‘N‐in‐one’ strategy for metabolite identification using a liquid chromatography/hybrid triple quadrupole linear ion trap instrument using multiple dependent product ion scans triggered with full mass scan

Abstract

This paper describes a new strategy that utilizes the fast trap mode scan of the hybrid triple quadrupole linear ion trap (QqQ(LIT)) for the identification of drug metabolites. The strategy uses information-dependent acquisition (IDA) where the enhanced mass scan (EMS), the trap mode full scan, was used as the survey scan to trigger multiple dependent enhanced product ion scans (EPI), the trap mode product ion scans. The single data file collected with this approach not only includes full scan data (the survey), but also product ion spectra rich in structural information. By extracting characteristic product ions from the dependent EPI chromatograms, we can provide nearly complete information for in vitro metabolites that otherwise would have to be obtained by multiple precursor ion scan (prec) and constant neutral loss (NL) analysis. This approach effectively overcomes the disadvantages of traditional prec and NL scans, namely the slow quadrupole scan speed, and possible mass shift. Using nefazodone (NEF) as the model compound, we demonstrated the effectiveness of this strategy by identifying 22 phase I metabolites in a single liquid chromatography/tandem mass spectrometry (LC/MS/MS) run. In addition to the metabolites reported previously in the literature, seven new metabolites were identified and their chemical structures are proposed. The oxidative dechlorination biotransformation was also discovered which was not reported in previous literature for NEF. The strategy was further evaluated and worked well for the fast discovery setting when a ballistic gradient elution was used, as well as for a simulated in vivo setting when the incubated sample (phase I metabolites) was spiked to control human plasma extract and control human urine.

General unknown screening procedure for the characterization of human drug metabolites: Application to loratadine phase I metabolism

“…Only one study has demonstrated so far, using a similar approach to that described herein, that the combination of the EMS survey and EPI-dependent scan modes could be applied to drug metabolite profiling [11]. Both this and the present methods took advantage of the so-called , dynamic background subtraction script' of Analyst software to improve the S/N and peak resolution and guarantee the detection of minor metabolites.…”

“…In contrast to standard protein precipitation methods, it is suitable for both in vivo and in vitro biological samples with minor changes in the sample working procedure. It probably represents an important advantage as compared to the procedure used by Li et al [11], which consisted in protein precipitation followed by evaporation and reconstitution of the dried extracts in the mobile phase. As pointed out by the authors themselves, such a procedure may result in concentrating the interfering compounds (especially from complex matrices) and thus in an increased background noise which may prevent minor metabolites to trigger dependent scans.…”

“…However, the detection of unexpected or secondary metabolites is not possible with such , targeted' analysis. Only one recent publication demonstrates that DDA combining the Q3 and the product ion scan modes could represent a powerful approach in drug metabolites profiling [11]. Their so-called , N in one' strategy is very similar to the general unknown screening (GUS) procedure we formerly published for the detection and identification of a priori unexpected compounds in biological samples [14].…”

Determination of species-difference in microsomal metabolism of amitriptyline using a predictive MRM–IDA–EPI method

“…To evaluate species differences in the metabolite profile of AMI, metabolite identification was performed on the same samples used for the metabolic stability test by LC-QTRAP-MS operated in the information-dependent acquisition (IDA) mode [21,22]. Simultaneous collection of MS/MS spectra for identification, as well as semi-quantitative analysis of metabolites of AMI in liver microsomes of the four species, were achieved using predicted MRM (pMRM) transitions to cover the most common phase I and II as survey scans; the MRM transitions selected by the IDA criteria were then used to trigger the corresponding EPI scan.…”

General unknown screening procedure for the characterization of human drug metabolites in forensic toxicology: Applications and constraints

“…Ming et al [11] recently combined multiple ion monitoring (MIM), MRM, enhanced mass scan (EMS) and EPI in the study of ritonavir metabolism. Li et al [12] also published a so-called "N in one" strategy for metabolite identification using a combination of EMS as survey scan and EPI as dependent scan. This strategy, similar to the General Unknown Screening procedure we formerly reported [13], was used for nefazodone, allowing the detection and characterization of unexpected metabolites.…”