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J. Sep. Sci. 2014, 37, 219–227 1 ¨ Sanna T. Pynnonen 1,2 Tuula A. Tuhkanen 1 Department

of Chemistry and Bioengineering, Tampere University of Technology, Tampere, Finland 2 Department of Biological and Environmental Science, ¨ ¨ University of Jyvaskyl a, ¨ ¨ Finland Jyvaskyl a,

Received May 8, 2013 Revised October 31, 2013 Accepted November 12, 2013

Research Article

Simultaneous detection of three antiviral and four antibiotic compounds in source-separated urine with liquid chromatography An analytical method for the simultaneous screening of three antiviral agents (nevirapine, zidovudine, lamivudine), four antibiotics (sulfamethoxazole, trimethoprim, ciprofloxacin, rifampicin) and one reference compound (carbamazepine) in human urine was developed. Separation was achieved with a Kinetex XB-C18 (75 × 4.6 mm, 2.6 ␮m) column after the extraction of pharmaceuticals from urine with SPE. Gradient elution with a mobile phase consisting of acetonitrile and 10 mM KH2 PO4 (pH 2.5), and diode array detection with monitoring at 210 and 264 nm was applied. The developed method was validated in terms of selectivity, linearity, stability and sensitivity. Repeatability (n = 3) and between-day precision (n = 3) revealed RSD 5 ␮L caused less symmetric peaks and the resolution between different peaks suffered. Thus, a 5 ␮L injection volume was selected. In addition, the effect of the eluent flow rate was tested. At a flow rate of 2.0 mL/min the elution of the last peak (RMP) was delayed markedly. Gradient elution was set to take place in 0.5 min steps to decrease the analysis time. Subsequently, the flow rate was adjusted to 2.5 mL/min, which further reduced the analysis time to 4 min. DeStefano et al. [40] have demonstrated that the flow rate of the mobile phase can be substantially increased and the separation time decreased with only a small decrease in the column efficiency and separation resolution when utilising new sub-3 ␮m particle columns. Figure 2 presents the order of the peak elution for the stock compounds and for the urine sample (sample taken from elution with 50:50:2 v/v/v, ACN/MeOH/acetic acid). Between peaks g and h there is a smaller peak (x), which is caused by RMP. Different gradient slopes and ACN endconcentrations were tested but they did not have an effect on the retention of CBZ and RMP. The retention times (tR ), the calibration curve R2 values, ␣ (selectivity), k’ value (capacity factor), and LOD and LOQ of the instrument for each compound can be found in Supporting Information Table S1. Supporting Information Table S1 also describes the standard deviations of the calibration curves slope and intercept. The R2 values were all ≥0.9992 and k and ␣ were acceptable. The standard deviations of the retention times were all < ± 0.02 min. The LOD and LOQ varied between 21–536 ␮g/L and 71–1786 ␮g/L, respectively, and depending on the compound.  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The linear range of the calibration varied depending on the compound. The number of calibration points was seven for 3TC, eight for CBZ and CIP, nine for TRI, SMX and NVP, ten for RMP and 11 for ZDV. The capacity factor (k’) of a peak should preferably be