MSACL 2016 EU Abstract

Quantification of Total Homocysteine in Human Plasma or Serum by LC-MS/MS

Sergio Indelicato (Presenter)
Thermo Fisher Scientific

Authorship: Sergio Indelicato(1), Irene Doering(2), Radouane Nagim(2)
(1) Thermo Fisher Scientific, Les Ulis, France (2) RECIPE Chemicals+Instruments GmbH, Munich, Germany

Short Abstract An analytical method for clinical research for the quantification of total homocysteine in human plasma or serum is reported. The method involves a fast and simple sample preparation followed by injection onto a Thermo Scientific™ Transcend™ II system. Mass spectrometric detection is performed by single reaction monitoring on a Thermo Scientific™ TSQ Endura™ triple quadrupole mass spectrometer using heated electrospray ionization in positive mode. The method was analytically validated using the MS2000 ClinMass® Complete Kit Homocysteine in Plasma / Serum from RECIPE and limit of quantification, linearity range, accuracy and intra- and inter-assay precision were evaluated.

Long Abstract

Introduction

An analytical method for clinical research for the quantification of total homocysteine in human plasma or serum is reported. The method, based on the MS2000 ClinMass Complete Kit Homocysteine in Plasma / Serum from RECIPE, involves a fast and simple sample preparation followed by injection onto an LC system using a Thermo Scientific™ Transcend™ II system; a Thermo Scientific™ TSQ Endura™ triple quadrupole mass spectrometer with heated electrospray ionization is used for detection by single reaction monitoring (SRM) using homocysteine-d4 as the internal standard. Implementation of the kit included the evaluation of limit of quantification, linearity range, accuracy and intra- and inter-assay precision for the analyte of interest.

Methods

This analytical method for clinical research is based on the MS2000 ClinMass® Complete Kit Homocysteine in Plasma / Serum from RECIPE and allows for the quantification of homocysteine in human plasma or serum. The kit includes calibrators and controls at four and two different levels, respectively, covering a concentration range between 5.87 and 50.7 µmol/L. Homocysteine-d8 is used as the internal standard, but it is reduced during sample preparation and therefore be used as homocysteine-d4 for the quantification. Sample clean-up is performed by a simple reduction and protein precipitation step followed by chromatographic separation by gradient elution on a Transcend II system operated in UHPLC mode using mobile phases and analytical column provided with the kit. Detection was performed by SRM on a TSQ Endura triple quadrupole mass spectrometer with heated electrospray ionization operated in positive mode. Two SRM transitions were included in the acquisition method for quantification and confirmation, respectively.

Results

A full analytical validation was performed; the assay proved to be linear in the calibration range covered by the kit, with a limit of quantification (LOQ) of 0.117 µmol/L, an upper limit (ULOQ) of 50.7 µmol/L and a correlation factor (R2) above 0.999. Accuracy for the assay was evaluated in terms of trueness of measurement using Standard Reference Material® 1955 from NIST prepared and analyzed on 5 different days in single runs each day; the percentage bias between nominal and average back-calculated concentration for these control samples was between 3.7 and 5.2%. Intra-assay precision was evaluated in terms of percentage coefficient of variation (%CV) using the kit controls at both levels in replicates of eight (n=8); the %CV for intra-assay precision was 2.0% and 1.4% for the lower and upper control, respectively. Inter-assay precision was evaluated on the same controls in replicates of three (n=3) prepared and analyzed on five different days; the %CV for inter-assay precision was 3.1% and 3.3% for the lower and upper control, respectively.

Conclusions

A liquid chromatography tandem mass spectrometry method for clinical research for the quantification of total homocysteine in human plasma or serum using the MS2000 ClinMass® Complete Kit Homocysteine in Plasma / Serum from RECIPE was implemented and analytically validated on a Transcend II system connected to a TSQ Endura triple quadrupole mass spectrometer. This analytical method meets research laboratory requirements, sensitivity and linearity of response covering analyte concentration ranges provided by the kit.

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