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J Chromatogr A. 2010 Mar 5;1217(10):1626-35. doi: 10.1016/j.chroma.2010.01.031. Epub 2010 Jan 18.
A rapid nanoLC-MS/MS method was developed and validated for the simultaneous determination of glutathionylated and cysteinylated precursors of 3-mercapto-hexan-1-ol (3MH) and 4-methyl-4-mercaptopentan-2-one in grape juice using stable isotope dilution assay (SIDA). The analytes were extracted from must using a cation exchange resin and purified on C18 cartridges. They were chromatographically separated on a reverse phase column and finally analyzed by tandem mass spectrometry in selected reaction monitoring mode (SRM) using deuterated analogues as standards except for glutathionylated conjugate of 4MMP which was analyzed by external calibration. The method was validated according to the International Conference on Harmonization recommendations by determining linearity, accuracy, precision, recovery, matrix effect, repeatability, intermediate reproducibility, LODs and LOQs. Calibration for each precursor was determined by performing Lack-of-Fit test and the best fitting for 3MH precursors was a quadratic model whereas a linear model was better adapted for 4MMP precursors. All calibration curves showed quite satisfactory correlation coefficients (R(2)>0.995 for SIDA quantification and R(2)>0.985 for external calibration). Quantification by SIDA and external calibration allowed a high level of accuracy since the averaged value ranged from 80 to 108%. Quantification of aroma precursors was accurate and reproducible over five days since intermediate precision (same analyst, same sample and same apparatus), which was evaluated by the calculation of RSD was inferior to 16%. Limits of quantification for G3MH and G4MMP were closed to 0.50 and 0.07 nmol/L and as 4.75 and 1.90 nmol/L for Cys3MH and Cys4MMP respectively. This method was applied to the quantification of precursors into several types of grape juices: Melon B., Sauvignon, Riesling and Gewurztraminer.
建立并验证了一种快速纳升液相色谱-串联质谱（nanoLC-MS/MS）方法，用于使用稳定同位素稀释分析（SIDA）同时测定葡萄汁中谷胱甘肽化和半胱氨酸化 3-巯基-1-己醇（3MH）和 4-甲基-4-巯基-2-戊酮前体。采用阳离子交换树脂从葡萄汁中提取分析物，并用 C18 小柱进行纯化。在反相柱上进行色谱分离，最后通过选择反应监测模式（SRM）进行串联质谱分析，除了 4MMP 的谷胱甘肽化缀合物外，均使用氘代类似物作为标准进行分析，而 4MMP 的谷胱甘肽化缀合物则通过外部校准进行分析。该方法按照国际协调会议的建议进行了验证，包括线性、准确性、精密度、回收率、基质效应、重复性、中间精密度、LOD 和 LOQ 的测定。通过进行失拟检验确定了每个前体的校准，3MH 前体的最佳拟合模型为二次模型，而 4MMP 前体的最佳拟合模型为线性模型。所有校准曲线的相关系数均令人满意（SIDA 定量的 R²>0.995，外部校准的 R²>0.985）。通过 SIDA 和外部校准进行定量可获得较高的准确度，因为平均值范围为 80-108%。通过计算 RSD 评估的中间精密度（同一分析人员、同一样品和同一仪器）表明，5 天内的定量重现性较好，精度低于 16%。G3MH 和 G4MMP 的定量下限接近 0.50 和 0.07 nmol/L，Cys3MH 和 Cys4MMP 分别为 4.75 和 1.90 nmol/L。该方法应用于几种类型的葡萄汁中前体的定量：甜瓜 B.、长相思、雷司令和琼瑶浆。
