QuEChERS结合超高效液相色谱-串联质谱法同时测定畜禽肉中44种食源性兴奋剂和6种孕激素

利用QuEChERS结合超高效液相色谱-串联质谱法建立了畜禽肉中44种兴奋剂和6种孕激素的检测技术。样品粉碎均质后加入内标,依次加入水和含0.5%乙酸的乙腈溶液振荡提取后,加入氯化钠和无水硫酸镁脱水离心,上清液采用PSA、C₁₈、中性氧化铝和无水硫酸镁分散固相萃取材料进行净化,净化液经氮气吹干,复溶后用超高效液相色谱-串联质谱仪测定。被测物采用ACQUITY BEH C₁₈(100 mm×2.1 mm, 1.7 μm)色谱柱,以0.1%甲酸-5 mmol/L乙酸铵水溶液和甲醇为流动相分离,在电喷雾正离子模式下,以多反应监测(MRM)方式采集,采用基质匹配标准曲线内标法定量分析。50个被测物包含6类化合物:β₂-激动剂类(19个)、β-阻断剂类(3个)、蛋白同化激素类(11个)、糖皮质激素类(8个)、利尿剂类(3个)、孕激素(6个)。所有被测物在各自的范围内线性关系良好,相关系数>0.99, β₂-激动剂类和β-阻断剂类的线性范围为0.1~20 μg/L,糖皮质激素类的线性范围为0.5~200 μg/L,蛋白同化激素类、孕激素类、利尿剂类的线性范围为0.2~50 μg/L。方法的定量限范围为0.1~0.4 μg/kg。在低、中、高3个浓度水平下的加标回收率试验中,50种目标化合物在鸡肉、猪肉、牛肉、羊肉中的平均回收率范围为50.3%~119.9%,相对标准偏差(RSD, n=6)范围为0.42%~15.1%。采用该法和国标方法(GB/T 21981-2008)同时对市售的9个肉样品(包括3个牛肉、3个猪肉、2个鸡肉、1个鸭肉)进行了比对检测,对检出的氢化可的松、可的松含量采用t检验进行统计学分析,结果表明,两种方法测得的数据没有显著性差异。采用该法测定了12个来自某养殖场的牛肉样品,结果共检出4种食源性兴奋剂,其中氢化可的松的含量范围为3.3~22.6 μg/kg,检出率为100%;可的松的含量范围为1.5~2.1 μg/kg,检出率为67%;雄烯二酮含量范围为0.7~1.2 μg/kg,检出率为17%;睾酮含量范围为0.6~1.5 μg/kg,检出率为42%。该法操作简单,准确灵敏,重复性好,适用于不同种类畜禽肉中食源性兴奋剂和孕激素的检测。

Determination of 44 foodborne stimulants and 6 progestogens in meat by QuEChERS and ultra-performance liquid chromatography-tandem mass spectrometry

To ensure the success of large-scale sporting events, prevent the contamination of food by prohibited substances, and evaluate the risk of foodborne stimulants and other hormones in food, it is necessary to establish a high-throughput, rapid, and accurate detection method for foodborne stimulants and other hormones. In this study, a QuEChERS method is proposed for the simultaneous determination of 44 foodborne stimulants and 6 progestogens using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The analyzed foodborne stimulants include 19 β₂-agonists, 3 β-blockers, 11 anabolic agents, 8 glucocorticoids, and 3 diuretics. A meat sample was crushed and homogenized, following which the internal standard was added. Subsequently, the sample was shaken and extracted with water and an acetonitrile solution containing 0.5% acetic acid, then dehydrated and centrifuged with sodium chloride and anhydrous magnesium sulfate. The supernatant was purified by PSA, C₁₈, neutral alumina, and anhydrous magnesium sulfate. It was then dried with nitrogen and concentrated. The concentrated extracts were separated using an ACQUITY BEH C₁₈ column (100 mm×2.1 mm, 1.7 μm) with gradient elution using 0.1% formic acid-5 mmol/L ammonium acetate solution and methanol as mobile phases. The target compounds were detected by ultra-performance liquid chromatography-tandem mass spectrometry with electrospray ionization and positive ion scanning (ESI⁺) in the multiple reaction monitoring (MRM) mode, and quantified by the internal standard method. The linear ranges of β₂-agonists and β-blockers were 0.1-20 μg/L, the linear ranges of glucocorticoids were 0.5-200 μg/L, and those of the others were approximately 0.2-50 μg/L. The linear relationships of 50 compounds were good, with correlation coefficients >0.99 in the linear ranges, and limits of quantification (LOQs) in the range of 0.1-0.4 μg/kg. The recoveries of the 50 target compounds spiked in chicken, pork, beef, lamb samples at three levels ranged from 50.3% to 119.9%, while the relative standard deviations (RSDs, n=6) ranged from 0.42% to 15.1%. Nine meat samples (including 3 beef, 3 pork, 2 chicken, and duck samples) were tested by this method and the national standard method (GB/T 21981-2008). The t test was used for statistical analysis of the hydrocortisone and cortisone contents, and no significant difference was found between the results obtained by the two methods. The developed method was used to analyze 12 beef samples from a farm. In all, 4 compounds were detected, while the other 46 were not detected. The content ranges and detection rates of the compounds were as follows: hydrocortisone: 3.3-22.6 μg/kg, 100%; cortisone: 1.5-2.1 μg/kg, 67%; androstenedione: 0.7-1.2 μg/kg, 17%; and testosterone: 0.6-1.5 μg/kg, 42%. In conclusion, the proposed method is simple, accurate, and sensitive, and hence, is suitable for the detection of foodborne stimulants and progestogens in different kinds of raw meat.