NanoLC, when compared to conventional UHPLC, is supposed to provide increased sensitivity up to approximately 50-fold and improved ionization efficiency [27, 28]. When compared to conventional ESI, nanoESI MS was demonstrated to provide considerably less variations in ionization efficiencies for different compounds and more likely similar response factors [29, 30]. After simple quantification by directly integrating peak areas for several compounds and their metabolites, concentrations were never overestimated or underestimated by more than factor three compared to radioactivity-based quantification [31]. Therefore, nanoLC coupled to a nanoESI source was used for metabolism studies and later estimation of parent/metabolite ratios in authentic human urine samples.

Identifying the main human urinary metabolites is of particular interest for nitrobenzodiazepines, as standard immunoassay screening usually fails to detect low dose intake (0.5 mg), and evaluation of metabolite ratios can be used to estimate the time of intake [32]. After a single dose intake, flunitrazepam itself was only detected in very low concentrations in urine samples, whereas the metabolites 7-aminoflunitrazepam and 7-aminodesmethylflunitrazepam could be detected for up to 10 days [32]. In another study, 7-aminoflunitrazepam was detected even up to 28 days after intake of 2 mg flunitrazepam [33].

So far, there is one report describing the determination of meclonazepam by capillary gas chromatography coupled to electron capture detection [34]. Peak plasma concentrations in the range of 10–100 μg/L were reported after a single intravenous dose of 1 mg and oral administrations of 1 and 4 mg.

The nanoLC-HRMS/MS spectra of clonazolam, meclonazepam, and nifoxipam, and their phase I and II metabolites, are given in Figs., and. The proposed chemical structures, accurate masses of ions, calculated elemental formulas, and calculated mass error values in parts per million (ppm) are summarized in Tables, and. Mass errors less than 5 ppm were considered acceptable for the full scan and TMSexperiments. The spectra shown in Figs., andand the data in Tables, andare taken from representative subjects.

The tentative identification of metabolites was done in a two-step procedure. First, reconstructed ion chromatograms of the exact masses of expected phase I and phase II metabolites were built, after full-scan acquisition. Positive findings were then added into the inclusion list of the TMS 2 experiments, to acquire the respective MS 2 spectra. The structures of the urinary metabolites were deduced from their MS/MS spectra, in relation to the spectra of the parent compounds. The fragmentation patterns were postulated by general rules [ 35 ] and previous fragmentation studies on flunitrazepam and its metabolites [ 36 , 37 ]. However, as already discussed [ 35 ], benzodiazepines hardly show any class-specific fragmentation and their fragmentation appears to be mainly determined by compound-specific moieties. Hence, a comparison to previous studies is difficult.

Proposed fragmentation patterns for identification of phase I and II metabolites by nanoLC-HRMS/MS

In the following, fragmentation patterns of the mass spectra of clonazolam, meclonazepam, nifoxipam, and their phase I and II metabolites are discussed in detail. All masses are the calculated (exact) masses. In general, the glucuronic acid conjugates showed the same fragment ions as the underlying phase I metabolites, after the typical conjugate loss of −176.0321 u for glucuronide.

Clonazolam The mass spectra of clonazolam and its metabolites are shown in Fig. 1. The most abundant fragment ion of m/z 340.0722 in the MS2 spectrum of the parent compound was most likely formed after radical loss of N, similar to the loss of the HNCH 2 in the case of flunitrazepam and hydroxyflunitrazepam [35, 36]. However, such a loss is very unusual. The fragment ion of m/z 308.0823 was based on the loss of the NO 2 radical. Besides the fragment ion of m/z 324.1011, corresponding to the protonated molecule, the mass spectrum of 7-aminoclonazolam (CM1) was dominated by the fragment ion of m/z 220.0869 at high collision energies. This ion resulted from the fragmentation pathways described for diazepam and alprazolam, based on the loss of a Cl radical and the opening of the triazole ring [35, 36]. Further fragmentation of m/z 220.0869, via phenyl elimination and opening of the pyrimidine part under loss of CN, led to the fragment ion of m/z 119.0604. The accurate mass of this ion was however outside the accepted 5 ppm deviation from the exact mass (Fig. 1 and Tables 1, 2, and 3). When applying lower collision energy (NCE 35), m/z 324.1011 together with m/z 296.023 was instead the most abundant fragment ion, most likely based on the radical loss of NCH 2 . The fragmentation of 7-acetaminoclonazolam (CM2) was similar to that of 7-aminoclonazolam, considering a shift of +42.0105 u due to the acetyl part. The spectrum also contained m/z 324.1011 corresponding to the protonated 7-aminoclonazolam after neutral loss of C 2 H 2 O. For hydroxyclonazolam (CM3), one isomer was detected but, based on the mass spectrum, no detailed position of the hydroxy group could be deduced. However, in relation to other triazolobenzodiazepines, hydroxylation is expected to occur mainly at the triazole ring. This is also supported by the mass spectrum, as there was a dominant elimination of water which is less likely after hydroxylation at the aromatic ring systems. For hydroxyclonazolam glucuronide, two isomers (CM4 and CM5) were observed. The second isomer should be derived from the hydroxyclonazolam isomer, as the fragmentation was similar to that shown in Fig. 1. The mass spectrum was dominated by the fragment ion of m/z 370.0701, corresponding to the loss of 176.0321 u, but a signal at m/z 546.1022 indicating the protonated molecule was also present. The other isomer is probably hydroxylated at the aromatic ring system, as water elimination was not very prominent in the mass spectrum. However, the actual position of the glucuronic acid for both isomers remains unclear and might be elucidated in further studies using experiments based on labeling or site-specific derivatization. As already stated, the mass spectra of 7-aminoclonazolam glucuronide (CM6) and 7-acetaminoclonazolam (CM7) glucuronide showed similar fragment ions as the MS2 spectra of the corresponding phase I metabolite, including the m/z indicating loss of 176.0321 u (glucuronide). However, only the spectrum of 7-aminoclonazolam glucuronide also contained the signal of the protonated molecule. Both glucuronides, but at least 7-acetaminoclonazolam glucuronide, are most likely quaternary N-glucuronides, as assumed for alprazolam [38].

Meclonazepam The mass spectra of meclonazepam and its metabolites are shown in Fig. 2. In addition to the protonated molecule of m/z 330.0640, the spectrum showed characteristic fragment ions of m/z 316.0609 and 284.0711 resulting from contraction of the diazepine ring and elimination of the nitro group, respectively. The fragment ion of m/z 316.0609 was again based on the unusual radical loss of N and the fragment ion of m/z 284.0711 on loss of the NO 2 radical. The spectrum of 7-aminomeclonazepam (MM1) included, besides the protonated molecule of m/z 300.0898, the characteristic m/z of 282.0793, which is most likely due to elimination of H 2 O under contraction of the diazepine ring, the m/z of 255.0684, and the m/z 236.1182. The m/z signal of 255.0684 is presumably due to loss of CH 2 NO and m/z of 236.1182 due to loss of Cl and CH 2 O, from the [M + H]+ ion. Elimination of Cl was also reported for other benzodiazepines such as diazepam [36]. A neutral loss of ammonia (NH 3 ) from the fragment ion with m/z 236.1182 results in the formation of the fragment ion of m/z 219.0917 and further degradation of the dihydro quinazoline ring in the fragment ion with m/z 236.1182 led to the fragment ion of m/z 195.0917. Another pathway, elimination of phenyl and opening of the dihydro quinazoline ring, led to the fragment ion of m/z 135.0917. The fragments observed in the mass spectrum for 7-acetaminomeclonazepam (MM2) corresponded closely to those seen for 7-aminomeclonazepam and were formed after neutral loss of CH 2 O, as described for 7-acetaminoclonazolam. In contrast, the formation of a fragment ion after neutral loss of CH 2 O from the protonated molecule was not observed, but a fragment ion of m/z 297.0789 was observed after neutral loss of CH 3 NO, probably as formamide. Further elimination of CH 2 O led to the fragment ion of m/z 255.0684.