Instrument Multiplexing: Amplified Throughput for Measurement of Vitamin B₁ in Whole Blood Using Parallel Liquid Chromatography Systems Interfaced to a Single Mass Spectrometer

Abstract

Background

Thiamine diphosphate (TDP), the active form of vitamin B₁, plays an essential role in energy metabolism. TDP is analyzed in the clinical laboratory to assess the nutritional status of individuals at risk of deficiency. In recent years, demand for vitamin B₁ testing has increased dramatically, prompting implementation of a high-throughput assay. We developed a method using rapid sample preparation and multiplex electrospray LC-MS/MS analysis.

Methods

Whole blood samples were deproteinized using trichloroacetic acid after the addition of isotope-labeled analyte (TDP-d₃). TDP was separated by reversed-phase chromatography on extended pH, trifunctional silane-bonded C₁₈ columns and analyzed using positive electrospray ionization and multiple reaction monitoring mass spectrometry. The system consisted of 4 LC instruments plumbed to a single mass spectrometer. TDP eluted in 3.15 ± 0.08 min with a run time of 9.0 min for a single stream; results for 4 streams were produced every 2.25 min. Passivation of the system was required to optimize sensitivity and peak quality.

Results

The method was linear from 20 to 1000 nmol/L. Spike-recovery experiments showed an accuracy of ±15%. The intra- and inter-day assay imprecision was ≤3%. Repeated injections of calibrators and QC materials across the four LC streams showed excellent parity (<2% imprecision). No carryover was detected. Each plate produced 81 results in 4.5 h.

Conclusions

An accurate, specific, and high-throughput LC-MS/MS method was developed and validated to measure TDP in whole blood. Simple, fast sample preparation was employed for adaptation to a staggered injection, multiple LC-stream platform, which minimized mass spectrometer idle time significantly and improved efficiency.