The assay was validated according to the regulatory guidelines on bioanalytical method validation of the FDA (2018) and the EMA (2012) [21,22].

Complete calibration curves over the concentration range of 0.25–40 µg/mL were analyzed on three correlative days. A linear regression with a weighting factor of 1/concentration was used to plot the peak area of ceftaroline (response) versus the corresponding concentration. Slopes, y-intercepts, correlation coefficient (R), the relative error (RE, %) from the nominal level of each standard and the coefficient of variation (CV, %) of the response factors (chromatographic area/concentration) were calculated. The correlation coefficient (R) had to be greater than or equal to 0.99 and the RE within 15%, except for the lower limit of quantification (LLOQ). The CV of the response factor should was intended to be within 15%.

Selectivity was determined using plasma samples of six healthy individuals. Additionally, plasma samples from six critically ill patients not receiving ceftaroline were used. Specificity was evaluated by testing both matrices regarding interference near the retention time of ceftaroline under the used chromatographic conditions.

The precision and accuracy of the method were determined using QC samples. Over 3 days, five QC samples at the three concentration levels (0.75, 15 and 30 μg/mL) were analyzed. Intra- and inter-day precision were calculated as the coefficient of variation (CV, %) within a single run and between the three assays, respectively; for the calculation of intra- and inter-assay accuracy, the RE of the nominal concentration was calculated. Analytical series were considered approved if the RE and CV did not exceed ±15%.

As with the QC samples, the intra- and inter-assay precision and accuracy of the LLOQ were also calculated. The LLOQ was considered the lowest level included in the calibration curves. The RE and CV were intended not to exceed ±20%.

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