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目的:建立HPLC-柱后光化学衍生法测定蛤蚧定喘丸中黄曲霉毒素G_2、G_1、B_2、B_1的含量,并建立LC-MS/MS确证方法。方法:采用SPOLAR C_(18)柱(4.6 mm×250 mm,5μm)色谱柱,以甲醇(A)-乙腈(B)-水(C)为梯度洗脱流动相进行洗脱,流速1.1 mL·min~(-1),柱温35℃;采用柱后光化学衍生法,柱后光化学衍生器波长为254 nm;荧光检测器激发波长为365 nm,发射波长为450 nm,增益值为15。用LC-MS/MS进行确证,流动相为水(0.1%甲酸+5 mmol·L~(-1)甲酸铵)-乙腈,离子源:电喷雾电离源(ESI),扫描模式:多反应监测(MRM)。结果:黄曲霉毒素G_2、G_1、B_2、B_1分别在0.001 18~0.029 6 ng(r=0.999 8)、0.004 18~0.104 6 ng(r=0.999 9)、0.001 23~0.030 8 ng(r=1.0000)、0.004 267~0.106 7 ng(r=1.0000)范围内线性关系良好;黄曲霉毒素G_2、G_1、B_2、B_1回收率(n=9)分别为101.2%(RSD=7.10%)、103.2%(RSD=3.91%)、92.5%%(RSD=2.91%)和93.5%(RSD=7.95%)。结论:所建立的HPLC-柱后光化学衍生法操作简便、可靠,结果准确,灵敏度高,重现性好,可作为该产品中黄曲霉毒素检测质量控制的方法。
OBJECTIVE: To establish a method for the determination of aflatoxins G_2, G_1, B_2, B_1 in Gejie Ge Dingchuan Pills by HPLC-column photochemical derivatization and establish the LC-MS / MS confirmation method. METHODS: The mobile phase was eluted with a gradient of methanol (A) - acetonitrile (B) - water (C) using a SPOLAR C 18 column (4.6 mm × 250 mm, 5 μm) min -1 and the column temperature was 35 ℃. The post-column photochemical derivatization wavelength was 254 nm. The excitation wavelength of the fluorescence detector was 365 nm, the emission wavelength was 450 nm and the gain was 15. The mobile phase consisted of water (0.1% formic acid + 5 mmol·L -1 ammonium formate) -acetonitrile, ion source: electrospray ionization source (ESI), scanning mode: multiple reaction monitoring (MRM). Results: The levels of aflatoxin G_2, G_1, B_2 and B_1 in the range of 0.001 18 ~ 0.029 6 ng (r = 0.999 8), 0.004 18 ~ 0.104 6 ng (r = 0.999 9) and 0.001 23 ~ 0.030 8 ng ), 0.004 267 ~ 0.106 7 ng (r = 1.0000). The recoveries of aflatoxins G_2, G_1, B_2 and B_1 were 101.2% (RSD = 7.10%) and 103.2% RSD = 3.91%), 92.5 %% (RSD = 2.91%) and 93.5% (RSD = 7.95%). Conclusion: The established HPLC-column photochemical derivatization method is simple, reliable, accurate, sensitive and reproducible. It can be used as a method for quality control of aflatoxins in this product.