Upper limit of quantification

Upper limit of quantification (ULOQ) the highest amount of an analyte in a sample that can be determined quantitatively with precision and accuracy. 

Lower Limit of Quantification (LLOQ) The lowest concentration of the analyte of interest in a matrix that can be quantitatively determined using the standard curve with acceptable precision and accuracy. The LLOQ is usually defined as the lowest concentration at which the assay imprecision does not exceed 20%. Upper Limit of Quantification (ULOQ) The highest concentration of an analyte in a matrix that can be quantitatively determined using the standard curve with an acceptable precision and accuracy. If the analyte concentrations in the postdose samples are higher than the ULOQ, then a dilution QC is needed to cover the highest anticipated dilution. 

Typically, the relationship in the assay between the detection signal and the concentration of the mAb is nonlinear. A simple regression analysis is, therefore, not possible. Usually, a third-to-fifth degree polynomial is used to relate the detection signal to the concentration. Because of this relationship, a typical sample dilution is not possible and careful evaluations are necessary if the concentration of the antibody is above the upper limit of quantification of the assay. As a consequence, the assay should ideally cover the whole concentration range of all samples measured. 

A series of unknown samples is usually measured together with two sets of calibration standard samples (covering the concentration range for the assay, usually two sets of six or more calibration standards) and two or more sets of quality control samples. The calibration standard samples will be used to establish the calibration for the unknowns. Quality control samples (usually at least 5 % of number of unknowns) are matrix samples of known concentration, which are equally distributed over the analytical run (usually two sets of three different concentration levels 2-3 times the LOQ, mid concentration range and close to the upper limit of quantification). They establish a set of control samples in order to verify the assay performance within the run. Typically, the calibration standards and quality control samples should be within +/-15% of the nominal value. However, in typical assays, it is considered to be acceptable, if 75 % of the standards are within the +/-15 % criteria. Outliers will not be used for the calculation of the calibration curve. Not all standards at one concentration should be excluded. A similar criteria is applied for the quality control samples 2 out of 3 of the quality control samples should be within +/-15 % of their nominal value. 

The precision and accuracy of the method was assessed by analyzing batches on three occasions (days). Each run included a duplicate set of calibration standards (CS) and a set of quality control samples (5 QC levels in 5 replicates). The following QC levels have been used 1, 3, 100, 800 2500 ng/mL in plasma and 0.25, 0.6, 25.2, 40 and 128 ng/mL in urine. The highest QC level was above the upper limit of quantification (ULQ). These samples were analyzed after 5-fold dilution in human plasma/urine prior to analysis in order to evaluate the possibility of diluting samples with concentrations above ULQ. 

The anal5d ical range of immunoassays is defined as the range between the lower limit of quantification (LLQ) and upper limit of quantification (ULQ) for which interpolated results have acceptable levels of accmacy and precision... 

The upper limit of quantification is the maximum analyte concentration of a sample that can be quantified with acceptable precision and accuracy (bias). In general the ULOQ is identical to the concentration of the highest calibration standard [10]. 

If not already available, a bioanalytical chemistry method needs to be defined and characterized for the quantification of the lead in physiological fluids. This assay can then support experiments in some of the other scientific disciplines involved in assessing the developability of the lead and, after appropriate validation, the preclinical, nonclinical, and clinical development of a selected drug candidate. For preliminary studies, a bioanalytical chemistry method should be characterized to demonstrate the range of reliable results, the lower and upper limits of quantification, specificity, accuracy, and precision. In addition, evaluations on the matrix to be used (blood, plasma, serum) should be conducted and the stability of the lead in each matrix should be determined. 

A totally different situation arises when covariates are missing because of the value of the observation, not because the covariate wasn t measured. In such a case the value is censored, which means that the value is below or above some critical threshold for measurement. On the other hand, a covariate may be censored from above where the covariate reported as greater than upper limit of quantification (ULOQ) of the method used to measure it. In such a case the covariate is reported as >ULOQ, but its true value may lie theoretically between ULOQ and infinity. The issue of censored covariates has not received as much attention as the issue of censored dependent variables. Typical solutions include any of the substitution or imputation methods described for imputed missing covariates that are not censored. 

Recommended analysis and acceptance criteria apply to each sample concentration HQC = high-quality control sample LLOQ — lower limit of quantification sample LQC = low-quality control sample MQC — middle quality control sample ULOQ — upper limit of quantification sample) %CV — percent coefficient of variation %RE — percent relative error [4]... 

Assay range LLOQ to upper limit of quantification (ULOQ)... 

The limits of quantification of an analytical procedure are the lowest and largest amounts of the targeted substance in the sample that can be quantitatively determined under the prescribed experimental conditions with well-established measurement error (analytical bias and precision). Consequently, the dynamic range of an analytical procedure is the range between the lower and the upper limits of quantification within which a measured result is expected to have acceptable levels of bias and precision. 

The measurement error profile is within the acceptance limits across the entire concentration range tested during prestudy validation. In other words, the tolerance intervals calculated at different concentration levels are all within the acceptance limits. In such a case, the method is declared valid across the entire range. The lower and upper limits of quantification are defined, respectively, by the lowest and highest concentrations of this tested range (see Fig. 5.1). 

Upper limit of quantification (ULOQ) is the highest concentration limit of the assay that can be reported with acceptable accuracy and precision. ULOQ is established after >3 assay runs using results from the standard curve. Generally, an ULOQ that demonstrates a %CV 20% and %recovery of 80-120% is considered acceptable. Samples with concentrations that fall at or above the ULOQ require sufficient dilution to bring concentrations into the report-able or dynamic range of the assay. Table 41.2 shows an example of an assessment of the precision and accuracy of a standard curve including the selection of LLOQ and ULOQ limits. 

Upper Limit of Quantification Quality Control (ULOQ QC) QC samples prepared at the same concentrations as the ULOQ standard. 

---