A QTRAP LC/ESI/MS/MS system can be used to detect and screen for many drugs of abuse (Table 2) that might be found in postmorten whole blood samples (43). One approach to drug screening is to search for an extended but finite group of drugs by monitoring reactions characteristic of each drug in the multiple reaction monitoring mode of a QTRAP LC/MS/MS system operated as a triple quadrupole. A partial list of such reactions for specific compounds and the instrumental parameters used to monitor them is provided in Table 2 (the complete list employed in (43) included 100 drugs). For each drug, the m/z value of the [M + H]+ ion and of a characteristic fragment ion produced upon CID are tabulated. Other tabulated parameters include the collision energy (CE) at which CID was performed, the dwell time during which the reaction was monitored, the relative retention time (RRT) on liquid chromatography compared to an internal standard, and the limit of detection (LOD).
The system was operated in an information-dependent acquisition (IDA) mode (another manufacturer uses the term data-dependent scanning to describe a similar experiment). That means that in response to certain specified data acquired in real-time, the instrument will alter the way and/or the type of information it gathers. In this case, if sufficient ion current for a target transition is observed in multiple reaction monitoring (MRM) mode while the instrument is operated as a triple quadrupole, the data system causes the instrument to obtain an enhanced product ion (EPI) spectrum for the precursor (parent) ion in the transition. This involves isolating that precursor (parent) ion in Q1, accelerating it into the collision cell (Q2) to undergo CAD, and then trapping the product ions in Q3, which is now operated as a linear ion trap rather than as a quadrupole mass filter. Once a sufficient number of product ions accumulate in the trap, it is operated in sequential m/z instability mode to “scan” the product ion population and obtain a mass spectrum. After the EPI spectrum is obtained, the system returns to MRM mode and operates as a triple quadrupole until the ion current for another target transition exceeds some threshold level to trigger the switch back to EPI data acquisition mode.
At the completion of the LC run and collection of all LC/MS data for a particular sample, an “extracted ion chromatogram” is constructed retrospectively that displays the ion current for a given transition as a function of LC retention time. In this way both the retention time of the compound relative to the internal standard (RRT) and the full EPI mass spectrum is compared to those of standards to achieve definitive identification of the drug in question.
The total ion current (TIC) from all MRMs in such an analysis of one sample is illustrated in Figure 36; the sample contained the internal standard (mepivacaine) and the drugs benzoylecgonine (BEG), cocaine, cocaethylne, and trazodone. Figure 37 illustrates the information-dependent acquisition (IDA) approach to drug identification. The left panel is the TIC of all MRM. Among the MRM detected was that forBEG, and the center panel illustrates the extracted ion chromatogram for that transition, which is compatible with the RRT expected for BEG. The right panel is the enhanced product ion (EPI) obtained for the precursor (parent) ion of the observed reaction, and it is consistent with the full mass spectrum of BEG.
Another approach to general unknown drug screening using a QTRAP LC/MS/MS system involves simply obtaining survey mass spectra rather than monitoring a specific set of reactions (44). Here, the survey scans are obtained in enhanced mass spectrum (EMS) mode, and this involves operating Q1 and Q2 as Rf-only transmission devices over a wide range of m/z values, e.g., 100-1100, that include the [M + H]+ ions of most conventional heterocyclic drugs. Ions accumulate in the trap until it contains a sufficient number to produce a high quality mass spectrum (rather than obtained “on-the-fly” as in a triple quadrupole, for example).
Once the trap accumulates an adequate number of ions, it is operated in sequential mass instability mode to scan the m/z values of the trapped ions. This triggers an IDA sequence in which the three most intense ions in each enhanced mass spectrum (EMS) are sequentially selected in Q1, subjected to CID in Q2, and then trapped in Q3 until a sufficient number of ions accumulate. Then an enhanced product ion (EPI) scan is obtained, and it is then compared to a library (in this case of over 1000 drugs) to determine if any spectra are “matched” to a sufficient degree to achieve drug identification. In addition, an extracted ion chromatogram can be constructed retrospectively for the [M + H]+ ion observed in the enhanced mass spectrum (EMS) survey scan so that the RRT of the compound in question can be compared to that of standards in the library. This sequence is illustrated in Figure 38, which is an LC/MS/MS analysis of a mixture of antipsychotic drugs. Panel A is the TIC of the enhanced mass spectrum (EMS) survey scans. Panel B is the enhanced mass spectrum (EMS) at retention time 7.3 min (indicated by the arrow in Panel A), and it contains an ion at m/z 370.1, consistent with the [M + H]+ ion of the drug amisulpride. Panel C is the extracted ion chromatograph for m/z 370.1 and indicates a RRT time consistent with standard amisulpride. Panel D is the enhanced product ion (EPI) scan of m/z 370.1 and is consistent with the CID spectrum of amisulpride.
This group compared the ability of this procedure to identify drugs in 36 clinical specimens (3 gastric contents, 6 serum, 8 whole blood, and 19 urine samples) to GC/MS and HPLC with diode array detection (DAD) procedures (Figure 39). Among all 3 methods, there were 130 positive results that identified 89 different compounds. LC/MS/MS identified 93.8% of all positive results, and 19.1% were identified only by LC/MS/MS. GC/MS identified 63.8% of all positives, and 1.5% were identified only by GC/MS. HPLC/DAD identified 54.6% of all positives, and 1.5% were identified only by HPLC/DAD. All 3 procedures identified 34.6% of positives. LC/MS/MS and GC/MS (but not HPLC/DAD) identified 24.6% of positives, and LC/MS/MS and HPLC/DAD (but not GC/MS) identified 15.4% of positives. The LC/MS/MS procedure thus performed better than did either GC/MS or HPLC/DAD or the combination of GC/MS and HPLC/DAD (44).
A Triple Stage Quadrupole (TSQ) LC/ESI/MS/MS system can also be used to detect and screen for drugs in forensic analyses of low concentrations of benzodiazepines and their metabolites in urine. Another approach is to conduct multiple reaction monitoring (MRM) for an extended but finite set of target drugs on a triple stage quadrupole and to monitor two reactions for each analyte (45). One reaction is used for quantitation relative to an internal standard, and the second is used as a qualifier reaction to determine whether the ratio of quantitator to qualifier reaction ion current falls within an acceptable ratio typical of the standard drug at the appropriate RRT. Table 3 provides a partial list of drugs examined in one study and it tabulates the two reactions monitored for each agent and specifies which reaction is quantitator and which qualifier (45). Figure 40 provides an illustration of an LC/MS/MS chromatogram for a sample that provides retention times and reactions monitored for the benzodiazepines identified. Figure 41 provides another example of LC/MS/MS on a TSQ with multiple reaction monitoring (MRM) for a single reaction for each target analyte used to screen for drugs of abuse in oral fluid. Here, [2Hx]-labeled analogs of tetrahydrocannabinol (THC), amphetamine, morphine, and other drugs were included to permit quantitation (46).
A Quadrupole/Time-of-Flight (QTOF) LC/ESI/MS/MS system can also be used for general unknown screening of drugs of toxicologic significance in blood. Information-dependent acquisition (IDA) approaches on a Micromass QTOF LC/MS/MS system have also been applied to general unknown drug screening. In this case, survey scans are performed by TOF, and when ion current exceeds a threshold, a switch to MS/MS is performed (47). Up to 3 of the most intense ions in the TOF spectrum that triggered the switch are selected sequentially in Q1 and subjected to CID in Q2. Product ions are then analyzed by TOF, and product ion spectra are compared to a library. This approach is illustrated in Figure 42. Panel A is the survey MS TIC. Panel B is the extracted ion chromatogram for [M + H]+ ion of methadone. Panels C and D are MS/MS scans of the CID spectra of the methadone [M + H]+ ion at two different collision energies, and Panel D contains the TIC for the 3 MS/MS channels.Accurate mass measurement by Fourier Transform MS or Time-of-Flight MS can be used to screen for drugs. Time-of-flight MS can achieve mass accuracy of 20-30 ppm, and Fourier Transform MS can achieve mass accuracy of 3-5 ppm. The latter is generally enough to assign the elemental composition of the ion, which greatly narrows the set of drugs that the ion might represent, sometimes to a single member. Isomers cannot be distinguished but might often exhibit similar pharmacologic properties. This is illustrated in Table 4; accurate mass measurements on urine drug screens were searched against a database of 7640 compounds (48).