A novel approach to evaluate the extent and the effect of cross-contribution to the intensity of ions designating the analyte and the internal standard in quantitative GC-MS analysis

Bud-Gen Chen 1 6 Chiung Dan Chang 1 4 Chia-Ting Wang 1 5 Yi-Jun Chen 1 2 Wei-Tun Chang 1 3 Sheng-Meng Wang 0 1 Ray H. Liu 1 6 0 Department of Forensic Sciences, Central Police University , Taoyuan, Taiwan 1 Address reprint requests to Professor Ray H. Liu, Department of Medical Technology, Fooyin University , 151 Ching-Hsueh Road, Ta-Liao Hsiang, Kaohsiung Hsien 831-02, Taiwan 2 Department of Pathology, Chang Bing Show Chwan Memorial Hospital , Changhua, Taiwan 3 Department of Criminal Investigation, Central Police University , Taoyuan, Taiwan 4 Department of Laboratory, Yang Ming Hospital , Chiayi, Taiwan 5 Department of Laboratory, Ben Tang Cheng Ching Hospital , Taichung, Taiwan 6 Department of Medical Technology, Fooyin University , Kaohsiung, Taiwan In gas chromatography-mass spectrometry methods of analysis adopting the analyte's isotopic analog as the internal standard (IS), the cross-contribution (CC) phenomenon contribution of IS to the intensities of the ions designating the analyte, and vice versa has been demonstrated to affect the quantitation data. A novel approach based on the deviations of the empirically observed concentrations of a set of standards was developed to assess the accuracy of the empirically derived CC data. This approach demonstrated that normalization of ion intensities derived from the analyte and the IS generates reliable CC data. It further demonstrated that an ion-pair (designating the analyte and the IS) with 5% or higher CC will result in a very limited linear calibration range. (J Am Soc Mass Spectrom 2008, 19, 598 - 608) 2008 American Society for Mass Spectrometry IFederal Workplace Drug Testing Program, mandatn 1984, guidelines were established for the U.S. ing (1) specific cutoff concentrations as positive/ negative criteria, and (2) certain concentration-related quality control and method validation requirements [1]. Accurate quantitation of drugs/metabolites in biological specimens has since, in addition to being a scientific pursuit, evolved into a legal issue. Selected ion monitoring (SIM) has long been established as the most effective approach for data collection where gas chromatography-mass spectrometry (GCMS) is used for the quantitation of various categories of analytes. Among various calibration approaches applied to SIM GC-MS protocols, internal standard (IS) method using isotopically-labeled analog (ILA) of the analyte as the IS has been well studied [2-7] and now widely adopted in forensic, clinical, and environmental laboratories. With ILA as the IS, one area of concern is the ion intensity cross-contribution (CC) between the analyte and the IS. Cross-contribution is defined as the contribution of the IS to the intensities of the ions designating the - analyte and vice versa. Since the measured ion intensities are used for the quantitation of the analyte, adopting an ion-pair with significant CC to designate the analyte and the IS will generate inaccurate analyte concentrations. For example, when the contribution of the IS to the intensity of the ion designating the analyte is more significant, the observed apparent analyte concentration will be higher than its true value. This error will become more significant as the analytes concentration is lowered. On the other hand, the observed apparent analyte concentration will be lower than its true value when the analytes contribution to the intensity of the ion designating the IS is more significant. Similarly, this error will become more significant as the analytes concentration is increased. Theoretical considerations [4] and approaches involving high-resolution ion monitoring [5] and computer programming for deconvoluting mass spectral peak abundance [6, 7] have been reported. The need to address this phenomenon in real world data was also highlighted by the inclusion of a section entitled, Corrections for Contamination and Isotope Spillover, in a 2006 book by Duncan et al. [8]. In their book, the authors illustrated a nonlinear relationship (Figure 8.4) between the monitored response and the analyte concentration, and further demonstrated (Figure 8.3) that a linear relationship can be expected by removing the portion of the intensity of the ions designating the analyte that was cross-contributed by (spillover from) the IS (and vice verse). Our interest in this area includes empirical measurements of the CC data [9 11], characterization of the effect of CC on the calibration curve [12], and the generation of favorable ion-pairs for designating the analyte and the IS, mainly through various chemical derivatization (CD) routes [13]. The CC phenomenon has long been recognized and, as mentioned above, many correction approaches have been reported. However, to the best of our knowledge, assessing the accuracy (trueness) of the empirically determined CC data, which could have been affected by systematic and random errors, has not been addressed. This study develops a novel approach to evaluate empirically-derived CC values, advancing current knowledge in this important analytical parameter. Standards and Reagents The following analytes and deuterated ISs (in 1 or 0.1 mg/mL methanol solution) were purchased from Cerilliant Corp., Austin, TX: 3,4-methylenedioxyamphetamine (MDA), hydromorphone (HM), MDA-d5, and hydromorphone-d6 (HM-d6). Derivatization reagents, N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide (with 1% t-butyldimethyl-chlorosilane) and N,O-bis(trimethylsilyl)trifluoroacetamide with (1% ttrimethylchlorosilane), were purchased from Pierce Chemical Co., Rockford, IL. All other common chemicals and solvents were of HPLC grade. Sample Preparation and Derivatization Procedure For full-scan and SIM data collection, the analytes (MDA and HM) and the ISs (MDA-d5 and HM-d6) solutions were prepared individually. For example, for the run including only MDA, 5 L of the MDA standard (1 mg/mL in methanol) was transferred into a 16 100-mm glass tube. For the run including only MDA-d5, 50 L of the MDA-d5 standard (0.1 mg/mL methanol solution) was used. Thus, an equal amount of the MDA and MDA-d5 was used in these two parallel experiments. The procedures described below were then followed to form the t-butyldimethylsilyl (t-BDMS) or the trimethylsilyl (TMS) derivatives of the analytes and the ISs. The 16 100-mm glass tube containing the analyte or the IS as prepared in the last paragraph was evaporated to dryness under a stream of nitrogen at 50 C. To the dried residue was added 50 L acetonitrile and 50 L of the selected derivatization reagent; the tube was capped, mixed, and incubated for 20 min at 90 C in a heating block [9]. The mixture was cooled for GC-MS analysis. The structures of the derivatized analytes and ISs are shown in Figure 1 along with their mass spectra. Instrumentation, Analytical Parameters, and Data Collection Procedure GC-MS analysis was performed on an Agilent 6890 GC interfaced to an Agilent 5975 MSD (Agilent, Palo Alto, CA). A 12-m H (...truncated)