超高效液相色谱-三重四极杆质谱联用法测定血浆和尿液中米酵菌酸和异米酵菌酸

建立了超高效液相色谱-三重四极杆串联质谱法(UPLC-MS/MS)测定生物样品中米酵菌酸和异米酵菌酸的方法。将米酵菌酸在2 mol/L KOH溶液中100℃加热反应2 h制备异米酵菌酸标准品,并通过与对照品的质谱、色谱、紫外光谱比较而确认。血浆样品用含0.5%氨水的乙腈溶液-甲醇(9∶1,V/V)沉淀除去血浆蛋白,在酸性条件下用正己烷萃取净化;尿液样品使用正己烷在酸性条件下萃取净化。选择Acquity BEH C18色谱柱,以0.05%甲酸-乙腈溶液(V/V)-0.05%甲酸水溶液(V/V)为流动相进行洗脱,电喷雾负离子多离子监测模式(MRM)检测,基质加标标准曲线外标法定量。结果表明:血浆和尿液中米酵菌酸和异米酵菌酸的线性范围均为0.05~10.0μg/L,相关系数大于0.998,平均加标回收率在92%~106%之间,相对标准偏差均为2.4%~13%(n=6),方法的检出限(S/N=3)均为0.02μg/L。该方法简单、灵敏、准确,可用于鉴定椰毒假单胞菌引起的中毒。

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.