A proposal for comparing methods of quantitative analysis of endogenous compounds in biological systems by using the relative lower limit of quantification (rLLOQ)

Accuracy, precision and lower limit of quantification (LLOQ) are experimentally achievable key analytical factors by which the quality of analytical methods can be ascribed and objectively evaluated. Endogenous substances (endobiotica) are physiologically present in biological fluids and tissues at varying basal concentration (C0,Ln). Formally, the definition of accuracy and LLOQ is same for xenobiotica and endobiotica. However, these analytical factors must be determined differently, notably by considering the C0,Ln value of endobiotica. Often, the impact of the endogeneity on the analytical method is underestimated. This especially applies to the LLOQ, because the LLOQ values for endobiotica are regularly not fixed measures due to the varying C0,Ln value in biological samples. In order to circumvent these difficulties and for a more reliable and objective evaluation and comparison of analytical methods for endobiotica, this work proposes the use of the relative lower limit of quantification, i.e., rLLOQ. The rLLOQ is defined as the percentage ratio of the LLOQ value, i.e., CLLOQ to C0,Ln: rLLOQ = (CLLOQ:C0,Ln) × 100. Thus, the rLLOQ describes that fraction of C0,Ln that can be still determined with acceptable values for accuracy (e.g., recovery of 100 ± 20%) and precision (e.g., RSD ≤ 20%) or with a total error (i.e., recovery + precision) of ≤30%. Examples from the quantitative analysis of selected endogenous compounds by previously validated GC–MS, GC–MS/MS and LC–MS/MS methods support the appropriateness and expressiveness of the rLLOQ in the quantitative analysis of endobiotica.