MSACL 2019 EU Abstract

Validation of a LCMS Assay for Citrulline Suitable for Daily Routine Through an Original MRM Approach

Claude Hercend (1) , Audrey Huguet (2), Anne Barnier (1), Alexandre Nuzzo (3), Kevin Guedj (3), Olivier Corcos (2, 3) Katell Peoc’h (1, 4)
(1) APHP HUPNVS, UF de biochimie Clinique, Hôpital Bichat, 75018, Paris, France 2) (2) APHP HUPNVS, SURVI, Service de Gastroentérologie, Hôpital Beaujon, 92110, Clichy, France 3) (3) Université de Paris, UMRs INSERM LVTS, 75018, Paris, France 4) (4) Université de Paris, UFR de Médecine de Bichat, et UMRs Inserm 1149, 75018, Paris, France

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Claude Hercend (Presenter) Aphp Hupnvs, UF de Biochimie Clinique, Hopital Bichat

Presenter Bio: Fund of analytical science since my pharmacy studies, it is quite recently that i started to get interested into LC MS theory and practice. In my clinical routine, in the lab, LC MS plays an ever increasing role. My main interested are amino acids, steroids and tryptophan metabolism.

Relevant Financial Disclosures (within past 24 months) Grant/Research Support foundation MSD Avenir Conflict mitigation NOT REQUIRED.

OBJECTIVES:
Citrulline is a nonproteinogenic amino acid synthesized from glutamine by enterocytes of the small intestine, considered as a functional marker of the enterocyte mass. We aimed to evaluate the analytical and clinical performances of an UPLC-MS method within a cohort of patients with acute abdomen, including patients with acute mesenteric ischemia (AMI).

DESIGN & METHODS:
Citrulline was measured after protein precipitation on Ostro™ Plates by UPLC-MS on a Xevo-TQS (Waters) on a BEH Column. We evaluated the analytical performances according to the CLSI EP10-A3E. We evaluated precision, linearity, carry-over, and matrix interference.
We integrated the sample composition in the development of the method through MRM and chromatographic separation of Phospholipids (PL), Triglycerides (TG) and Bilirubin. We admixed plasma samples of increasing concentrations of TG, Bilirubin to low and high quality controls (QC). Citrulline was measured in eighty patients samples with acute abdominal pain, including 40 patients with AMI.

RESULTS:
Intraday imprecision was lower than 8% for low and high QC samples of citrulline (12.4 and 62.4 µmol/L, N=30). The linearity ranged from 0.5 and 120 μmol/L compatible with the reference values previously published. The low limit of quantification was at 0.1 µmol/L with a CV of 5 % and the upper limit of quantification superior to 500 µmol/L with a CV < 1%.
The carry over performed by mixing low and high QC (10 and 60 µmol/L respectively) was acceptable with a negative 7% bias at 10 µmol/L.
For the matrix interference study, we confirmed the removal by the Ostro plate of both PL and lysoPL. For the sample composition study, we calculated the bias on low high QC (n=3) mixed with plasma samples with increasing concentration of bilirubin (82-320 µmol/L) and TG (5-20 mmol/L).
For the bilirubin interference, the bias for the low QC ranged from -1.4 to 8.6%, and for the high QC, from 1.8 to 11.7%.For the TG interference the bias for the low QC low ranged from 18.5 to -27%, and for the high QC from 7.9 to 10.2%.
We obtained results for 40 patients with AMI and 40 patients with abdominal acute pain. Median of citrulline in AMI patients was 16.2 µmol/L (IQ: 12.3-29.8) compared to 23.1 µmol/L (IQ: 19.4-29.6) in other etiologies.
CONCLUSION:
Hence we present a robust method for everyday clinical practice based on an original MRM approach for citrulline measurement that presents promising results in patients with AMI.