Abstract：
Newborn screening (NBS) laboratories in the United Kingdom adhere to common protocols based on single analyte cutoff values (COVs); therefore, interlaboratory harmonization is of paramount importance. Interlaboratory variation for screening analytes in UK NBS laboratories ranges from 17% to 59%. While using common stable isotope internal standards has been shown to significantly reduce interlaboratory variation, instrument set-up, sample extraction, and calibration approach are also key factors.
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.
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.
摘要：
英国的新生儿筛查（NBS）实验室遵守基于单一分析物截止值（COV）的通用协议；因此，实验室间的协调是至关重要的。在英国NBS实验室中筛选分析物的实验室间差异范围为17％至59％。虽然使用常见的稳定同位素内标已被证明可以显著减少实验室间的差异，仪表设置,样品提取，和校准方法也是关键因素。
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。
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。
