MSACL 2023 Abstract

Introduction:
Concentrations of Vitamin B1 (Thiamine Diphosphate, TDP) and B6 (Pyridoxal-5-Phosphate, PLP) in whole blood are measured clinically to help measure various metabolic functions. Accurate concentrations of vitamins B1 and B6 are difficult to obtain due to ion suppression found in LCMS analysis. Phospholipids and proteins are a main cause of the ion suppression. Currently, laboratories are struggling on creating accurate, precise and robust sample preparation methods focused on removal of these interferences.

Objectives:
The goal of this study is to provide a sample preparation method providing accurate and precise concentrations of vitamin B1 and B6 through the removal of interfering phospholipids and proteins. This study further aids in accuracy and precision by use of automation.

Methods:
The Biotage ISOLUTE® PLD+ protein and phospholipid removal plate is used in conjunction with the Biotage Extrahera sample preparation workstation to automate the extraction of vitamins B1 & B6 from whole blood. Vitamins B1 & B6 are highly water soluble (logP= -5.9, -2.2 respectively) and are extremely light-sensitive. An acidic, aqueous crash solvent was used for whole blood in order to extract the vitamins. MRM of three ions each were monitored for quantitation of vitamins B1 & B6. Phospholipid and protein removal is measured by monitoring the 184-product ion in precipitated samples compared to the samples prepared with ISOLUTE® PLD+.

Results:
Five replicate extractions were performed on whole blood samples which were precipitated alone and five samples which were precipitated with the ISOLUTE® PLD+ protein and phospholipid removal plate. Recoveries for vitamins B1 & B6 were 94% and 82% respectively with a %RSD < 2. Phospholipids and proteins were reduced by over 90%. Percent recovery is based upon the calculated recovery of the vitamins B1 & B6 by precipitation alone, highlighting any analyte loss due to the use of the ISOLUTE® PLD+ sample preparation plate.

Conclusion:
Matrix phospholipids and proteins were successfully removed (>90%) from whole blood vitamin B1 & B6 analysis using the ISOLUTE® PLD+ protein and phospholipid removal plate. Recoveries for vitamins B1 & B6 were 94% and 82% respectively with a %RSD < 2. Additional studies will be performed to determine if this method is suitable for other biological matrices as well.