In this paper, a calibration procedure for LC/MS-based bioanalysis methods, termed “A/B fortification”, is proposed. The concept relies on the post-extraction fortification (B-spike) of an aliquot of the injection-ready sample extract for the determination and compensation of specific signal suppression or enhancement effects compared to matrix-free extract prepared in buffer or mobile phase. Conventional analyte recovery, observed due to the incomplete extraction of analytes from the sample or losses during a cleanup, is determined by the conventional pre-extraction fortification (A-spike) of a blank sample that belongs to the same type of matrix as the sample with the unknown analyte concentration. This approach permits a higher throughput than conventional sample fortification strategies. The results obtained by utilizing the A/B fortification concept were extensively compared against conventional methods (representative bank matrix fortification, sample fortification and internal standard). The proposed concept (based on the pre-fortification of a reference matrix and post-fortification of the sample) was found to be significantly less biased than internal standard-based techniques. The A/B fortification indicated a better accuracy than the sample fortification or representative blank matrix fortification approach and, most importantly, produced significantly fewer outliers. This was linked to the fact that in the case of the A/B fortification, the uncertainty of the subtraction of two peak areas (fortified minus unfortified sample) is reduced, because fortifications are not made prior to the extraction step but are made into the final injection-ready sample extract. Fortification into an injection-ready aliquot eliminates all sample processing-related differences (procedural errors), which can affect conventional sample fortification-based quantifications.

在本文中，提出了一种基于LC / MS的生物分析方法的校准程序，称为“ A / B强化”。与在缓冲液或流动相中制备的无基质提取物相比，该概念依赖于准备注射样品提取物的等分试样的提取后强化（B峰）。常规分析物的回收率是由于空白样品的常规萃取前强化（A钉）确定的，而常规分析物的回收率是由于样品中分析物的提取不完全或清理过程中的损失所致，而空白样品与样品属于同一类型的基质分析物浓度未知。与传统的样品强化策略相比，该方法可实现更高的通量。通过使用A / B设防概念获得的结果与常规方法（代表性银行矩阵设防，样品设防和内标）进行了广泛的比较。与基于内部标准的技术相比，发现提出的概念（基于参考矩阵的强化和样品的强化）的偏见明显减少。A / B设防显示出比样品设防或代表性空白矩阵设防方法更好的准确性，最重要的是，产生的异常值明显减少。这与以下事实有关：在A / B设防的情况下，减少了两个峰面积（强化减去未强化样品）相减的不确定性，因为强化不是在提取步骤之前进行的，而是被制成最终的可注射样品的提取物。将其强化成可立即进样的等分试样，可消除所有与样品处理相关的差异（过程错误），这些差异可能会影响基于常规样品强化的定量。