Dried blood spot (DBS) extraction processes, instrument set-up, mobile-phase composition, sample introduction technique, and calibration approach of flow injection analysis-tandem mass spectrometry (FIA-MS/MS) methods were optimized. Inter- and intralaboratory variation of methionine, leucine, phenylalanine, tyrosine, isovaleryl-carnitine, glutaryl-carnitine, octanoyl-carnitine, and decanoyl-carnitine were determined pre- and postoptimization, using 3 different calibration approaches.
Optimal recovery of analytes from DBS was achieved with a 35-min extraction time and 80% methanol (150 μL). Optimized methodology decreased the mean intralaboratory percentage relative SD (%RSD) for the 8 analytes from 20.7% (range 4.1-46.0) to 5.4% (range 3.0-8.5). The alternative calibration approach reduced the mean interlaboratory %RSD for all analytes from 16.8% (range 4.1-25.0) to 7.1% (range 4.1-11.0). Nuclear magnetic resonance analysis of the calibration material highlighted the need for standardization. The purities of isovaleryl-carnitine and glutaryl-carnitine were 85.13% and 69.94% respectively, below the manufacturer\'s stated values of ≥98%.
For NBS programs provided by multiple laboratories using single analyte COVs, harmonization and standardization of results can be achieved by optimizing legacy FIA-MS/MS methods, adopting a common analytical protocol, and using standardized calibration material rather than internal calibration.
干血斑(DBS)提取工艺,仪表设置,流动相组成,样品导入技术,并对流动注射分析-串联质谱(FIA-MS/MS)方法的校正方法进行了优化。蛋氨酸的中间和内部变异,亮氨酸,苯丙氨酸,酪氨酸,异戊酰基肉碱,戊二酰肉碱,辛酰基肉碱,并在优化前和优化后测定了癸基肉碱,使用3种不同的校准方法。
用35分钟的提取时间和80%的甲醇(150μL)从DBS中实现了分析物的最佳回收。优化的方法将8种分析物的平均流内百分比相对SD(%RSD)从20.7%(范围4.1-46.0)降低到5.4%(范围3.0-8.5)。替代校准方法将所有分析物的平均实验室间%RSD从16.8%(范围4.1-25.0)降低至7.1%(范围4.1-11.0)。校准材料的核磁共振分析强调了标准化的必要性。异戊酰基肉碱和戊二酰基肉碱的纯度分别为85.13%和69.94%,低于制造商规定的≥98%的值。
对于多个实验室使用单一分析物COV提供的NBS程序,可以通过优化传统的FIA-MS/MS方法来实现结果的协调和标准化,采用通用的分析方案,并使用标准化的校准材料而不是内部校准。