Determination of Bongkrekic Acid and Isobongkrekic Acid in Plasma and Urine by Ultra-Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry

Bongkrekic acid (BA) and isobongkrekic acid (IBA) are produced by the bacterium Burkholderia gladioli pv. Cocovenenans (B. cocovenenans) associated with outbreaks of foodborne diseases involving coconut and corn-based products in Indonesia, China and Mozambique. Bongkrekic acid and isobongkrekic acid are littleknown mitochondrial toxins that inhibit adenine nucleotide translocase (ANT). The latency period after exposure to BA and IBAcontaminated foods is reported to be 224 hours. The symptoms of poisoning include discomfort of digestive system and nervous system. Serious patients will die from multiple organ failure, such as liver, brain and kidney. The mortality rate from past outbreaks in China was about 40%, and that in Indonesia was about 60%. The method of ultraperformance liquid chromatographic method coupled with triple quadrupole tandem mass spectrometry (UPLCMS/MS) was established for determination of bongkrekic acid and isobongkrekic acid in plasma and urine. Isobongkrekic acid was prepared by treating of bongkrekic acid with 2 mol/L KOH for 2 h at 100 ℃, and confirmed with the reference substance by mass spectrometry, chromatography and ultraviolet spectroscopy. The main factors including methods of sample pretreatment, separation column types, compositions of mobile phases, and instrumental conditions of mass spectrometry were optimized. The bongkrekic acid and isobongkrekic acid in plasma were ultrasonically extracted with acetonitrilemethanol (9∶1, V/V) solution containing 05% (V/V) ammonia solution, and then the extract was centrifuged to remove the impurities, such as proteins. After acetonitrile and methanol in extract were removed, the analytes in the residues were extracted by hexane under pH 1520. The bongkrekic acid and isobongkrekic acid in urine were directly extracted by hexane under pH 1520. The chromatographic analysis was separated on an Acquity BEH C18 column (21 mm×100 mm×17 μm) with gradient elution of using mobile phases of acetonitrile and water both containing 0.05% (V/V) formic acid. A triple quadrupole mass spectrometer, equipped with electrospray ionization (ESI) in the negative ion mode, was used to detect bongkrekic acid and isobongkrekic acid in multiple reaction monitoring (MRM) mode. Bongkrekic acid and isobongkrekic acid were quantitated by external standard of matrix working curve. The linear ranges of the analytes were from 005 μg/L to 10 μg/L with the correlation coefficients greater than 0998. The limits of detection (LODs) of bongkrekic acid and isobongkrekic acid in plasma and urine were 002 μg/L, and the limits of quantification (LOQs) of them were 005 μg/L. The average recoveries were 92%106% with the relative standard deviations of 24%13%. The method is simple, sensitive and accurate, and can be used for the detection of bongkrekic acid and isobongkrekic acid in plasma and urine poisoned by Burkholderia gladioli pv. Cocovenenans.