Purpose: Although major adverse effects associated with nonsteroidal anti-inflammatory drugs (NSAIDs) are gastric injury, assessment of NSAIDs-induced gastrointestinal adverse effects is mostly dependent on endoscopy due to the lack of plasma biomarkers. Several amino acids associated with collagenase activity and gastric mucosal mass have been suggested as plasma biomarker candidates for gastric injury. Therefore, this study aimed to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the plasma biomarker candidates, i.e., O-acetylcarnitine, proline, hydroxyproline, citrulline, and arginine and evaluate their potential as a biomarker for NSAIDs-induced gastric injury.
Methods: The method utilized simple protein precipitation with acetonitrile as a precipitation solvent and D4-citrulline as an internal standard (IS). The calibration standard samples were prepared by using 4% bovine serum albumin (BSA) as a surrogate plasma matrix. Chromatographic separations were performed on a HILIC column by using a binary gradient mobile phase composed of 0.1% formic acid and 20 mM ammonium formate in distilled water and 0.1% formic acid and 20 mM ammonium formate in 90% acetonitrile. The mass spectrometer was operated in positive, multiple reaction monitoring (MRM) mode.
Results: The assay resulted in the lower limit of quantification (LLOQ) of 0.1 μg/mL for acetylcarnitine and 1 μg/mL for proline, hydroxyproline, citrulline, and arginine in the surrogate blank plasma. The intra- and inter-day accuracy ranged 82.5 - 111.2% for acetylcarnitine, 95.4 - 103.3% for proline, 98.9 - 106.4% for hydroxyproline, 99.5 - 103.5% for citrulline, and 87.4 - 105.3% for arginine. The precision was within 6.17%, 3.63%, 6.20%, 6.31%, and 6.17% for acetylcarnitine, proline, hydroxyproline, citrulline, and arginine, respectively. Following oral administrations of aceclofenac (200 mg/kg/day) for seven days, plasma concentrations of proline, hydroxyproline, citrulline, and arginine were decreased compared to those obtained after single administration of the aceclofenac administration in Beagle dogs.
Conclusion: An LC-MS/MS method for simultaneous determination of acetylcarnitine, proline, hydroxyproline, citrulline, and arginine was developed and validated. The assay was successfully applied to monitor changes in these amino acids concentrations in the plasma following aceclofenac administrations in Beagle dogs. The present assay could be utilized to monitor the changes of these amino acids as potential indicators for various physiological conditions.
SEUNG EUN CHUNG– Master's Course, Sungkyunkwan university, Suwon-si
JUNYOUNG LIM– Master's Course, Sungkyunkwan University, Suwon-si
DAYOUNG LEE– Sungkyunkwan University
CHANG HO Song– Researcher, Sungkyunkwan University, Kyonggi-do
BEOM SOO Shin– Sungkyunkwan University, Kyonggi-do