超高效液相色谱法测定水产品中甲基睾酮残留物

Determination of Methyltestosterone Residues in Aquatic Products by Ultra-High Performance Liquid Chromatography

将5.000g样品加入乙腈-甲酸(199+1)混合液15mL,匀浆30s,振荡提取10min后离心,下层固体再用乙腈-甲酸(199+1)混合液8mL重复提取1次,合并上清液于平行蒸发管中,于55℃平行蒸发至干。用甲醇3mL溶解残渣,再加入水7mL,置于冰箱冷冻30min后冷冻离心,取甲醇溶液层采用poly-sery HLB固相萃取柱净化,用20%(体积分数,下同)甲醇溶液6mL淋洗,用90%甲醇溶液4mL洗脱,洗脱液在50℃下用氮气吹干,用乙腈(1+1)溶液1mL溶解残渣,过0.22μm微孔滤膜。滤液在BEH C_(18)色谱柱(50mm×2.1mm,1.7μm)上分离,流动相为乙腈(1+1)溶液,采用紫外检测器,检测波长为254nm。甲基睾酮质量浓度在0.05~1.00mg·L^(-1)内与对应的峰面积呈线性关系,检出限(3S/N)为5.0μg·kg^(-1)。在10.0,50.0,100μg·kg^(-1)浓度水平上进行加标回收试验,回收率为73.6%~91.4%,测定值的相对标准偏差(n=5)小于9.0%。

The sample （5.000 g） was added into 15 mL of acetonitrile-formic acid （199＋1） solution, and the mixture was homogenized for 30 s, then extracted by shaking for 10 min and centrifuged. The lower solid was then extracted with 8 mL of acetonitrile-formic acid （199＋1） aolution. The supernatant was merged in a parallel evaporation tube and evaporated to dryness at 55℃. The residue was dissolved with 3 mL of methanol, and 7 mL of water was added. The solution was freezed in the freezer for 30 min and then centrifuged. The methanol solution layer was purified with poly-sery HLB solid phase extraction column, using 6 mL of 20% （φ） methanol solution for rinse and 4 mL of 90% （φ） methanol solution for elution. After the eluate was dried at 50℃ by nitrogen blowing, the residue was dissolved in 1 mL of acetonitrile （1＋1） solution and the solution was filtered with a 0.22 μm microporous membrane filter. The filtrate was separated on a BEH C18 column （50 mm×2.1 mm, 1.7 μm） with acetonitrile （1＋1） solution as the mobile phase. Methyltestosterone was detected by a UV detector at the wavelength of 254 nm. Linear relationship was found between peak area and mass concentration of methyltestosterone in the range of 0.05-1.00 mg·L^-1, with detection limit （3S/N） of 5.0 μg·kg^-1. Test for recovery by standard addition method was carred out at the spike level of 10.0, 50.0, 100 μg·kg^-1, giving results in the range of 73.6%-91.4%, and RSDs （n=5） were less than 9.0%.