Developing methods for the analysis of drugs of abuse has become a priority for forensic toxicology and law enforcement. The large variety of illicit drugs and new and so-called “designer drugs” has made method development for these compounds a significant undertaking. GC/MS/MS using the isotope dilution technique, which uses isotopically labeled analogs of target compounds as internal standards, is widely used for precise quantitation in drug assays. However, in many cases, when using deuterium-labeled analogs, the mass spectra differ only slightly from the corresponding unlabeled compounds. This becomes complicated when the native and labeled compounds completely or partially co-elute, as they often do, and the spectra overlap.
Operation of the triple quadrupole GC/MS/MS in the multiple reaction monitoring (MRM) mode provides exceptional sensitivity, selectivity and specificity for the detection and quantitation of targeted drugs in the presence of background interferences. The specificity of unique MRM transitions for close-eluting native and labeled analogues, combined with the sensitivity of triple quadrupole MRM transitions, is a powerful technique for unambiguous, quantitative determination of this important compound class.
This article discusses instrument configuration, operating parameters and analytical results for the analysis of a common narcotic, hydrocodone, using isotope dilution paired with the specificity of the MRM analysis mode of a triple quadrupole GC/MS/MS. Internal standard calibration of codeine and oxycodone was also included in the study.
Experimental
Figure 1 – GCMS-TQ8030 triple quadrupole GC/MS/MS.Analyses were conducted using a GCMS-TQ8030 triple quadrupole GC/MS/MS (Shimadzu Scientific Instruments, Columbia, Md.) (Figure 1) operated in MRM mode with optimized collision energy (CE) for each MRM transition providing high sensitivity. Instrument operating conditions are shown in Table 1.
Six MRM transitions were selected for both hydrocodone-d3 and hydrocodone, most of which had unique precursor ions paired with common product ions (Table 2). This approach allowed evaluation of any potential mass spectral interference, or cross-talk, between the transition pairs of the two coeluting compounds. Three transitions were selected for codeine and oxycodone since they were chromatographically resolved from the other compounds, and there were no isotopically labeled internal standards used.
Table 1 – Instrument operating conditions for analysis of opioid drugs
Table 2 – MRM transition details with optimized collision energies (CE)
Calibration standards were prepared in methanol, and data for a five-point calibration were acquired over the range of 25–200 ng/mL (parts-per-billion, ppb). Isotope dilution was used to generate the calibration curve for hydrocodone. Concentration of the internal standard, hydrocodone-d3, was held constant at 100 ng/mL. The concentration range of the calibration was sufficient to satisfy the requirements of the specific application. The chromatographic conditions chosen were intended to fit into a larger scheme for analysis of numerous drug classes; thus optimization of the chromatographic conditions for efficiency was not considered in this study.
Results and discussion
Chromatography
The total ion chromatogram (TIC) acquired in the MRM mode for the opioid drug mix is shown in Figure 2. The chromatographic peaks for hydrocodone-d3 and hydrocodone partially overlap, with the deuterium-labeled analog eluting first. In the MRM mode, the TIC is the sum of the signal for each MRM transition for that particular analyte, so the appearance of the chromatogram is slightly different than the typical TIC chromatogram from full scan analysis.
Figure 2 – Total ion chromatogram acquired in MRM mode for the opioid drug mix.Mass spectral results in full scan (Q3 scan) mode
The full scan mass spectra of hydrocodone-d3 and hydrocodone are shown in Figure 3a and b. Notable features of these mass spectra are the prominent molecular ions for the labeled and unlabeled compounds at m/z 302 and 299, respectively, with the difference of 3 m/z units associated with the isotopically labeled n-methyl group on hydrocodone-d3.
Figure 3 – Full scan mass spectra of hydrocodone-d3 and hydrocodone.Common fragment ions are present in both spectra at m/z 242, 214, 199, 185 and 115 (indicated with a vertical arrow [↓] in Figure 3a and b). These fragments represent loss of a fragment, which includes the labeled n-methyl group from hydrocodone-d3 and the corresponding unlabeled n-methyl group from hydrocodone, to form identical fragment ions from the two compounds.
Fragment ion pairs in the spectra for the labeled/unlabeled compounds can be seen at m/z 287 and 284, 273 and 270, 231 and 228, 99 and 96, and 62 and 59 (indicated with an asterisk [*] in Figure 3a and b). In this case, the corresponding fragments are offset by a difference of 3 m/z units (i.e., 287 and 284) and represent the loss of the same nonlabeled group from hydrocodone-d3 and hydrocodone, respectively.
The full scan spectra of hydrocodone-d3 and hydrocodone were used to select precursor and product ions for the MRM transitions. Three transitions were selected for each compound based on their unique molecular ions and common product ions (i.e., 302 → 242 and 299 → 242). To illustrate the unique specificity of the MRM mode, a second set of three transitions was defined using the molecular ions and unique product ions for hydrocodone-d3 and hydrocodone (i.e., 302 → 273 and 299 → 270). The ions selected for MRM transitions are tabulated in Table 2.
Mass spectral results in MRM mode
Operation of the GCMS-TQ8030 in the MRM mode provides enhanced selectivity for analysis of trace-level contaminants in complex matrices, such as drugs of abuse in biological fluids, because the co-extracted matrix interferences are significantly minimized. The specificity that can be achieved by using unique MRM transitions for each compound, even when they have common product ions, is illustrated in Figure 4. Six overlaid MRM chromatograms for hydrocodone-d3 and six for hydrocodone are shown. Note that the chromatograms corresponding to the MRM transitions for hydrocodone-d3 and hydrocodone are uniquely defined for each of the analytes and do not interfere with one another, even for the three transitions that have common MRM product ions. The non-interfering chromatograms illustrate the power of the MRM mode and the specificity that can be achieved when unique transitions are selected for close-eluting compounds with similar mass spectra.
Figure 4 – MRM chromatograms for hydrocodone-d3 and hydrocodone.Cross-talk
Cross-talk, a phenomenon unique to triple quadrupole mass spectrometry, occurs when residual ion fragments are not fully swept from the collision cell at the end of a cycle and are detected as “ghost fragments” in subsequent transitions. The slowing of product ions in the collision cell results from interactions with the collision-induced dissociation (CID) gas. In some cases, a small portion of the residual product ions that have slowed down may not be completely swept from the collision cell during the transition, resulting in cross-talk, which then can appear as “ghost” mass spectral fragment peaks in subsequent transitions.
Cross-talk is virtually eliminated in the GCMS-TQ8030 using UFsweeper technology, which alters the pseudo-potential surface within the collision cell, shortening the path on which ions must travel and accelerating them through the cell and toward Q3. This process completely clears the collision cell with each transition and eliminates cross-talk from one transition to the next. The overlaid chromatograms in Figure 4 clearly show that there was no indication of cross-talk.
Calibration results
Five calibration standards were prepared for the opioids over the 25-500 ng/mL (ppb) range and were transferred to autosampler vials with limited-volume inserts for analysis; hydrocodone-d3 was used as the internal standard and was held at a constant concentration of 100 ng/mL. The calibration standards were analyzed using the instrument conditions outlined above. The electron multiplier was adjusted to give acceptable response at the lowest calibration level and avoid saturation at the highest calibration level.
Response factors were calculated and percent relative standard deviation (%RSD) was determined using GCMSsolution software (Shimadzu Scientific Instruments). Calibration precision was evaluated using the %RSD of the response factors and the correlation coefficient (r) for each of the calibration analytes. The %RSD and correlation coefficient values for the multipoint calibration are shown in Table 3. The linear, multipoint calibration curve for hydrocodone is illustrated in Figure 5. Calibration results demonstrate linearity for each analyte.
Table 3 – Result of the five-point calibration for three opioids from 25 to 200 ng/mL using MRM analysis mode
Figure 5 – Linear, multipoint calibration curve for hydrocodone.Conclusion
The above results demonstrate the power and specificity of the MRM analysis mode when using unique transitions for close-eluting compounds such as hydrocodone-d3 and hydrocodone, even when they have similar mass spectra and common product ions. The GCMS-TQ8030 fast scanning and UFsweeper technologies completely cleared the collision cell with each transition and eliminated cross-talk. There was no interference from cross-talk or the close-eluting deuterium-labeled internal standard, as evidenced by the fact that multipoint calibration for hydrocodone was linear and passed through zero.
Richard Whitney is GC/MS product specialist, and Laura Chambers is GC/MS product manager, Shimadzu Scientific Instruments, 7102 Riverwood Dr., Columbia, Md. 21046, U.S.A.; tel.: 410-381-1227; e-mail: [email protected]; www.ssi.shimadzu.com