Human saliva quantitative monitoring of roxithromycin (ROX) at picomolar-level by flow injection (FI) chemiluminescence (CL) analysis is described for the first time, to our knowledge. Monitoring was based on the CL intensity from luminol-BSA reaction, which can be quenched in the presence of ROX, with the decreasing CL intensity linearly proportional to the logarithm of the ROX concentration, ranging from 0.6 to 1000 pmol·L<sup>-1</sup>. The detection limit of the proposed method for the determination of ROX was as low as 0.2 pmol·L<sup>-1</sup> (3?), and the relative standard deviations were less than 4.0% (<i>n</i> = 7). A complete analytical process, including sampling and washing for ROX determination, conducted at a flow rate of 2.0 mL·min<sup>-1</sup>, was performed completely within 30 s, yielding a sample efficiency of 120 h<sup>-1</sup>. The proposed method was successfully applied to the determination of ROX in human saliva and serum samples with recoveries from 90.9% to 110.1%. The continuous monitoring of ROX in human saliva after oral intake showed that the total elimination ratio was 87.1% during 24 h, and the pharmacokinetic parameters were 0.97 ± 0.18 h<sup>-1</sup> for the absorption rate constant <i>K<sub>a</sub></i>, 0.082 ± 0.010 h<sup>-1</sup> for the elimination rate constant <i>K<sub>e</sub></i>, and 8.56 ± 1.11 h for the elimination half-life time <i>t</i><sub>1/2</sub>. It was also found that ROX in human saliva and urine simultaneously reached the maximum at 2 h with the concentration correlate ratio of 0.97.

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