-

suring interval (LLMI) of 0.1 ng/mL. A spike analysiswas performed using 4 pools of UV-depleted hu-man serum spiked with vitamin K1 (Cerilliant CRM)to a concentration of 0.1 ng/mL. A total of 48 rep-licates were analyzed in 2 different run sequenceswith two different SFC-MS/MS instruments.

Imprecision and trueness

Repeatability data were obtained by analysis of10 replicates of prepared validation solution at 2concentration levels in the same run sequence.Reproducibility data were obtained by routineanalysis of 2 replicates within each series of thein-house QC samples at 2 concentration levelsfor a period of 2 months using 2 SFC-MS/MSinstruments.

Table 2. MRM transitions and MS settings for vitamin K1 and vitamin K1-d7.

CompoundMonoisotopicmass, Da

Precursorion, Da

Production,Da

Dwelltime, s

Cone,V

Collision energy,eV

Vitamin K1 (quantifier) 450.35 451.3 187.2 0.023 45 22Vitamin K1 (qualifier) 450.35 451.3 128.2 0.023 45 65Vitamin K1-d7 (IS) 457.74 458.3 194.1 0.023 45 22

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The validation solutions and QC samples usedto determine imprecision were prepared froman independent vitamin K1 stock solution (stocksolution 2, Table 1) and used to address true-ness by calculating the % bias of the measuredmean concentration compared to the spikedvalue.

Method comparison

Patient samples (n = 89) were used for compar-ison between the established method and an ex-isting LC method.

Statistics

All statistical calculations were performed withMicrosoft Excel 2010. LLMI was assessed by calcu-lating the mean, SD, %CV, % bias, and total error(%CV × 2 + % bias) of 48 replicates. Repeatabilitywas assessed by calculating the mean, SD, and%CV of 10 replicates within the same run se-quence. Reproducibility was assessed by ANOVAcalculating the within-run (1 run daily) andbetween-run variances based on pooled resultsfrom the 2 instruments. The method comparisonwas performed using weighted Deming regressionanalysis.

RESULTS

SFC-MS/MS

Extracted ion chromatograms of vitamin K1 andIS in prepared samples and neat heptane areshown in Fig. 2. There are several peaks originatingfrom the serum matrix in the vitamin K1 m/z tran-sition, of which all are separated from vitamin K1,with an analysis time of <4 min.

Analytical specificity and interferencestudies

Interfering compounds, e.g., other fat-soluble vi-tamins, were not observed in the retention timewindow of vitamin K1 (Fig. 2).

The use of plasma as an alternative to serumwas investigated by collecting blood in plasma(EDTA and Li-heparin) containers. Compared to se-rum, the plasma showed no significant bias (n = 6,CV <7%, bias <5%) or interferences.

Recovery

In the spiking experiment presented in Fig. 3A,the measured recovery of vitamin K1 was found tobe about 60%. However, as shown in Fig. 3B, therecovery of the calculated concentration using theanalyte/IS peak area ratios was 90%.

Matrix effects

The good linearity and almost identical slopes ofthe analyte/IS peak area ratios in spiked extractedblank matrix and neat heptane in Fig. 3B showedthat matrix interference was low. In the postcol-umn infusion experiment, none of the samplesshowed impact on the vitamin K1 or IS signal (seeFig. 3 in the online Data Supplement), confirming alow degree of matrix interferences in the method.

Linearity and lower limit of the measuringinterval

The method was found to be linear in the con-centration range of 0.1–50 ng/mL with a correla-tion coefficient of R2 >0.999.The aim of an LLMI (28) of 0.1 ng/mL was

achieved. The measured mean (n = 48) was 0.116ng/mLwith a SD of 0.01 ng/mL and CV of 9.0%. Thebias compared to the spiked value of 0.100 ng/mLwas 16%, hence a total error of 34%.

Imprecision and trueness

Imprecision measurements are presented inTable 3. Repeatability and total reproducibilitydata were satisfactory with CV <3% and CV <6%,respectively.The validation solutions andQC samples used to

determine imprecision were also used to address

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trueness by calculating the bias of the measuredmean concentration compared to the spiked(= true) value. Both validation solutions and in-house QC samples showed good compliance withthe true value, with a bias <10% (Table 3).

Method comparison

Satisfactory correlation between our methodand the existing routine method was found for pa-tient sampleswith a regression of y=1.18x−0.031.The difference in ng/mL between themethodswas−0.01 (95% CI, −0.05 to 0.03) at 0.1 ng/mL and 0.37(95% CI, 0.21 to 0.54) at 2.2 ng/mL.

DISCUSSION

Reversed-phase liquid chromatography is stillthe most common among the chromatographicbioanalytical methods today (24). However, the lowviscosity and high diffusion rates of SFC mobilephases allow for high speed and efficient separa-tions (22–25), and thus SFC is attractive forhigh-throughput methods. SFC methods are con-sidered especially suitable for nonpolar com-pounds, such as fat-soluble vitamins (29, 30), andthe use of SFC could simplify the sample prepara-tionwork flow, since the elution strength of organic

Fig. 2. Extracted ion chromatograms of vitamin K1.(A), standard in heptane (<0.1 ng/mL); (B), blank matrix; (C, D), patient sample (0.1 ng/mL); and (E) IS, chromatographed on a2.1 × 100 mm Torus 1-AA column with precolumn. The mobile phase was CO2 with a linear modifier gradient from 0.1% to15% methanol (MeOH) in 1.3 min, at 1.3 mL/min (45 °C, 1650 psi).

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solvents (e.g., heptane) is similar to that of the CO2

mobile phase, making direct injection possiblewithout the need of evaporation and reconstitu-tion (22, 25, 31).Several columns designed for UPC2 were inves-

tigated during the initial method development. Allwere 10-mm-long Acquity UPC2 columns fromWa-ters, including BEH, BEH 2-EP, HSS C18 SB, CSHFluoro-Phenyl, Torus 1-AA, and Torus 2-PIC. Whencomparing columns with inner diameter of 2.1 and3.0mm, the 2.1-mm columns provided higher sen-sitivity (larger peak area), as expected, since ESI MSis concentration sensitive. The choice of Torus1-AA (2.1 × 100mm) as the separation columnwas

mainly based on the peak shape and retention ofvitamin K1, but also the chromatography of retinol,α-tocopherol, 25-OH-vitamin D2, 25-OH-vitaminD3, menaquionone-4, and menaquinone-7, sincethe other fat-soluble vitamins may be included inthe method in the future. Chromatograms areshown in Fig. 1 in the online Data Supplement.The ABPR pressure was optimized by injecting

heptane standards in the ABPR pressure range of1500–2500 psi. Instabilities in spray and peakshape were observed above 2200 psi. Largestpeak area was observed at 1650 psi. Column tem-peratures in the range 40–60 °C were investi-gated. Lower temperatures gave shorter retention

Fig. 3. Linear regression plots used to calculate recovery and matrix effects.(A), Based on peak area of vitamin K1. (B), Based on vitamin K1/IS peak area ratio.

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times and better peak shape; hence, 45 °C waschosen (results not shown).Because of the strong lipoprotein binding of vi-

tamin K1 and its low endogenous concentrations(32, 33), extraction of vitamin K1 from serum sam-ples is challenging. Most of the publishedmethodsfor vitamin K1 determination use a combination ofPPT, liquid-liquid extraction (LLE), and/or SPE withlarge solvent volumes (9–16, 34, 35). Aiming at afast and simple routine method, the possibility touse RLH and 96-DWP limited sample volume (≤250μL). Before vitamin K1 extraction, PPT was re-quired to break the lipoprotein binding, and avariety of organic solvents such as ethanol, meth-anol, 2-propanol, and acetonitrile were investi-gated, neat or in combination with additives suchas formic acid, phosphoric acid, trichloroaceticacid, and zinc sulfate. A combination of methanol/ethanol (50/50, v/v) provided good recovery for vi-tamin K1 and least breakthroughwhen loading thesupernatant on the SPE plate. An isotope-labeledIS is preferred when using mass spectrometry de-tection (36, 37), hence the commercially availablevitamin K1-d7 (5,6,7,8-d4, 2-methyl-d3) was chosenas IS and added to the PPT solvent.LLE was briefly examined as the extraction

method for vitamin K1, but large volumes of

sample and extraction solvents were needed. Al-though not common, RP SPE of vitamin K1 inplasma was reported (14, 16, 17). SPE plates fromdifferent vendors were investigated; all were poly-meric sorbents with chemistry designed to retainboth hydrophilic and lipophilic analytes: SOLAμHRP (Thermo Scientific), EVOLUTE express ABN(Biotage), Oasis HLB μElution (Waters), and OasisHLB PRiME μElution. All the SPE plates providedsufficient cleanup and recovery of vitamin K1. Thenovel sorbent Oasis HLB PRiME was suitable foruse without needing conditioning and equilibra-tion of the plate or evaporation and reconstitutionof the elute fraction andwas therefore chosen. Thewash step of the SPE method was optimized pur-suing phospholipid removal while retaining vita-min K1, resulting in the choice of methanol/water(80/20, v/v) as wash solution. The elution solventwas neat heptane. Heptane is fully compatible withSFC and gave narrow chromatographic peaks. Thefinal sample preparation method was imple-mented on an RLH as a semiautomated one-platemethod and has a total sample preparation time ofapproximately 2 h. One-plate setup includes cali-brators, QCs, blank, and 80 patient samples. Thetotal SFC-MS/MS analysis time is about 6 h for 1plate; hence, the SFC-MS/MS is the time-limiting

Table 3. Imprecision (%CV) and trueness (% bias) of vitamin K1 measurements.

Concentrationlevel (true value)

Measuredmean,ng/mL SD, ng/mL

Imprecision,%CV

Trueness, %bias fromtrue value

Repeatability (n = 10) Level 1 (0.25 ng/mL) 0.268 0.008 3.0 7.2Level 2 (1.75 ng/mL) 1.80 0.03 1.7 2.9

Imprecision, within-run (duplicatesin each series)

Level 1 (0.32 ng/mL) 0.322 0.017 5.3 0.6

Imprecision, between-run 0.0a 0.0a

Reproducibility (n = 86 samples) 0.017 5.3Imprecision, within-run (duplicatesin each series)

Level 2 (2.46 ng/mL) 2.27 0.11 4.8 −7.7

Imprecision, between-run 0.0a 0.0a

Reproducibility (n = 86 samples) 0.11 4.8a The calculated between-run variance component was negative, since the within-run imprecision was greater than the between-run imprecision.Therefore, the between-run imprecision was set to zero.

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step in the method. Established methods for de-termination of vitamin K1 often uses more exten-sive sample preparation, and the run time of thechromatographicmethods are oftenmore than 10min (10–16, 34, 35). Thus, in comparison, ourmethod is rapid. With our sample preparationmethod and SFC conditions, there was no sign ofpressure buildup on the analytical column. Hence,the precolumn could be removed and >6000 sam-ples have been injected on 1 column.In prevalidation experiments, both human se-

rum and BS were spiked in various concentrationsto compare the 2 matrices. No significant differ-ences were observed regarding matrix effects, re-covery, or accuracy. Spiking into human serumobtained almost identical slopes as that of BS, andthe measured concentrations corresponded wellwith spiked values. Since BS is commercially avail-able, it is preferable to use this for calibrators andQC in routine analysis. The method shows goodlinearity within the measurement range and has atotal imprecision of 5.4% at 0.3 ng/mL and 4.9% at2.3 ng/mL. Although the absolute recovery isabout 60%, inclusion of a stable isotope-labeled ISmitigated this shortcoming and afforded themethod satisfactory sensitivity and trueness.The LLMI of the method (0.1 ng/mL) is compara-

ble to that of existing methods (9–15, 34, 35) andcompared to a total allowable error of 46% (38), atotal error of 34% was acceptable. Method com-parison showed negligible difference at LLMI. De-spite the difference at 2.2 ng/mL, our method wasconsidered satisfactory to determine adequate

supplementation, which together with uncoveringdeficiencies, is the major clinical use of themethod. We have been participating in the KEQASscheme since October 2015, and the measure-ments with our method shows no systematic biascompared to other laboratories (see Table 2 in theonline Data Supplement for more details).In a period of 3 months, the method has been

used to analyze >5000 serum samples from abroad group of outpatients, of which >99% have avitamin K1 concentration within the method'smeasurement range of 0.1–50 ng/mL (see Fig. 4 inthe online Data Supplement). The reference rangefor vitamin K1 found in Tietz Clinical Guide to Labo-ratory Tests are 0.1–2.2 ng/mL (39), and Harringtonet al. suggest a reference range of 0.33–3.44nmol/L (0.15–1.5 ng/mL) (40).In conclusion, a rapid and reliable routine

method for determination of vitamin K1 in serumhas been developed. The method is selective andsuitable for determination of vitamin K1 in themeasurement range of 0.1–50 ng/mL using a sam-ple size of 0.25 mL serum. With a SFC-MS/MS anal-ysis time of <4 min and a semiautomated samplepreparation in the 96-DWP format, the method issuitable for high-throughput use. For 1 plate, thetotal analysis time is about 8 h, of which the SFC-MS/MS analysis is the time-limiting step. Themethod has been applied for analysis of patientsamples and KEQAS samples. The short total anal-ysis time makes it practical for this method to be-come a routine test for vitamin K1 in serum foraverage clinical laboratories.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and havemet the following4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b)drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable forall aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriatelyinvestigated and resolved.

Authors’ Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

Role of Sponsor: No sponsor was declared.

Acknowledgments: The authors thank AS Vitas for performing the LC method analysis in the method comparison.

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