Lower limit of quantitation. LLOQ

Accuracy, precision, and linearity of standards — The linear dynamic range was established as 10.22 pg/mL to 2.037 ng/mL with coefficient of determination (r2) below 0.996190 when using 1/x2 weighing for three consecutive accuracy and precision runs. The lower limit of quantitation (LLOQ) was accurate (inter-run mean bias = 1.1%) and precise (inter-run mean CV = 14.1%). Three levels of QCs were prepared. The inter-run mean bias varied from -4.3 to 1.0% and the inter-run mean CV varied from 4.6 to 6.6% for all QC levels. 

Other applications include bioequivalent measurements of bromazepam, an anticonvulsant, in human plasma. The lower limit of quantitation (LLOQ) was 1 ng/mL (Gongalves et al. 2005). Kuhlenbeck et al. (2005) studied antitussive agents (dextromethorphan, dextrophan, and guaifenesin) in human plasma LLOQ values were 0.05, 0.05, and 5 ng/mL, respectively. Other compounds studied were nucleoside reverse transcriptase inhibitors, zidovudine (AZT) and lamivudine (3TC) (de Cassia et al. 2004) and stavudine (Raices et al. 2003) in human plasma, and paclitaxel, an anticancer agent, in human serum (Schellen et al. 2000). 

The recent trend of decreasing available sample volumes and requiring lower limits of quantitation (LLOQs) means better sample preparation procedures are under consideration. Further improvements MS sensitivity will eventually impact sample preparation strategies and sample throughput. 

Second, an internal standard must have adequate purity. Preferably, the contribution of an internal standard to any analyte should be less than 20 % of the corresponding lower limit of quantitation (LLOQ) of the analyte. Otherwise, the significant amount of analyte from the added internal standard can bias the reported signal to noise (S/N) ratio at the LLOQ and cause larger variability at low concentrations. The interference of an internal standard to other cointemal standards in a multianalyte method is rare, but it should be also evaluated. Though there are no reported criteria for this, it should be at least less than 15 % of the concentration of a cointemal standard in a multianalyte method. In addition, an internal standard should not correspond to any in vivo metabolic products of the analyte (e.g., hydrox-ylated metabolite, N-dealkylation metabolite). 

Lower limit of quantitation (LLOQ) The lowest concentration of an analyte in a test sample that can be determined quantitatively with suitable accuracy and precision (ICH term limit of quantitation). 

The selectivity is the ability of an analytical method to differentiate and quantify the analyte (protein) in the presence of other components in the sample. Selectivity investigations focus on reliable quantitation of the analyte against a background of interferences from endogenous matrix components. Measurements are assessed by spiking the analyte into the biological matrix (e.g. serum) from a representative number of individual subjects (at least six) at concentrations near the lower limit of quantitation (LLOQ, see below), [37]. 

During method development (Chapter 9) and validation (Chapter 10), QCs are used for several purposes including checks on precision and accuracy, lower limit of quantitation (LLOQ), recovery and method robustness and ruggedness (Section 9.8.4), as well as stability studies of various kinds (Sections 10.2.7 and 10.2.8), studies of inter-day validation within a specified laboratory and cross-validations in inter-laboratory method transfer (Section 10.2.11). QC samples are also used during method development to assess the final method prior to validation experimental runs that use QCs for this purpose are often referred to as assay prequalifications or pre-study assay evaluations (PSAE). 

The lower limit of quantitation (LLOQ, Section 8.4.2) is strictly a figure of merit for an integrated analytical method. It refers to the lowest concentration of analyte in a particular complex matrix that can be analyzed quantitatively by the analytical method with specified levels of accuracy and precision. Sometimes, if not critical for the end purpose of the analysis, the LLOQ can be estimated as the lowest concentration that provides a S/B ratio of 10 1 (Section 8.4.2). 

A related problem concerns a scientifically defensible definition of the lower limit of quantitation (LLOQ). This is sometimes referred to simply as the LOQ but in fact the upper limit of quantitation (ULOQ) can also be an important parameter for validated analytical methods (see Chapter 9). A very important definition of LLOQ is that of the US FDA with regard to bioan-alytical data used in human clinical pharmacology and in bioavailabihty and bioequivalence studies requiring pharmacokinetic evaluation (FDA 2001) this guidance also applies to bioanalytical methods used for non-human pharmacology-toxicology studies and preclinical studies. According to this protocol, the LLOQ is accepted as the lowest standard on the cahbration curve provided that (a) the analyte response for this concentration (amount)... 

Sensitivity strictly refers to the slope of the calibration curve for the method but is widely used (including in this book) in a more general sense of a low value for the limit of detection (LOD, the lowest concentration of an analyte that the bioanalytical procedure can reliably differentiate from background and noise, see discussion in Section 8.4.1). Sensitivity can also refer to the lower limit of quantitation (LLOQ, the lowest amount of an analyte in a sample that can be quantitatively determined with acceptable precision and accuracy. Section 8.4.2). While it is true that a higher sensitivity in the strict sense can lead to lower values of LOD and LLOQ, the latter parameters also vary with the level of chemical background and statistical noise (see discussion of Figure 8.10 in Section 8.4). While the LLOQ is usually the more important parameter in quantitative analyses, the LOD can be significant for some applications (e.g. dioxin analysis. Section 11.4.1). 

An LC-MS-MS method for the simultaneous quantitative determination of actinomycin-D (Act-D) and vincristine (VCR), which are cytotoxic agents commonly used in the treatment of pediatric cancers. Following sohd-phase extraction, plasma samples are separated and analyzed using electrospray ionization (ESI). Lower limit of quantitation (LLOQ) for both Act-D and VCR 0.5 ng/ml. Analytical accuracy for detection of both Act-D and VCR <90%. Analytical precision, as estimated hy the coefficient of variation <6% for Act-D and <11% for VCR. Useful in clinical monitoring. 

---