Articles

Nine important 1,4-benzodiazepines and zolpidem were characterized by liquid chromatography-mass spectrometry using a multimode ionization source able to generate ions using both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), and a single quadrupole mass analyzer. An optimum chromatographic separation was applied for all target compounds in less than 8 minutes using a Zorbax Eclipse Plus column (100 x 4.6 mm, 3.5 μm) kept at 35°C and a 0.3% HCOOH/ ACN/IPA (61:34:5) mobile phase pumped at 1 ml/min. Optimization of LC-MS method generated low limit of quantitation (LOQ) values situated in the range 0.3–20.5 ng/ml. Comparison between differences in method sensitivity, under specified chromatographic conditions, when using ESI-only, APCI-only, and simultaneous ESI-APCI ionization with such a multimode source was discussed. Mixed ESI-APCI(+) mode proved to be the most sensitive ionization generating an average 35% detector response increase compared to ESI-only ionization and 350% detector response increase with respect to APCI-only ionization. Characterization of the nine benzodiazepines and zolpidem concerning their MS fragmentation pathway following ‘in-source’ collision-induced dissociation is discussed in detail and some general trends regarding these fragmentations are set.

Non-linear van’t Hoff plots were observed for several benzodiazepines over the usual temperature interval used in RP-LC (10–60 C), when acetonitrile was added in the mobile phase. Such deviation from linearity was not observed when methanol or isopropanol was added to the mobile phase. Polynomial regressions were applied to fit the experimental data when acetonitrile was used as organic additive in mobile phase. The second-order polynoms may allow finding out the maximum retention depending on temperature, which can be within or out of the normal temperature range used in RP mechanism. A thermodynamic model deriving from the partition of two or more tautomers of the same analyte was proposed to explain this deviation from linearity of van’t Hoff plots.