高效液相色谱-串联质谱检测牛奶中的甲状腺素

建立了高效液相色谱-串联质谱(HPLC-MS/MS)检测牛奶中甲状腺素3,3′,5,5′-四碘-L-甲腺原氨酸(T4), 3,3′,5-三碘-L-甲腺原氨酸(T3)和3,3′,5′-三碘-L-甲腺原氨酸(rT3)的方法。样品用乙腈提取,离心,上清液经氨水碱化和Cleanert PAX固相萃取小柱净化,在Zorbax Eclipse XDB-C18 (150 mm×2.1 mm, 3.5 μm)色谱柱上以0.1%(v/v)乙酸水溶液和甲醇为流动相等度洗脱分离,以电喷雾正离子(ESI+)模式电离,多反应监测(MRM)模式质谱检测,内标法定量。结果表明,甲状腺素的检出限(LOD)不大于0.03 ng/g,定量限(LOQ)不大于0.1 ng/g;在考察的浓度范围内线性关系良好(r2＞ 0.998);回收率为80.61%～101.7%,相对标准偏差(RSD)为1.48%～9.70%。室温下样品溶液中的甲状腺素保持稳定。对5个牛奶样品的测定结果显示,T3含量为0.59～1.30 ng/g, RSD为2.06%～7.70%; T4和rT3未检出。该方法具有样品处理简单,灵敏度高,重现性好,定性和定量结果可靠等特点,为牛奶中甲状腺素的测定和相关质量安全评价提供了可靠手段。

Determination of thyroid hormones in milk by high performance liquid chromatography-tandem mass spectrometry

A method of high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed for the determination of thyroid hormones in milk including 3,3′,5,5′-tetraiodothyronine (T4), 3,3′,5-triiodothyronine (T3) and 3,3′,5′-triiodothyronine (rT3). The sample was extracted with acetonitrile and centrifuged, then the up layer was alkalized with concentrated ammonia water and cleaned up with a Cleanert PAX cartridge. The chromatographic separation was carried out on a Zorbax Eclipse XDB-C18 column (150 mm×2.1 mm, 3.5 μm) with the mobile phase of 37% water containing 0.1% (v/v) acetic acid and 63% of methanol with an isocratic elution mode at a flow rate of 0.3 mL/min. The mass spectrometry detection was performed in positive electrospray ionization and monitored using multiple reaction monitoring (MRM) mode (m/z 652.0/605.5 and 652.0/478.6 for T3 and rT3; m/z 777.7/731.7 and 777.7/604.9 for T4; m/z 784.0/737.9 and 784.0/639.4 for T4-13C6). The analytes were identified by their retention times and relative abundance ratios of the characteristic product ions, and quantified by internal standard method. The method was linear with the correlation coefficients (r2) greater than 0.998 in the concentration ranges investigated. The limits of detection (LODs) were not more than 0.03 ng/g, and the limits of quantification (LOQ) were not more than 0.1 ng/g for the analytes. The recoveries were 80.61%-101.7% with the relative standard deviations (RSDs) of 1.48%-9.70%. The results of five real milk samples showed that the contents of T3 were 0.59-1.30 ng/g with the RSDs of 2.06%-7.70%, and T4 and rT3 weren’t found. The method presents many merits including simple sample pretreatment, high sensitivity, good reproducibility and unequivocal confirmation and quantification for the analytes. It could be used to monitor the levels of thyroid hormones in the milk for safety quality evaluation.