Defendenti C, Bonacina E, Mauroni M, Gelosa L
Presidio Multizonale di Igiene e Prevenzione, Unità di Riferimento Regionale per la Micologia, Milano, Italy.
Forensic Sci Int. 1998 Mar 2;92(1):59-68. doi: 10.1016/s0379-0738(98)00006-1.
The objective of the present study was to develop and validate a liquid chromatographic method with electrochemical detection to measure alpha amanitin concentrations in urine after sample pretreatment with double mechanism (reversed phase/cation exchange) solid-phase extraction cartridges. The urine samples (10 ml) were purified and concentrated to 1 ml with elimination of matrix contaminants. The extracts were then separated by isocratic reversed-phase chromatography using a C18 column (4.6 mm x 25 cm) with a mobile phase composed of 0.005 M phosphate buffer (pH 7.2) and acetonitrile (90:10). Coulometric detection was performed by applying an oxidation potential of +500 mV to a porous graphite electrode in a low-volume analytical cell. The limit of quantitation was 10 ng/ml with a signal-to-noise ratio = 25. The linearity studied on spiked urine was satisfactory (r = 0.9966) from 10 ng/ml to 200 ng/ml. The average extraction recovery of alpha amanitin was 78%, determined using spiked urine samples ranging from 10-300 ng/ml. The intra-assay precision was checked at 10, 50 and 100 ng/ml levels (n = 10) in spiked urine samples, with resulting coefficients of variation of 3.6%, 2% and 1.5%, respectively.
本研究的目的是开发并验证一种采用电化学检测的液相色谱方法，用于在使用双机制（反相/阳离子交换）固相萃取柱对样品进行预处理后测量尿液中的α-鹅膏毒肽浓度。将尿液样品（10毫升）进行纯化并浓缩至1毫升，同时去除基质污染物。然后使用C18柱（4.6毫米×25厘米）通过等度反相色谱法分离提取物，流动相由0.005 M磷酸盐缓冲液（pH 7.2）和乙腈（90:10）组成。通过在低体积分析池中对多孔石墨电极施加+500 mV的氧化电位进行库仑检测。定量限为10 ng/ml，信噪比 = 25。在加标尿液中从10 ng/ml至200 ng/ml进行的线性研究结果令人满意（r = 0.9966）。使用浓度范围为10 - 300 ng/ml的加标尿液样品测定α-鹅膏毒肽的平均提取回收率为78%。在加标尿液样品中，在10、50和100 ng/ml水平（n = 10）下检查批内精密度，所得变异系数分别为3.6%、2%和1.5%。
