Furosine is often used both domestically and internationally as an indicator of the degree of heating to evaluate milk quality. However, in actual detection, the complexity of the milk matrix may lead to the inaccurate quantification of furosine in liquid milk. Therefore, in this study, an efficient and accurate method based on high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF/MS) was established to determine furosine in liquid milk. A 2.00 mL milk sample was hydrolyzed with 5 mL 12.00 mol/L hydrochloric acid solution and 1 mL water at 110 ℃ for 12 h. After hydrolysis, vortex-mixing and filtration were performed. The filtrate was diluted six times with 6.00 g/L ammonium acetate solution and then analyzed. Gradient elution was performed with 0.20% formic acid aqueous solution and acetonitrile solution as mobile phases, followed by chromatographic separation on an AQ-C18 column (150 mm×3.5 mm, 5 μm). The data were collected by Q-TOF/MS with an electrospray ionization source operated in positive-ion mode. The accuracy of the quantification of furosine in milk was assessed by investigating the effects of the hydrochloric acid concentration (0.30, 1.25, and 3.00 mol/L) in the furosine solution on the MS response. The results showed that high hydrochloric acid concentrations inhibited the response signals. A good linear relationship was obtained in the mass concentration range of 0.05-2.00 mg/L, with a correlation coefficient (r) of 0.994. The limit of detection of the method was 0.50 mg/100 g, which meets the requirements of actual sample detection. The average recoveries of furosine ranged from 79.9% to 119.7% at three spiked levels of 1.52, 3.03, and 15.17 mg/100 g, with relative standard deviations of 1.4%-2.6%. The method was applied to detect 303 samples from 101 batches of pasteurized milk sold in the market, and the contents of furosine in these samples ranged from 5.1 to 11.9 mg/100 g. The proposed method is characterized with high efficiency, recovery, sensitivity, and accuracy. Thus, it can be used for the determination of large quantities of samples and provides technical support for the continuous promotion of the high-quality development of the whole dairy industry chain.
国内外常使用糠氨酸含量作为牛奶受热程度的指标用于评价牛奶品质,但在实际检测时,由于牛奶基质复杂可能造成液态乳液中糠氨酸定量不准,因此,本研究基于高效液相色谱-高分辨四极杆飞行时间质谱法建立了高效准确的液态乳中糠氨酸的检测方法。取2.00 mL牛奶样品,加入5 mL 12 mol/L盐酸溶液和1 mL水后在110 ℃条件下水解12 h,水解完成后涡旋混匀过滤,滤液经6.00 g/L的乙酸铵溶液稀释6倍后进样分析,以0.20%甲酸水溶液和乙腈溶液为流动相进行梯度洗脱,AQ-C18色谱柱(150 mm×3.5 mm, 5 μm)进行分离,在高分辨四极杆飞行时间质谱仪的电喷雾电离正离子模式下采集数据。为确保牛乳中糠氨酸定量的准确性,考察了糠氨酸溶液中盐酸浓度(0.30、1.25、3.00 mol/L)对质谱响应的影响,结果显示,盐酸浓度过高会抑制质谱响应信号。糠氨酸在0.05~2.00 mg/L范围内线性关系良好,相关系数(r)为0.994,方法检出限为0.50 mg/100 g,满足实际样品检测需求。在1.52、3.03、15.17 mg/100 g 3个添加水平下对糠氨酸进行加标回收试验,平均回收率为79.9%~119.7%,相对标准偏差(RSD, n=6)为1.4%~2.6%。应用该方法对某市售巴氏杀菌乳101批次303个样品进行检测,样品中糠氨酸含量在5.1~11.9 mg/100 g之间。该方法高效快速,回收率高,灵敏度好,分析准确,可进行大批量的样品测定,为持续推动奶业全产业链高质量发展提供技术支持。
Keywords: furosine; high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF/MS); high resolution mass spectrometry (HRMS); liquid milk.