纸喷雾离子化质谱法快速筛查保健食品中违禁添加的磷酸二酯酶5抑制剂

目的建立纸喷雾离子化质谱(PSI-MS)法快速检测保健食品中3种非法添加的磷酸二酯酶5(PDE5)抑制剂(西地那非、伐地那非和他达拉非)的方法。方法利用PSI-MS法对样品中非法添加物进行初步定性鉴别,并通过内标法进行半定量分析。结果 24种包括胶囊、片剂、丸剂、粉末、酒剂、糖浆及口服液等剂型在内的待测市售保健品的PSI-MS检测结果与HPLC-UV检测结果基本一致。各目标物在一定范围内线性关系良好,相关系数r~2均0.99,检出限均在1.0 mg/L以下,相对标准偏差为20%~24%。结论本方法快速、准确、专属性强,适用于大批量复杂基质样品中PDE5抑制剂的快速筛查。     

同时检测伐地那非及其类似物的免疫学方法

伐地那非是用于治疗阳痿的磷酸二酯酶(phosphodiesterase type-5,PDE-5)抑制剂药物。违法添加到保健品和中成药的伐地那非及其结构类似物对公众健康构成了威胁。单克隆抗体4B9被鉴别为能够同时检测伐地那非及其类似物。以这株抗体建立的间接竞争ELISA方法,对伐地那非的检测限为5.0 ng/mL,线性范围为5.0~40 ng/mL(R2=0.952),IC50值为18.2 ng/mL。对20个中成药进行检测没有发现假阳性,伐地那非最低检出浓度为0.08 mg/g;添加回收率76%~116%,批内差异为9.7%~16.2%。     

Sildenafil and analogous phosphodiesterase type 5 (PDE-5) inhibitors in herbal food supplements sampled on the Dutch market

Herbal food supplements, claiming to enhance sexual potency, may contain deliberately added active pharmacological ingredients (APIs) that can be used for the treatment of erectile dysfunction (ED). The aim of this study was to determine whether herbal food supplements on the Dutch market indeed contain APIs that inhibit phosphodiesterase type 5 (PDE-5) inhibitors, such as sildenafil and analogous PDE-5 inhibitors. Herbal food supplements intended to enhance sexual potency (n = 71), and two soft drinks, were sampled from 2003 up to and including 2012. In 23 herbal supplements, nine different PDE-5 inhibitors were identified; in a few cases (n = 3), more than one inhibitor was indentified. The presence of these APIs was however not stated on the label. The concentrations of PDE-5 inhibitors per dose unit were analysed. Furthermore, the potential pharmacologically active properties of the detected PDE-5 inhibitors were estimated by using data from the scientific and patent literature regarding (1) in vitro PDE-5 activity, (2) reported effective doses of registered drugs with PDE-5 inhibitor activity and (3) similarity to other structural analogues. It was concluded that 18 of the 23 herbal food supplements, when used as recommended, would have significant pharmacological effects due to added APIs. Adequate use of existing regulation and control measures seems necessary to protect consumers against the adverse effects of these products.     

Structure determination of new analogues of vardenafil and sildenafil in dietary supplements

New analogues of vardenafil and sildenafil illegally added to dietary supplements were detected by high-performance liquid chromatography (HPLC) analysis with a photodiode array detector (PDA). These compounds were isolated and their structures elucidated by mass spectrometry (MS), infrared (IR) spectroscopy, one- and two-dimensional nuclear magnetic resonance (NMR). One of the new analogues given the trivial name pseudovardenafil (compound 1) was structurally elucidated and shown to be 1-[[3-(1,4-dihydro-5-methyl-4-oxo-7-propylimidazo[5,1-f][1,2,4]triazin-2-yl)-4-ethoxyphenyl]sulfonyl]-piperidine. It was a vardenafil analogue isolated from a dietary supplement capsule. Compared with vardenafil, the piperidine ring was substituted for the ethylpiperazine group. The second new analogue, trivially named hydroxyhongdenafil (compound 2), was separated from bulk powder used as a raw material for a dietary supplement. The piperazine and phenyl groups were connected through an acetyl group instead of a sulfonyl group, and hydroxyethylpiperazine was substituted for the methylpiperazine of sildenafil. It was structurally elucidated as 5-[2-ethoxy-5-[[4-(2-hydroxyethyl)-1-piperazinyl]acetyl]phenyl]-1,4-dihydro-1-methyl-3-propyl-7H-pyrazolo[4,3-d]pyrimidin-7-one.     

超高效液相色谱-串联质谱法同时测定保健品中三种违禁成分

建立固相萃取-超高效液相色谱-串联质谱/质谱法(SPE-UPLC-MS/MS)测定保健品中伐地那非、西地那非、他达那非的分析方法。试样经甲醇或盐酸溶液提取,HLB固相萃取柱净化后,以乙腈和0.1%甲酸水溶液作为流动相,C18色谱柱分离,采用电喷雾离子源,正离子扫描多反应监测(MRM)模式进行检测。伐地那非、西地那非、他达那非在10~200μg/L浓度范围内呈良好线性关系,相关系数(r2)均大于0.99。空白样品加标回收率均大于75%,相对标准偏差小于8%。胶囊样品中伐地那非、西地那非、他达那非的方法检出限分别为10μg/kg、10μg/kg、20μg/kg;定量限分别为40μg/kg、40μg/kg、80μg/kg。口服液中分别为3μg/L、3μg/L、6μg/L,定量限分别为10μg/L、10μg/L、20μg/L。研究了基质效应的影响,样品基质对伐地那非、西地那非、他达那非的响应表现为抑制作用,伐地那非受到抑制作用最明显。     

检测伐地那非的单克隆抗体的制备方法

为了能够获得检测伐地那非的单克隆抗体,采用戊二醛作为连接剂,将伐地那非连接到载体蛋白上,使伐地那非药效必需基团暴露在人工抗原表面作为主要抗原决定簇。经条件优化,偶联比为6.8:1 的抗原免疫小鼠后产生的抗体滴度最高。通过杂交瘤技术获得了4 株能够同时识别伐地那非及其类似物Piperidenafil 的单克隆抗体。