Bioanalytical quantitation method for phytonadione isomers in human plasma using LC/MS/MS

L Ramalingam, Senior Manager, QPS Bioserve India, Hyderabad, Dr Anoop Kumar, Application Support Manager and Dr Manoj Pillai, Director, Application support, SCIEX India, talk about the objective to develop a rapid and sensitive method that allows cis-and trans-vitamin K1 extracted from human plasma to be simultaneously determined by a chromatography-mass method

Vitamin K1, 2-methyl-3-[(2E,7R,11R)-3,7,11,15-tetramethyl-2-hexadecenyl]-1,4-naphthalenedione, is a nutrient the human body uses to form coagulation factors that aid in normal blood clotting and bone formation [1]. Vitamin K1 exists naturally only in the trans-form. Trans- and cis-isomers are formed during UV-light exposure or synthetic production of vitamin K1. This cis-isomer is considered to have low bioactivity. Therefore, inactive cis-vitamin K1 is necessary to measure individually.  The objective of this work was to develop a rapid and sensitive method that allows cis-and trans-vitamin K1 extracted from human plasma to be simultaneously determined by a chromatography – mass method.

Sample preparation

To 500 µL Spiked Plasma add 1mL of Ethanol and vortex for 1minute to precipitate. Add 2mL of n-hexane and vortex for another one minute. Centrifuge at 4000rpm for five min. Take the organic layer, dry in nitrogen stream at 50ºC and reconstitute in Mobile phase and inject in LC-MS/MS system.

Chromatography

LC System: GL Sciences LC 800 System
Column: CHIRALPAK IA3 (50 mm X 4.6 mm, 3u)
Column Temp: 40 °C
Injection: 25µL
Flow Rate: 1.000 ml/min
Mobile phase:
A) 0.1% v/v Formic Acid in Water
B) 0.1% v/v Formic Acid in Acetonitrile
Isocratic Mode – 4: 96 v/v (A:B v/v)

Mass spectrometry

Analysis of Cis and Trans Phytonadione was carried out in APCI mode and required different mass spectrometric settings. The MRM transition monitored for Phytonadione was 451.3/186.9. Dwell time was kept 200ms.

LC-MS/MS System: SCIEX Triple Quad 6500 System
Interface: IonDrive Turbo V Source in positive ion mode

Conclusions

A highly sensitive and high – throughput bioanalytical method was developed for the quantitation of low levels of the phytonadione cis and trans isomers in human plasma on the SCIEX Triple Quad 6500 LC/MS/MS System.

Method sensitivity for phytonadione detection was exceptional, and demonstrated high-reproducibility and cost effectiveness with good precision and accuracy.

Total run time for each sample was only six minutes, using a flow rate rapid enough for high-throughput analysis in the bioanalytical laboratory.