高效液相色谱-二极管阵列检测器技术在助眠保健品非法添加化学药物检测中的应用

Application of high performance liquid chromatography with diode array detector technology in the detection of illegally added chemical drugs in sleep aid dietary supplements

目的利用高效液相色谱-二极管阵列检测器(HPLC⁃DAD)技术,建立助眠保健品中可能添加的舒必利、乙琥胺、拉莫三嗪、酒石酸唑吡坦、褪黑素、盐酸丁螺环酮、苯巴比妥、马来酸咪达唑仑、氟哌啶醇、卡马西平、苯妥英钠、奥沙西泮、硝西泮、阿普唑仑、氯硝西泮、艾司唑仑、盐酸氟哌噻吨、硫喷妥、盐酸硫利达嗪和地西泮等20种化学药物的检查方法。方法采用Agilent Zorbax C18色谱柱(250 mm×4.6 mm,5μm),以乙腈⁃0.01 mol/L磷酸二氢铵水溶液(用磷酸调pH值至3.0)为流动相,梯度洗脱,流速0.8 ml/min,柱温25℃,检测波长为220和280 nm。从专属性、系统适应性、线性关系、检测限、定量限、回收率、精密度、稳定性和重复性等方面对HPLC⁃DAD技术在检测20种非法添加化学药物中的作用进行评估。结果HPLC⁃DAD技术在20种化学药物成分的检测专属性好,在一定质量浓度范围内与峰面积的线性关系良好(相关系数r≥0.9998);20种化学药物低、中、高3个浓度9次进样的平均回收率为98.68%~99.85%,峰面积变异度的相对标准偏差(RSD)为0.57%~1.16%;检测20种化学药物的精密度、重复性和稳定性良好,检测限为0.05~1.59μg/ml,定量限为0.16~5.09μg/ml。应用该方法对6种助眠保健品样品进行检测,其中1种检出硝西泮。结论该方法准确、简便、易于推广,可用于助眠类保健品中20种可能非法添加化学药物的检测。

Objective To develop the method of high performance liquid chromatography with diode array detector(HPLC⁃DAD)technology for detection of 20 chemical drugs possibly added into sleep aid dietary supplements such as sulpiride,ethosuximide,lamotrigine,zolpidem tartrate,melatonine,burspirone hydrochloride,phenobarbital,midazolam maleate,haloperidol,carbamazepine,phenytoin sodium,oxazepam,nitrazepam,alprazolam,clonazepam,estazolam,fupentixol dihydrochloride,thiopental,thioridazine hydrochloride and diazepam.Methods An Agilent Zorbax C18 column(250 mm×4.6 mm,5μm)was used for the separation,with gradient elution of acetonitrile and ammonium dihydrogen phosphate aqueous solu⁃tion(adjust pH to 3.0 with phosphoric acid)as the mobile phase at the flow rate of 0.8 ml/min.The column temperature was 25℃,and the detection wavelength was set at 220 and 280 nm.The specificity,system adaptability,linear relationship,limit of detection,limit of quantitation,recovery,precision,stability and repeatability of the HPLC⁃DAD technology in detection of the above 20 chemical drugs was evaluated.Results The HPLC⁃DAD technology had good specificity in detection of 20 chemical drugs,and the drug concentration had good linear relationship with peak area within certain ranges(correlation coefficients r≥0.9998).The average recoveries of 9 injections of 20 chemical drugs at low,medium,and high concentra⁃tions were 98.68%⁃99.85%,and the relative standard deviations of peak area variation were 0.57%⁃1.16%.The precision,repeatability,and stability of the method in detecting 20 chemical drugs were good.The limits of detection and quantitation were 0.05-1.59μg/ml and 0.16-5.09μg/ml,respectively.The established method was used to test 6 samples,and nitrazepam was detected in 1 sample.Conclusions The establishedmethod is accurate,easy to operate,and easy to promote.It can be used for the detection of 20 chemical drugs that may be added into sleep aid dietary supplements.