固相萃取-超高效液相色谱-串联质谱法测定发酵食品中的曲酸

Determination of kojic acid in fermented foods by solid-phase extraction coupled with ultra performance liquid chromatography-tandem mass spectrometry

曲酸由曲霉属和青霉属真菌好氧发酵产生,天然存在于发酵食品中,且常作为防腐添加剂滥用于食品工业中,监测其在发酵食品中的含量及评估其暴露水平带来的健康风险具有重要意义。本研究针对发酵食品基质复杂、基质干扰严重这一关键瓶颈,通过溶剂转换、固相萃取净化,有效地去除了基质干扰。通过系统优化固相萃取条件和仪器条件,建立了发酵食品中曲酸含量的固相萃取-超高效液相色谱-串联质谱(SPE-UPLC-MS/MS)测定方法。样品经0.1%甲酸无水乙醇溶液超声提取,氮吹至近干后用超纯水复溶,过PRiME HLB固相萃取小柱,在ACQUITY UPLC■ BEH C_(18)色谱柱(100 mm×2.1 mm,1.7μm)上分离,以甲酸-5 mmoL/L乙酸铵(1∶999,v/v)溶液和甲酸-乙腈(1∶999,v/v)溶液作为流动相进行梯度洗脱,电喷雾正离子模式下以多反应监测扫描方式测定,内标法定量。在优化的测定条件下,曲酸在5 min内可获得有效分离,在5.0~100.0μg/L内线性关系良好,相关系数(r)为0.9994,方法检出限为2~5μg/kg,定量限为6~15μg/kg。在低、中、高3个加标水平下的回收率为86.8%~111.7%,日内精密度为1.0%~7.9%(n=6),日间精密度为2.7%~10.2%(n=5)。通过建立的方法对酱油、醋、酒、酱、豆豉、腐乳等重点食品中的曲酸含量进行测定,发现醋的检出率最高,其次为酒、酱、酱油、豆豉和腐乳,曲酸含量为5.69~2272μg/kg。该方法具有高效、准确、灵敏等特点,可为发酵食品中曲酸含量的日常监测提供可靠的技术支持。

Kojic acid naturally appears in fermented foods and can be formed during the aerobic fermentation process induced by Aspergillus and Penicillium fungi.It is widely used in the food industry because it exhibits antibacterial and antifungal properties and does not affect food taste.However,recent studies indicate that kojic acid may be a potential carcinogen.Therefore,assessing the health risks of kojic acid in fermented foods are of great importance,and developing a sensitive and accurate analytical method for this compound is a significant endeavor.Much efforts have been devoted to the detection of kojic acid using electrochemistry,high performance liquid chromatography(HPLC),gas chromatography-mass spectrometry(GC-MS),and high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).HPLC and HPLC-MS/MS are the analytical techniques most often employed for this purpose.Of these two methods,HPLC-MS/MS displays excellent sensitivity and is the optimal selective technique.Pretreatment is usually necessary for kojic acid determination because of the complex matrix effects of fermented foods.However,few researches on the determination of kojic acid in food are available,and,to the best of our knowledge,the determination of kojic acid using solid-phase extraction(SPE)pretreatment has not been reported yet.Herein,a convenient,sensitive,and accurate method was developed to determine kojic acid in fermented foods using solid-phase extraction-ultra performance liquid chromatography-tandem mass spectrometry(SPE-UPLC-MS/MS).The pretreatment conditions,such as the extraction solvent,cartridge,rinse solvent,and eluent,were systematically optimized.The samples,including soy sauce,vinegar,liquor,sauce,fermented soya bean,and fermented bean curd,were extracted with 0.1%formic acid-absolute ethyl alcohol and purified using a PRiME HLB cartridge.Kojic acid was separated using an ACQUITY UPLC■ BEH C_(18) column(100 mm×2.1 mm,1.7μm)with formic acid-acetonitrile(1∶999,v/v)and formic acid-5 mmol/L ammonium acetate(1∶999,v/v)solutions as the mobile phases under gradient elution mode.MS was performed in electrospray positive ionization(ESI^(+))and multiple reaction monitoring(MRM)modes.An internal standard method was used for quantification.Under optimized conditions,good linearity was achieved at mass concentrations of 5.0-100.0μg/L,with a correlation coefficient(r)of 0.9994.The limits of detection and quantification of the method for kojic acid were 2-5μg/kg and 6-15μg/kg,respectively.Good recoveries of 86.8%-111.7%,intra-day precisions of 1.0%-7.9%(n=6),and inter-day precisions of 2.7%-10.2%(n=5)were also obtained.The matrix effect was evaluated by establishing a matrix-matching calibration curve,and weak inhibitory effects were found in vinegar and liquor;moderate inhibitory effects in fermented bean curd,fermented soya bean,and soy sauce;and a strong inhibitory effect in sauce.The developed method was used to detect kojic acid in 240 fermented foods,and the results showed that the detection rate of vinegar was the highest,followed by liquor,sauce,soy sauce,fermented soya bean,and fermented bean curd,the contents were 5.69-2272μg/kg.Matrix interferences can be significantly reduced by optimizing the pretreatment and detection procedures.The proposed method is sensitive,accurate,and can be used to analyze kojic acid in fermented foods.