90 Chapter 5 Table 2. Summary of LC-MS/MS method validation performancea. Validation parameter Sample Expressed as Result Acceptanceb Within-run precision QC LLOQ RSD (%) 4.9 <20 QC Low 4.2 <15 QC Medium 2.4 <15 QC High 2.1 <15 Between-run precision QC LLOQ RSD (%) 7.4 <20 QC Low 4.5 <15 QC Medium 2.8 <15 QC High 3.4 <15 Accuracy QC LLOQ Bias (%) 6.1 <20 QC Low -4.1 <15 QC Medium -3.8 <15 QC High 1 <15 Selectivity in plasma Human samples #1−12 Max relative to LLOQ (%) 0.2 <20 LLOQ LLOQ of 4 µg/mL Signal/noise 88 >5× Linearity Standards 4, 8, 16, 32, 64, 128, 256, 512 µg/mL R2 0.999 >0.99 Freeze-and-thaw stability QC Low Bias (%) -4.5 <15 QC High 4.3 <15 Spiked recovery in plasma Human samples #1−12 low Min/max Bias (%) Min/max Bias (%) -1.1/11.8 <15 Human samples #1−12 high -6.3/1.2 <15 Autosampler stability Day 1 reinjected after 7 days Min/max Bias (%) -9.6/8.8 <15 Carry over Blank after highest standard Relative to LLOQ (%) 0.3 <20 Zero sample Pool human plasma with IS Relative to LLOQ (%) 0.2 <20 aSGG as signature peptide for SRM transition of 751.33 1100.46. bIn accordance with ‘Guideline on bioanalytical method validation’ of the European Medicines Agency [29]. Abbreviations: IS: internal standard; LLOQ: lower limit of quantification; max: maximum; min: minimum; RSD: relative standard deviation; QC: quality control. In addition to the analytical validation results, two plasma control samples from R2 Diagnostics were measured with the LC-MS/MS method. The assigned values of these controls were 26.6 and 78.3 µg/mL, and LC-MS/MS results were 25.9 and 79.2 µg/mL. Also, a sample with an unknown amount of emicizumab from a pilot External Quality Assessment study of the WFH (UK-NEQAS, Sheffield, UK. sample: WFH EMI 21:01, July 2021) was tested. The LC-MS/MS result was 59.0 µg/mL for a median of 57.5 µg/mL derived from 11 laboratories. Both these findings corroborate the results of the analytical performance of the LC-MS/MS method.
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