MSACL 2019 EU Abstract

Determination of Serum Triglyceride by Isotope Dilution LC-MS/MS

W. Zhou(1), C. Zhang(1), W. Chen(1), J. Dong(2), S. Wang(2), T. Zhang(1), J. Zeng(1), Y. Yan(1), J. Zhang(1).
(1)National Center for Clinical Laboratories, Beijing hospital, National Center of Gerontology, Beijing Engineering Research Center of Laboratory Medicine, Beijing, China,(2) The Key Laboratory of Geriatrics，Beijing Institute of Geriatrics，Beijing Hospital，National Center of Gerontology, Beijing, China

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Weiyan Zhou (Presenter) NCCL，Beijing Hospital

Presenter Bio: I graduated from Chinese Academy of Medical Sciences & Peking Union Medical College and got doctorate degree in medicine in 2010. Now I am working in the National Center for Clinical Laboratories, an organization who is responsible for the national EQA/PT scheme and medical laboratory quality management in China. As a director of clinical mass spectrometry, I am mainly responsible for the EQA/PT scheme of clinical mass spectrometry in China, and for standardization of some analytes in clinical chemistry based on mass spectrometry. Now I am responsible for some EQA/PT programs for MS, such as catecholamines, non-peptide hormone and so on. MS is becoming more widely used in the clinical laboratory around the world including China. More and more clinical laboratories attend our EQA/PT schemes using MS.
I hope I can have the chance to learn more about the technology of MS in MSACL 2019.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

Background: There are some reference methods to determine serum total glycerol and triglyceride all by isotope dilution gas chromatography mass spectrometry which are time consuming and complicated. A need exists for a simple reference method that can be easily adopted to verify the accuracy of serum triglyceride measurements, especially with different measurement principles. Just as serum triglyceride concentrations are generally determined from total glycerol with or without a subtraction of free glycerol. So candidate reference methods involving isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) for total glycerol and free glycerol were established. The triglyceride concentration was the difference between total glycerol and free glycerol.
Methods: An isotopically labeled internal standard, [13C3]-glycerol, was added to serum, protein precipitation, and derivatization by benzoyl chloride to prepare samples for LC/MS/MS analysis using electrospray for ionization (ESI). For total glycerol, hydrolysis was conducted after adding the internal standard. For separation, a Nova-Pak C18 column was used with a mobile phase consisting of 10 mmol/L ammonium formate in water–acetonitrile (20:80 by volume) for positive ions. The quantitative ion transitions of [M+NH4]+ at m/z 422.2→283.2 and m/z 425.2→286.2 were monitored for glycerol and [13C3]-glycerol, respectively. The qualitative ion transitions were at m/z 422.2→105.1 and m/z 425.2→108.1, respectively. The method was calibrated with linear regression using five-point calibration curves.
Results: The correlation coefficients between the peak area ratios and glycerol concentrations were 0.9999 and higher. The within-run coefficients of variation (CV) for serum total glycerol analysis averaged 0.52% (ranged 0.3%~1.02%) and the total CV 0.73% (0.49%~1.27%). Results on certified reference materials (SRM 909b Level I and Level II, SRM 1951b Level I and Level II, SRM 909c, GBW 09146 and GBW 09147) showed an averaged bias of 0.32% (0%~0.97%).
Conclusion: Isotope dilution LC/MS/MS method for serum triglyceride has been developed. This method was been used in international laboratory comparison including RELA (IFCC) and Cholesterol Reference Method Laboratory Network（CRMLN，US. CDC）. Results showed that this ID-LC/MS/MS method was well-characterized for serum glycerides with a theoretically sound approach, demonstrated good accuracy and precision, and low susceptibility to interferences qualifies as a candidate reference method. Use of this reference method as an accuracy base may reduce the apparent biases in routine methods along with the high interlaboratory imprecision.