Vitamin K1 in Serum using SelexION

Authors: Serge Auger, Pierre Picard and Jean Lacoursière
Themes: High-Throughput, Vitamin K1, Serum, LDTD®-MS/MS
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Vitamin K1 (Phylloquinone) is an important compound that plays a key role in the synthesis of several blood coagulation factors, in bone metabolism and in vascular calcifications.

Our goal for this application note is to use a combination of efficient sample preparation and ion mobility technique for the quantification of vitamin K1 in serum using the Luxon Ion Source®, based on the LDTD® technology.

LDTD-MS/MS offers specificity combined with an ultra-fast analysis for an unrivaled quantification method. To develop this application, we focused on performing an efficient sample preparation. Vitamin K1 is analyzed, and results are obtained in less than 12 seconds per sample.

Sample Preparation Method

Due to the instability of vitamin K1, stock solutions were prepared in ethanol (0.01% BHT). Pooled serum was exposed to UV light for at least 24h due to photodegradation of endogenous vitamin K. The pooled exposed serum was then spiked to generate a calibration curve and QC.


LDTD®-MS/MS Parameters


Model: Luxon S-960, Phytronix

Carrier gas: 7.5 L/min (air)

Laser pattern: 9-second ramp to 75% power

Ion Mobility

SelexION QTrap® System 5500, Sciex

DMS Temperature (DT): Low

Modifier: None

Separation Voltage (SV): 4200

Compensation Voltage (COV): 18

DMS offset (DMO): -3


MS model: QTrap® System 5500, Sciex

IonSpray Voltage: 4800

Temperature, GS1 and GS2 set to zero.

CUR: 30

CAD: 8

Scan Time: 100 msec

Analysis Method: Positive MRM mode


Table 1 – MRM transitions for LDTD-MS/MS
Transition CE (V)
Vitamin K1 451.2 → 187.1 35
Phylloquinone-d7 458.3 → 194.1 35


Results and Discussion

Validation Test

Calibration curves ranging from 200 to 5200 pg/mL were prepared in a photodegraded serum. Replicate extractions were deposited onto a LazWell™ plate and dried before analysis. The peak area against the internal standard (IS) ratio was used to normalize the signal.


The calibration curves were plotted using the peak area ratio and the nominal concentration of standards. For the linearity test, the following acceptance criteria was used:

Table 2 shows the inter-day correlation coefficients for Vitamin K1. Values greater than 0.995 are obtained. Figure 3 shows a typical calibration curve result for Vitamin K1.


Figure 3 – Vitamin K1 calibration curve


Table 2 – Inter-day calibration curve correlation coefficients
Vitamin K1
Curve 1 0.99811
Curve 2 0.99718
Curve 3 0.99714
Curve 4 0.99796
Curve 5 0.99835

Precision and Accuracy

For the accuracy and precision evaluation, the following acceptance criteria were used:

For the intra and inter-run precision and accuracy experiment, each QC was analyzed in sextuplicate, on five different days. Table 3 and 4 show the intra and inter-run precision and accuracy results for Vitamin K1. The obtained %CV was below 15% and the accuracy was within 15% of the nominal value.

Table 3 – Intra-Run Precision and Accuracy of Vitamin K1
Vitamin K1 QC-L QC-M QC-H
Conc (pg/mL) 700 2200 4200
N 6 6 6
Mean (pg/mL) 679.9 2186.1 4228.5
%CV 10.9 5.5 3.9
%Nom 97.1 99.4 100.7


Table 4 – Inter-Run Precision and Accuracy of Vitamin K1
Vitamin K1 QC-L QC-M QC-H
Conc (ng/mL) 700 2200 4200
N 30 30 30
Mean (ng/mL) 674.7 2156.7 4280.8
%CV 12.5 5.2 4.3
%Nom 96.4 98.0 101.9

Wet Stability of Sample Extracts

Following the extraction, sample extracts are kept at 4°C in closed containers. After 4 days, sample extracts are spotted on a LazWell™ plate, dried and analyzed. The precision and accuracy results of QCs are reported in Table 5. All the results are within the acceptable criteria range for 4 days at 4°C.

Dry Stability of Samples Spotted on LazWell™

Extracted samples are spotted onto a LazWell™ plate, dried and kept at room temperature for 1 hour before analysis. The precision and accuracy results of QCs are reported in Table 5. All the results are within the acceptable criteria range for 1 hour at room temperature.

Table 5 – Wet and Dry Stability of Vitamin K1
Parameters Dry Stability
(1 hour / RT)
Wet Stability
(4 days / 4°C)
Conc (ng/mL) 700 2200 4200 700 2200 4200
N 6 6 6 6 6 6
Mean (ng/mL) 682.5 2086.8 4181.2 649.6 2206.7 4191.0
%CV 5.3 7.4 6.3 4.0 3.7 1.9
%Nom 97.5 94.9 99.6 92.8 100.3 99.8

Cross Validation Study

Real patients’ serum samples (N=6) have been tested with this method to correlate with results obtained by traditional LC-MS/MS. The percentage of difference between the values are evaluated. A difference below 15% is obtained. Results are reported in Table 6.

Table 6 – Comparison between Vitamin K1 concentration values
LC (pg/mL) Luxon (pg/mL) %Diff (%)
M1 528.2 615.9 7.7
M2 641.2 604.8 -2.9
M3 324.2 362.1 5.5
M4 984.6 975.6 -0.5
M5 484.4 628.6 13.0
M6 810.8 788.9 -1.4


The Luxon Ion Source combined with Sciex QTrap® 5500 mass spectrometer system allows the ultra-fast (12 seconds per sample) analysis of Vitamin K1 in serum.