MSACL 2016 EU Abstract

A Method for the Quantification of PEth 16:0/18:1 in Human Blood Based on UHPLC and Orbitrap High Resolution Accurate Mass Spectrometry (HRAM)

Magnus Olin (Presenter)
Themo Fisher Scientific

Bio: Sales support expert clin/tox. 20 years industrial experience in small molecule LC-MS in biological fluids. Expertise in chromatography, nominal mass and HRAM mass spectrometry. Methods development, validation and sample preparation.

Authorship: Magnus Olin (1), Pernilla Eliasson (2), Kim Kultima (2,3), Torbjörn Åkerfeldt (2,3)
(1) Thermo Fisher Scientific, Hägersten, Sweden (2) Uppsala University Hospital, Uppsala, Sweden (3) Uppsala University, Uppsala, Sweden

Short Abstract

Phosphatidylethanol (PEth) is an abnormal phospholipid that is only formed in red blood cells membrane in the presence of ethanol and its usage as a potential alcohol biomarker is being investigated by the Swedish Healthcare system; PEth 16:0/18:1 is the most abundant form. An analytical research method based on liquid chromatography high resolution mass spectrometry for the quantification of PEth 16:0/18:1 in human blood is described. Mass spectrometric detection was performed by monitoring of the intact mass on a Thermo Scientific™ Exactive Plus™ high resolution mass spectrometer using heated electrospray ionization.

Long Abstract

Introduction

Phosphatidylethanol (PEth) is an abnormal phospholipid that is mainly located in the membrane of red blood cells. PEth is only formed in the presence of ethanol and its usage as a potential alcohol biomarker is being investigated by the Swedish Healthcare system. PEth 16:0/18:1 is the most abundant form, thus, we developed a clinical research method for the quantification of PEth 16:0/18:1 in human blood. The method is based on ultra-high pressure liquid chromatography (UHPLC) and monitoring of the intact molecular ion on a Thermo Scientific™ Exactive Plus™ high resolution accurate mass (HRAM) spectrometer.

Methods

Calibration samples covering a range of concentrations between 0.03 and 5 µM were prepared by spiking known amounts of pure standard PEth 16:0/18:1 as isopropanolic solution into PEth negative blood samples. External quality control samples were also used. Blood was quickly frozen in methanol/dry ice in order to haemolyze the blood cells. 400 µL of isopropanol containing a deuterated analog of PEth as internal standard was added to 100 µL of blood sample, followed by vortex-mixing and centrifugation. The supernatant was transferred to a clean vial and injected onto a UHPLC-HRMS system. Chromatographic separation was achieved on a Thermo Scientific™ Hypersil Gold™ C18 column (50 x 2.1 mm, 1.9 µm) at 60°C using an isocratic mobile phase consisting of 10 mM ammonium acetate and 0.1% acetic acid in water, acetonitrile and isopropanol. Detection was performed by monitoring the intact molecular ion on an Exactive Plus™ high resolution mass spectrometer using heated electrospray ionization in negative ion mode. An isolation window of 10 ppm was used for extracting chromatograms of both PEth 16:0/18:1 and its internal standard from the full scan data. Data was collected between 650 and 750 at a resolution of 70000 (at m/z 200).

Results

The lower limit of quantification (LLOQ) for the assay was 0.03 µM, below the lower cutoff suggested in Swedish Healthcare (0.05 µM); the method proved to be linear in the 0.03 – 5 µM calibration range specified above. Accuracy for the method was measured at 0.05 and 0.3 µM, the latter being the suggested discrimination level between moderate and heavy drinking, in terms of percentage bias (% Bias) between nominal and experimental concentration and was always within ± 15%. Precision at the same concentrations calculated as percentage coefficient of variation (% CV) was always below 15%. This high resolution method was cross-validated against a method based on a triple quadrupole mass spectrometer2 and a good correlation between the two was found (R2 = 0.98). The external quality assurance programme (EQUALIS) revealed an excellent agreement with the mean value from 13 other laboratories.

Conclusions

A liquid chromatography high-resolution mass spectrometry method for the quantification of PEth 16:0/18:1 and its deuterated internal standard in human blood has been developed on an Exactive Plus mass spectrometer. The instrument proved to have the proper sensitivity to satisfy the cutoff requirements in Swedish Healthcare, with a LLOQ of 0.03 µM and a covered linear range of concentrations up to 5 µM. Accuracy and precision for the method were within international guidelines1 and suggests the method could be suitable for clinical research applications.


References & Acknowledgements:

[1] “Guideline on bioanalytical method validation”, EMEA/CHMP/EWP/192217/2009 Rev. 1 Corr. 2

[2] Fast and robust LC-MS/MS method for determination of the alcohol biomarker phosphatidylethanol (PEth) in whole blood using an automated extraction procedure, Blomgren Anders, Hansson Therese, Isaksson Anders, Walther Lisa

Region Skåne, Medical Services, Laboratory Medicine, Clinical Chemistry Lund, Sweden


Financial Disclosure

DescriptionY/NSource
Grantsno
SalaryyesThermo Fisher Scientific
Board Memberno
Stockno
Expensesno

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above:

yes