Besides, the presence of various diuretics in the same sample is unlikely. Time of chromatography is 12 min under the optimal conditions.
3.2. Optimization of MS-MS Conditions
One of the major disadvantages of mass spectrometric methods is low selectivity in the analysis of biological objects. The identification of analytes in biological matrices is complicated due to misrepresentation of mass spectrum caused by concomitant substances. It can lead to appearance of false-positive results. In this case tandem mass spectrometry is more preferable method . The combination of mass selection of the precursor followed by potentially unique collision-induced dissociation and mass selection or scanning of the product ion provides higher specificity, since coincidence of spectrum of dissociated ion and ion with known structure is a conclusive proof of ions structure identity. The tandem mass spectrometry method can be used either in full scan acquisition mode or multiply reaction monitoring mode. The second technique is more sensitive in comparison with the first one. Typically, two transitions precursorproduct must be monitored, but in some cases the combination of a single precursor-product ion pair is sufficiently unique to be definitive.
For each diuretic molecular ions, product ions and collision energy (E) were chosen. Collision energy should ensure maximum yield of product ions . The data for transition reaction molecular ion—product ion are presented in Table 3.
Chromatograms of spiked with bendroflumethiazide and triamterene chosen as examples urine samples and negative urine samples at these conditions are presented in Figure 3.
It is shown that the compounds may be detected in each of the reactions. Besides, peaks with signal-to-noise ratio ≥ 3 at the time which corresponds to analyte retention time, are absent in chromatogram of negative sample. This result was observed for other diuretics and probenecid as well. This fact proves sufficient selectivity of method.
3.3. Method Validation
According to the requirements of WADA  the relative intensities of any of the ions in mass shall not differ by more than the amount in Table 4 from the relative intensities of the same ions acquired from spiked urine.
The retention times of all diuretics (except metolazone) do not differ by more than 1% from that of the same substance in a spiked urine sample. However, there are no peaks with signal-to-noise ratio ≥ 3 in chromatograms of six analyzed blanks for the following compounds: acetazolamide, furosemide, bumetanide, indapamide, chlorothiazide, hydrochlorothiazide, metolazone, bendroflumethiazide, trichlormethiazide, chlorthalidone, triamterene.
Table 3. The diagnostic reactions and optimal collision energies for diuretics determination in Multiple Reaction Monitoring mode.
Figure 3. The chromatograms of spiked (1-3) and negative urine sample (4-6), (a) Bendroflumethiazide; (b) Triamterene. С = 0.25 µg·ml–1.
Table 4. The maximum tolerance windowsa for relative ion intensities.
Peak of small intensity with signal-to-noise ratio ≥ 3 is observed for probenecid in chromatogram of negative urine sample (m/z 270→135, Table 3). This fact is possibly caused by the influence of matrix. But the integrated intensity of this peak is negligible which according to WADA recommendations  can not be a cause of false identification. Besides, there are no peaks with signal-to-noise ratio ≥ 3 in the chromatograms of negative urine sample obtained using other diagnostic ions.
The ratio of intensities of diagnostic ions of acetazolamide, chlorothiazide, furosemide, chlortha-lidone, bumetanide, hydrochlorothiazide, triamterene and probenecid in the standard solutions and spiked urine samples meets WADA requirements (Tables 3 and 5). In case of indapamide and trichlormethiazide this parameter does not fit the tolerance window. Moreover, significant change of relative intensities dependent on concentration is observed which obviously can be explained by matrix influence at relative intensity. Thereby, this method can not be recommended for confirmation of indapamide and trichlormethiazide in urine.
Table 5. Relative abundance of diagnostic ions in Multiple Reaction Monitoring mode, established during the method validation.
Limits of detection of acetazolamide, probenecid, furosemide, chlorthalidone, bumetanide, hydrochlorothiazide, triamterene, bendroflumethiazide, metolazone by tandem mass spectrometry are 0.050 µg·ml–1. Limit of detection of chlorothiazide which is 0.500 µg·ml–1 does not meet WADA requirements. High limit of detection may be explained by its partial dissociation at the column.
The developed method proves the suitability of gas chromatography and tandem mass spectrometry for confirmation of presence of 8 diuretics and probenecid in urine. It is selective towards acetazolamide, furosemide, bumetanide, hydrochlorothiazide, bendroflumethiazide, metolazone, probenecid, chlorthalidone and triamterene, since the components of urine do not interfere with the detection of diuretics. The method can not be recommended for determination of indapamide, trichlormethiazide and chlorothiazide.
The reduction of time for sample preparation and sufficient recovery of all diuretics merit the method proposed. Optimization of temperature program for diuretics determination at chromatographic column which wasn’t previously used for this purpose allowed to achieve chromatographic separation of all diuretics in short time. Selection of diagnostic fragmentation reactions and optimal collision energies allowed to achieve high selectivity.
Limits of detection of diuretics and probenecid by MS-MS are lower than that regulated by WADA and are 0.050 µg·ml–1.
The method was validated that proves the possibility of its application in anti-doping laboratories.