TY - JOUR
AU1 - McKenna, Josiah
AU2 - Jett, Rachel
AU3 - Shanks, Kevin
AU4 - Manicke, Nicholas E
AB - Abstract Immunoassays and high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) are both widely used methods for drug screening in toxicology. We investigated an alternative approach for rapid drug screening: paper spray MS (PS-MS). In paper spray, the biofluid sample is spotted onto a paper substrate. Upon application of a spray solvent and an electric potential, extraction and ionization occur directly from the paper without any need for additional sample preparation. We developed two paper spray high-resolution MS/MS targeted drug screening assays using a quadrupole-orbitrap mass spectrometer, one the positive ion mode and one in the negative ion mode. In the positive ion mode, over 130 drugs and drug metabolites were semi-quantitatively screened at sub-toxic concentrations in a single 2.5 min analysis. Limits of detection and calibration performances for each target compound are reported. The PS-MS/MS assay was tested on authentic postmortem specimens, and its screening ability and semi-quantitative performance were evaluated against independent LC–MS-MS screening and confirmation assays with good agreement. The paper spray MS/MS showed good qualitative agreement with LC–MS-MS; the true positive rate of paper spray MS/MS was 92%, and the true negative rate was over 98%. The quantitative results between the two methods were also acceptable for a screening application; Passing-Bablok regression yielded a slope of 1.17 and a Pearson’s correlation coefficient of 0.996. A separate PS-MS/MS negative ion screening method was also developed for a small panel of barbiturates and structural analogs, demonstrating its potential for acidic drug detection and screening. Introduction Forensic toxicology laboratories typically use a two-step process to detect toxicants in biological samples. The first step is screening, which employs a variety of analytical procedures to detect a broad range of targets. The best screening tests require little sample manipulation, cover a wide array of analytes, and are fast, inexpensive, sensitive and selective. Immunoassays (IAs), gas chromatography (GC), GC-mass spectrometry (MS) and high-performance liquid chromatography (HPLC) with spectroscopic or MS detection (1–4) are all commonly used methods for drug screening. Positive identifications during the initial drug screening step are confirmed and quantitated by an independent analytical procedure. LC–MS-MS is the most widely used method for drug confirmation and quantitation due to its excellent sensitivity and selectivity. According to the guidelines published by the Society of Forensic Toxicologists in conjunction with the American Academy of Forensic Science, a confirmatory test should be more specific than the screening test and based upon a different chemical principle (5). Immunoassays are widely used for drug screening of toxicological samples on whole blood, plasma/serum and urine specimens because they are inexpensive, simple, fast and automatable. Several types of IAs are utilized in casework, including fluorescence polarization immunoassays (FPIAs), radioimmunoassays (RIAs), enzymemultiplied immunoassay technique (EMIT), kinetic interaction of microparticles in solution (KIMS) and enzymelinked immunosorbent assays (ELISAs) (6). While IAs are a suitable screening tool in some scenarios, their limitations for drug screening are well-known, including inadequate sensitivity and selectivity, which can result in false negatives and unacceptably high false positive rates (7). In addition, the number of drugs that can be screened by IA is often insufficient even in multiplexed systems. As a result, IA screening alone is not acceptable for some applications including postmortem toxicology (8). GC-MS is widely used as a confirmatory method, and it has also been used in drug screening (9). GC gives good chromatographic resolution, and when coupled to mass spectrometry the specificity is excellent. The reproducible fragmentation arising from electron ionization (EI) facilitates the use of database searching for peak identification. A significant limitation of GC-MS is that most drugs and drug metabolites cannot be analyzed with adequate sensitivity by GC methods unless the targets are first derivatized (10). Analysis of blood and plasma samples therefore involves extensive sample cleanup and may require multiple derivatization reagents in order to obtain adequate detection limits and analyte coverage (9). HPLC-MS has good sensitivity and specificity, especially when high-resolution or tandem mass spectrometry is used. It is also able to screen for a broad spectrum of analytes and is not limited to volatile or thermally stable compounds like GC-MS. Because of this, HPLC-MS is rapidly gaining prominence in toxicology laboratories for confirmatory testing. As a screening method, however, HPLC-MS is still not ideal. While it is an improvement on GC-MS, HPLC-MS still requires sample preparation. HPLC systems also have a reputation for being less robust than GC systems. Leaks, fluctuating back-pressure, shifting retention times, column deterioration, dead volumes and carry-over are all technical issues that have persisted despite HPLC being a fairly mature technology. The instrument costs, the expertise required to maintain and operate the system, and the required sample preparation/handling all point to a need for an alternative approach for mass spectrometry-based drug screening. To simplify MS analyses, ambient or direction ionization methods have been developed. Beginning with DESI (desorption electrospray ionization) (11) and DART (direct analysis in real time) (12), these methods seek to analyze complex samples directly without sample preparation. This research area has been very active, and numerous techniques and applications have been reported in the literature. Paper spray mass spectrometry, first described in 2010, is one such method for performing rapid, direct analyses of complex samples spotted on paper (13, 14). Paper spray MS/MS analysis of drugs at low or sub-ng/mL levels with quantitative accuracy and precision has been reported in numerous studies (13, 15–35). In addition to quantitative analysis, some drug screening work has also been reported (36, 37). PS-MS has been widely investigated since its inception. Aside from analysis of drugs in biofluids, other applications have included food and agrichemical analysis (38–42), forensic applications (43–46), analysis of acylcarnitines (47, 48), chemical warfare agents (49), natural products (50–52), paper-based immunoassays (53, 54) and identification of bacteria (55, 56). Alternative spray substrates (15, 57–61) and cartridges with built-in sample preparation (18, 21) have also been investigated. Paper spray is performed by first spotting blood or other biofluid specimens are onto paper and allowing them to dry before analysis, although analysis of freshly deposited blood samples has also been demonstrated (30). A small volume of solvent (20–100 μL depending on the size of the paper substrate) is applied to the paper where it wicks through the substrate and sample by capillary action. The paper is cut to a sharp tip, which is positioned a few millimeters away from the atmospheric pressure inlet of a mass spectrometer. A high voltage (3–5 kV) is then applied directly to the paper, inducing an electrospray at the paper tip. The solvent evaporates from the charged droplets generated by the electrospray process, leaving gas-phase ions of the analyte molecules which can then be detected by a mass spectrometer (13). Chemicals which are both soluble in the extraction/spray solvent and ionizable—typically by protonation or cationization with sodium or ammonium—will be detected immediately by the mass spectrometer. The entire analysis takes about 60 s and does not require any offline sample preparation. Because of its speed, simplicity and low sample consumption, paper spray shows good promise for drug screening applications. Here, we report on the development of a semi-quantitative paper spray high-resolution MS/MS screening method for 137 drug and drug metabolites that are commonly encountered in routine toxicology casework. The entire drug panel can be screened in a single 2.5 min analysis, with cut-offs ranging from 1 ng/mL to over 1 μg/mL depending on the target. The screening and quantitative performance of the paper spray MS/MS assay was compared against independent toxicology laboratory results for 30 postmortem blood specimens. Furthermore, a separate negative ion method was also developed to screen for acidic compounds such as barbiturates. Materials and Methods Chemical materials Glacial acetic acid, Optima-grade ammonium hydroxide (NH4OH), and HPLC-grade methanol, acetonitrile, isopropanol and acetone were purchased from Fisher Scientific (Pittsburgh, PA, USA). Anhydrous carbon tetrachloride (CCl4) was purchased from Sigma Aldrich (St. Louis, MO, USA). Single donor human whole blood for calibrator preparation was obtained from Innovative Research (Novi, MI, USA) in K2EDTA tubes. Postmortem (PM) blood samples were obtained from banked material at Axis Forensic Toxicology (Indianapolis, IN, USA). Most of the 144 drugs and 12 stable isotope labeled internal standards (SIL ISTDs) used in the drug screens were purchased as analytical standards from Cerilliant (Round Rock, TX, USA) at concentrations of either 1.0 mg/mL or 100 μg/mL in methanol. Secobarbital and thiopental were purchased from Sigma Aldrich as 1.0 mg/mL standards. Acetaminophen, amlodipine, aripiprazole, benztropine, bupivacaine, carbamazepine, donepezil, etomidate, fluvoxamine, hydroxyzine, labetalol, metaxalone, methocarbamol, metoclopramide, papaverine and ropinirole were purchased as powders from Sigma Aldrich and dissolved in 95:5 methanol:water to create standard solutions. Calibration standards Five-point calibration curves for each analyte were prepared in drug-free human blood. The 137 analytes in the positive ion screen were divided up into separate groups of about 15 to maintain low organic content in the blood samples. Structural isomers were places in separate groups prevent structural isomers from interfering in calibration standards. Calibration curves extended to 50 times higher than the cut-off concentration for most analytes (see Table II for cut-off concentrations). The negative ion drug screen calibration standards were also prepared in blood, dividing up the analytes into two groups to maintain low organic content and separate the structural isomers amobarbital and pentobarbital. The concentrations of the analytes in the lowest calibrator were: 500 ng/mL for butabarbital, butalbital, amobarbital, pentobarbital and secobarbital; 1,000 ng/mL for phenobarbital and phenytoin; and 2,000 ng/mL for thiopental. A five-point calibration series was used for each, spanning up to 15 times these cut-off concentrations. Sample preparation All positive ion calibration standards and PM samples were mixed 1:3 (v:v) with an aqueous ISTD solution before spotting onto the paper spray cartridge. The relatively large volume of ISTD solution was used to dilute the blood because acetonitrile-based spray solvents did not penetrate dried undiluted blood quickly enough. The concentrations of each compound in the ISTD solution were: 65 ng/mL of alprazolam-d5 (A); 650 ng/mL of benzoylecgonine-d8 (B), cocaine-d3 (C) and methamphetamine-d11 (I); 260 ng/mL of flunitrazepam-d7 (D), hydrocodone-d3 (F) and trimipramine-d3 (J); 1,300 ng/mL of gabapentin-d10 (E); 2,600 ng/mL of metaxalone-d6 (G); 325 ng/mL of methadone-d3 (H); and 130 ng/mL of zolpidem-d6 (K). No analyte from any of the stable isotope labeled ISTDs was observed. For ease of reporting, the ISTDs—in alphabetical order—are given labels A-K. The internal standards were assigned to the analytes by hierarchical clustering analysis on logP and pKa, which were obtained from drugbank.ca. Alternative ISTDs were manually tested for targets in which poor calibration performance was obtained with the initial ISTD. For the negative ion screen, 5 μL of an 8 μg/mL ISTD solution of phenobarbital-d5 was spiked into 100 μL of each sample. Phenobarbital-d5 was used as the internal standard for each target. Paper spray ionization Paper spray was performed using Velox sample cartridges on the automated Velox 360 source from Prosolia, Inc. (Indianapolis, IN, USA). About 12 μL of the blood-ISTD mixture were spotted onto the paper contained within the cartridges and allowed to dry at room temperature for at least 90 min before analysis. The drying process can be accelerated if desired by heating (62) or blowing air over the cartridges. The extraction/spray solvent used was 85:10:5:0.01 acetonitrile:acetone:water:acetic acid for the larger positive ion screen. Acetonitrile was selected as the primary organic phase because it showed lower matrix effects for the PM samples, in agreement with earlier work (63). However, acetonitrile-based solvents were problematic because they did not quickly permeate and fully wet dried blood spots (DBSs), which completely stymies the spraying process. Acetone was added as a co-solvent in conjunction with dilution using the ISTD solution to ensure faster solvent penetration of the DBS. In total, 136 μL of solvent were gradually applied to each cartridge by the automated paper spray source; 6 μL were deposited directly onto the DBS followed by 130 μL into the rear solvent well of the cartridge. The method for negative ion mode detection of acidic compounds was adapted from recent work with organophosphonic acids (49). The spray solvent was 90:10:0.01 methanol:CCl4:NH4OH. Carbon tetrachloride was included to decrease corona discharge. In total, 142 μL of this solvent were added to the paper spray cartridge—12 μL first onto the DBS followed by 130 μL into the rear solvent well. Mass spectrometry All data were acquired on a Q-Exactive Focus orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) with the S-lens set to 75 and capillary temperature set to 320°C for both positive and negative ion mode. The instrument method for the positive ion drug screen was 2.43 min long, operating in positive ion mode at +5.0 kV for the first 1.6 min before turning the voltage off to 0 kV for the next 0.83 min. The voltage was turned off for 0.83 min at the end of the run to generate zero-intensity scans for each drug, which were required for automatic integration in Thermo’s TraceFinder. In the final 0.2 min, the instrument was switched to negative ionization at −4.0 kV to eliminate any charge buildup. The mass spectrometer was operated in MS/MS mode using an inclusion list with an isolation width of 1.0 m/z in the first quadrupole to filter precursor ions. A maximum injection time of 50 ms was used. In the cases where the precursor ions of two or more targets had similar or identical precursor m/z and the optimal collision energy were similar, a single scan event was used for both targets. The precursor ions, fragment ions and optimized collision energies (CEs) for all 137 drugs and the 11 SIL ISTDs in the positive ion screen can be found in Supplementary Table I. For nearly all of the analytes, the intact [M + H]+ ion predominated and was used as the precursor ion. The [M + Na]+ ion was used for carisoprodol and topiramate. Norpropoxyphene, norsertraline and propoxyphene all showed intense fragment ions in the full scan MS. Since these in-source fragment ions showed higher signal than the intact molecular ion, they were used as the precursor ions for MS/MS. The inclusion list for the smaller negative ion screen can be found in Supplementary Table II; each of the analytes generated [M−H]− precursor ions. Since this screen was significantly smaller than the positive ion screen in terms of the number of targets, a larger maximum injection time of 500 ms was used. The spray voltage was set to −4.0 kV. The lower spray voltage was used to minimize the risk of corona discharge. The voltage was left on for the first 1.1 min of the 1.4-min instrument method and was set to 0 kV for the final 0.3 min to acquire the zero-scans necessary for automated integration of the data. Data processing All data were automatically processed using TraceFinder v. 3.3 (Thermo Fisher Scientific). Peak detection was restricted to a 5 ppm m/z window around the target compound’s fragment ion. The area under the curve (AUC) of the analyte fragment ion over the entire analysis time was determined by automatic integration. The AUC of the analyte fragment ion was then divided by the AUC of the ISTD fragment ion. Each positive ion calibration point was run in duplicate, and the negative ion calibration points were run in triplicate. The ratios of analyte signal to ISTD signal were plotted against their known concentrations to generate the calibration curve, which was linearly fit using 1/x weighted least squares. The signal-to-blank ratio (S:B) for each target compound at the cut-off was determined to provide a rough estimate of the detection limit. For most drugs, no blank signal was detected from drug-free blood samples. To calculate S:B in those cases, the instrumental noise level was used. In the few cases in which there was detectable signal in the drug-free blood, the average signal obtained across all matrix blanks was used. PM sample drug screen All 30 unknown PM samples were run in triplicate alongside the calibration samples. The results of this PS-MS/MS drug screen were then compared to an LC–MS-MS confirmatory assay, which was run off-site and independently by Axis Forensic Toxicology. Results and Discussion Paper spray screening on a quadrupole-orbitrap mass spectrometer An example of the total ion chronogram acquired using the described instrument method is shown in Figure 1a. The term chronogram is used here rather than chromatogram because no separations are being performed. The chronograms are plotted in stick mode rather than smoothed so that individual scans can be seen. A full cycle of the inclusion list, encompassing all target compounds, was completed within ~0.3 min. Five or six scans were acquired for each MS/MS scan filter followed by a zero-intensity scan at the end, which was obtained by turning off the spray voltage 1.6 min into the acquisition. Such zero-intensity scans were required for each channel for automatic peak integration through the TraceFinder software. The TIC for the MS/MS of m/z 304.2 (corresponding to cocaine) is shown in Figure 1b, demonstrating the number and frequency of MS/MS scans as well as the zero-scan. The MS/MS spectra for a neat infusion of cocaine and a low concentration of cocaine spiked into drug-free blood are shown in Figure 1c and d, respectively. The cocaine MS/MS spectrum from blood shows a number of extraneous peaks owing to the complex chemical background. However, the diagnostic fragment ions for cocaine, such as m/z 182.117, were clearly detected with mass accuracy better than 5 ppm. Figure 1. View largeDownload slide (a) Total ion chronogram (all scans combined). (b) Extracted ion chronogram for cocaine’s MS/MS scans. (c) Tandem mass spectrum for a neat standard of cocaine at 200 ng/mL, infused via a commercial ESI source. (d) Tandem mass spectrum for blood spiked with 16 ng/mL cocaine (0.33 times its cut-off), ionized via paper spray. Figure 1. View largeDownload slide (a) Total ion chronogram (all scans combined). (b) Extracted ion chronogram for cocaine’s MS/MS scans. (c) Tandem mass spectrum for a neat standard of cocaine at 200 ng/mL, infused via a commercial ESI source. (d) Tandem mass spectrum for blood spiked with 16 ng/mL cocaine (0.33 times its cut-off), ionized via paper spray. When screening for a relatively large number of targets using an MS/MS inclusion list, the settings of the mass spectrometer must be adjusted to ensure an adequate number of scans are obtained for each target within the analysis time. The maximum injection time and the resolution of the orbitrap were set such that around five scans were obtained per target during the 90-s analysis time. The analysis is limited to 90 s primarily due to depletion of the extraction/spray solvent from the paper spray cartridge after analysis begins. Five scans were sufficient to give accurate m/z measurements and ion ratios and was deemed acceptable for semi-quantitative performance. The injection time of the orbitrap is analogous to dwell time on a triple quadrupole. The injection time is directly proportional to sensitivity unless the orbitrap is filled to capacity. In this case, the orbitrap rarely reached capacity. The injection time could therefore be increased to several seconds with a concomitant increase in sensitivity if desired. However, increasing the injection time would lead to an undesirably long acquisition time considering the number of target compounds. Resolution is another factor affecting both assay performance and scan time. The m/z resolution used here was 35,000, which lies in the middle of the range offered by this instrument. This resolution was considered an acceptable trade-off between selectivity and scan time. If fewer targets were included in the screening panel, the resolution and/or the injection time could be increased to improve selectivity and sensitivity, respectively. In this PS-MS/MS assay, target detection was based off the presence of a single fragment ion within a 5 ppm m/z window with adequate signal-to-blank (≥3). We opted for detection of only a single fragment ion in order to minimize the risk of false negatives. Because full MS/MS spectra were collected, additional fragment ions, with or without use of fragment ion ratios, can be added to the data analysis step to increase selectivity and decrease the rate of false positives. Relative matrix effects We compared the signal of the 11 ISTDs obtained for each of the 30 postmortem blood samples to the signal obtained from the fresh whole blood calibrators. The results, summarized in Table I, showed that there is no systematic difference between the postmortem blood and the fresh blood used for the calibration curves. The absolute signal of the ISTD varied somewhat more in PM samples than the calibrators, likely due to variations across the 30 PM samples. While the variation of the absolute signal was around 30%, the variation in the analyte:ISTD ratio was considerably less (<10% on average). Table I. The area under the curve (AUC) and relative standard deviation (RSD) for each of the internal standards across all calibrators and all PM samples during a single batch. The percent difference between the PM samples and calibrators is also shown ISTD Parameter PM Cal Percent difference Alprazolam-d5 AUC 3.5 × 108 3.1 × 108 +11% RSD 46% 26% Benzoylecgonine-d8 AUC 3.6 × 108 3.1 × 108 +15% RSD 28% 28% Cocaine-d3 AUC 6.8 × 108 5.7 × 108 +18% RSD 28% 24% Flunitrazepam-d7 AUC 4.0 × 107 2.3 × 107 +54% RSD 32% 27% Gabapentin-d10 AUC 3.5 × 107 3.6 × 107 −2% RSD 41% 31% Hydrocodone-d3 AUC 7.0 × 107 8.6 × 107 −20% RSD 30% 29% Metaxalone-d6 AUC 2.4 × 107 2.9 × 107 −19% RSD 32% 24% Methadone-d3 AUC 9.4 × 108 8.6 × 108 +9% RSD 50% 32% Methamphetamine-d11 AUC 6.9 × 108 9.0 × 108 −27% RSD 37% 35% Trimipramine-d3 AUC 6.2 × 108 5.8 × 108 +6% RSD 48% 27% Zolpidem-d6 AUC 2.3 × 107 1.9 × 107 +19% RSD 29% 26% ISTD Parameter PM Cal Percent difference Alprazolam-d5 AUC 3.5 × 108 3.1 × 108 +11% RSD 46% 26% Benzoylecgonine-d8 AUC 3.6 × 108 3.1 × 108 +15% RSD 28% 28% Cocaine-d3 AUC 6.8 × 108 5.7 × 108 +18% RSD 28% 24% Flunitrazepam-d7 AUC 4.0 × 107 2.3 × 107 +54% RSD 32% 27% Gabapentin-d10 AUC 3.5 × 107 3.6 × 107 −2% RSD 41% 31% Hydrocodone-d3 AUC 7.0 × 107 8.6 × 107 −20% RSD 30% 29% Metaxalone-d6 AUC 2.4 × 107 2.9 × 107 −19% RSD 32% 24% Methadone-d3 AUC 9.4 × 108 8.6 × 108 +9% RSD 50% 32% Methamphetamine-d11 AUC 6.9 × 108 9.0 × 108 −27% RSD 37% 35% Trimipramine-d3 AUC 6.2 × 108 5.8 × 108 +6% RSD 48% 27% Zolpidem-d6 AUC 2.3 × 107 1.9 × 107 +19% RSD 29% 26% Table I. The area under the curve (AUC) and relative standard deviation (RSD) for each of the internal standards across all calibrators and all PM samples during a single batch. The percent difference between the PM samples and calibrators is also shown ISTD Parameter PM Cal Percent difference Alprazolam-d5 AUC 3.5 × 108 3.1 × 108 +11% RSD 46% 26% Benzoylecgonine-d8 AUC 3.6 × 108 3.1 × 108 +15% RSD 28% 28% Cocaine-d3 AUC 6.8 × 108 5.7 × 108 +18% RSD 28% 24% Flunitrazepam-d7 AUC 4.0 × 107 2.3 × 107 +54% RSD 32% 27% Gabapentin-d10 AUC 3.5 × 107 3.6 × 107 −2% RSD 41% 31% Hydrocodone-d3 AUC 7.0 × 107 8.6 × 107 −20% RSD 30% 29% Metaxalone-d6 AUC 2.4 × 107 2.9 × 107 −19% RSD 32% 24% Methadone-d3 AUC 9.4 × 108 8.6 × 108 +9% RSD 50% 32% Methamphetamine-d11 AUC 6.9 × 108 9.0 × 108 −27% RSD 37% 35% Trimipramine-d3 AUC 6.2 × 108 5.8 × 108 +6% RSD 48% 27% Zolpidem-d6 AUC 2.3 × 107 1.9 × 107 +19% RSD 29% 26% ISTD Parameter PM Cal Percent difference Alprazolam-d5 AUC 3.5 × 108 3.1 × 108 +11% RSD 46% 26% Benzoylecgonine-d8 AUC 3.6 × 108 3.1 × 108 +15% RSD 28% 28% Cocaine-d3 AUC 6.8 × 108 5.7 × 108 +18% RSD 28% 24% Flunitrazepam-d7 AUC 4.0 × 107 2.3 × 107 +54% RSD 32% 27% Gabapentin-d10 AUC 3.5 × 107 3.6 × 107 −2% RSD 41% 31% Hydrocodone-d3 AUC 7.0 × 107 8.6 × 107 −20% RSD 30% 29% Metaxalone-d6 AUC 2.4 × 107 2.9 × 107 −19% RSD 32% 24% Methadone-d3 AUC 9.4 × 108 8.6 × 108 +9% RSD 50% 32% Methamphetamine-d11 AUC 6.9 × 108 9.0 × 108 −27% RSD 37% 35% Trimipramine-d3 AUC 6.2 × 108 5.8 × 108 +6% RSD 48% 27% Zolpidem-d6 AUC 2.3 × 107 1.9 × 107 +19% RSD 29% 26% Calibration curves and detection limits Calibration curves prepared in whole blood are summarized in Table II. Nearly all of the calibration curves had coefficients of determination (R2) above 0.9 and relative errors of the slope less than 15%. Buspirone stood out as having a relatively poor calibration curve with an R2 of 0.53 and a relative error of the slope of 33%. Although concentration determinations for this drug are not expected to be accurate, buspirone was nevertheless reliably detected throughout its calibration range with strong signal. The cut-off concentrations are also indicated in Table II. These values were set within or below the therapeutic or “safe” range for each target compound. In many cases, the drug could be detected at significantly lower concentrations if desired. The S:B of the primary fragment ion at the cut-off concentration is indicated in the table to provide a rough estimate of the feasible detection limits for each drug using this method. For example, the cut-off for carbamazepine was set at 1 μg/mL because the therapeutic levels of this drug are in the low μg/mL range, and toxic levels are above about 10 μg/mL (64). However, the magnitude of the S:B indicates that carbamazepine can feasibly be detected at concentrations less than 10 ng/mL if desired. Table II. Quantitative measurements for each of the analyte calibration curves ran concurrently with the PM samples in the PS-MS/MS drug screen Analyte ISTD Cut-off (ng/mL) S:B at cut-off Rel. error in slope (%) R2 6-Monoacetylmorphine F 20 5 3 0.993 7-Aminoclonazepam F 25 34 3 0.994 7-Aminoflunitrazepam F 20 52 3 0.992 9-Hydroxyrisperidone F 10 155 7 0.963 10,11-Dihydro-10-hydroxycarbamazepine J 500 19 6 0.976 Acetaminophen E 5000 4 9 0.942 Alfentanil K 50 257 7 0.961 Alpha-PVP K 50 214 9 0.936 Alprazolam A 5 3 2 0.996 Amitriptyline J 20 72 3 0.994 Amlodipine F 80 9 7 0.959 Amphetamine I 80 3 5 0.984 Aripiprazole H 50 111 8 0.951 Atenolol F 100 48 3 0.993 Baclofen E 250 11 7 0.961 Benzoylecgonine B 50 7 1 0.998 Benztropine H 10 303 6 0.972 Benzylpiperazine F 50 2 2 0.997 Brompheniramine H 25 83 13 0.877 Bupivacaine H 250 4,696 5 0.983 Buprenorphine H 10 6 4 0.986 Bupropion K 50 156 11 0.915 Buspirone H 6 69 33 0.528 Carbamazepine J 1000 5,925 9 0.940 Carbamazepine-10,11-epoxide J 500 194 2 0.995 Carisoprodol B 2000 10 7 0.961 Chlordiazepoxide K 50 120 5 0.981 Chlorpheniramine H 15 145 5 0.977 Chlorpromazine J 50 9 5 0.973 Citalopram H 10 63 10 0.926 Clomipramine J 20 35 3 0.993 Clonazepam A 30 3 4 0.984 Clozapine K 50 542 8 0.951 Cocaethylene C 50 136 2 0.997 Cocaine C 50 36 12 0.903 Codeine F 20 7 3 0.992 Cyclobenzaprine J 10 208 2 0.997 Demoxepam D 50 12 5 0.993 Desalkylflurazepam A 50 64 6 0.972 Desipramine J 20 21 4 0.991 Dextromethorphan H 10 78 8 0.952 Diazepam A 50 97 3 0.995 Diltiazem K 50 129 11 0.910 Diphenhydramine H 25 7 4 0.986 Donepezil H 45 47 4 0.989 Doxepin J 20 128 5 0.977 Doxylamine K 25 60 6 0.974 Duloxetine H 800 4 10 0.963 EDDP H 25 141 6 0.973 Ephedrine/Pseudoephedrine I 50 17 2 0.996 Etomidate A 100 9 5 0.982 Felbamate B 2500 4 12 0.889 Fentanyl C 1 10 4 0.988 Flecainide K 250 549 5 0.981 Flunitrazepam D 20 5 4 0.989 Flurazepam H 25 182 5 0.979 Fluvoxamine K 15 7 9 0.933 Gabapentin E 250 39 7 0.966 Haloperidol H 10 264 7 0.963 Hydrocodone F 20 35 2 0.998 Hydromorphone F 20 11 6 0.973 Hydroxychloroquine K 2000 336 12 0.899 Hydroxyzine K 10 82 4 0.985 Ketamine H 100 370 7 0.963 Labetalol F 45 50 5 0.983 Lamotrigine F 500 252 5 0.983 Levetiracetam G 2000 3 4 0.984 Lidocaine K 250 4303 3 0.993 Lorazepam D 25 8 6 0.972 MDA I 100 8 4 0.984 MDMA I 45 79 3 0.994 MDPV K 45 360 4 0.987 Meperidine K 25 137 5 0.980 Mephedrone I 45 64 5 0.983 Meprobamate B 1000 3 11 0.910 Mescaline F 100 3 6 0.971 Metaxalone G 1000 37 7 0.966 Methadone H 15 165 5 0.980 Methamphetamine I 45 70 2 0.995 Methocarbamol G 500 4 6 0.974 Methylone I 45 17 5 0.983 Methylphenidate K 20 464 2 0.997 Metoclopramide F 100 726 5 0.982 Metoprolol F 45 131 3 0.993 Midazolam K 45 65 4 0.987 Mirtazapine K 45 572 4 0.985 Morphine F 30 3 5 0.980 Naproxen B 14994 83 14 0.878 Norbuprenorphine H 10 3 8 0.9575 Norclomipramine J 36 276 2 0.998 Norclozapine J 45 140 3 0.993 Nordiazepam A 50 133 5 0.978 Nordoxepin J 20 65 4 0.985 Norfluoxetine H 20 7 2 0.989 Norketamine F 91 114 5 0.978 Normeperidine F 25 297 6 0.972 Norpropoxyphene (fragment) H 50 16 3 0.995 Norsertraline (fragment) H 50 113 3 0.993 Nortramadol K 2500 3 8 0.953 Nortriptyline J 20 66 3 0.994 Norvenlafaxine K 25 3 5 0.9879 o-/m-Chlorophenylpiperazine F 20 89 4 0.989 Olanzapine K 50 164 7 0.966 Oxazepam D 50 41 4 0.987 Oxycodone F 50 18 3 0.992 Oxymorphone F 15 5 5 0.981 Papaverine K 250 2,064 3 0.994 Paroxetine K 15 57 7 0.959 PCP H 250 7 12 0.901 Pentazocine H 50 425 4 0.987 Pregabalin E 250 7 7 0.958 Primidone B 750 3 11 0.910 Promethazine H 25 61 5 0.978 Propoxyphene (fragment) H 50 39 3 0.994 Propranolol K 50 194 2 0.996 Quetiapine K 50 579 5 0.983 Ranitidine F 250 231 5 0.981 Risperidone K 10 48 7 0.961 Ropinirole K 10 144 5 0.982 Sertraline H 100 18 6 0.971 Sildenafil F 100 14 3 0.994 Temazepam D 50 86 2 0.997 TFMPP K 50 386 3 0.992 Topiramate G 5000 4 14 0.889 Tramadol K 100 8 3 0.991 Trazodone K 100 539 3 0.995 Triazolam A 20 19 5 0.982 Trimipramine J 20 179 3 0.993 Vardenafil F 100 39 4 0.989 Venlafaxine F 50 3 4 0.984 Verapamil H 50 267 4 0.987 Zaleplon D 15 3 4 0.986 Ziprasidone H 40 49 9 0.944 Zolpidem K 10 169 1 0.999 Zonisamide A 750 3 13 0.875 Analyte ISTD Cut-off (ng/mL) S:B at cut-off Rel. error in slope (%) R2 6-Monoacetylmorphine F 20 5 3 0.993 7-Aminoclonazepam F 25 34 3 0.994 7-Aminoflunitrazepam F 20 52 3 0.992 9-Hydroxyrisperidone F 10 155 7 0.963 10,11-Dihydro-10-hydroxycarbamazepine J 500 19 6 0.976 Acetaminophen E 5000 4 9 0.942 Alfentanil K 50 257 7 0.961 Alpha-PVP K 50 214 9 0.936 Alprazolam A 5 3 2 0.996 Amitriptyline J 20 72 3 0.994 Amlodipine F 80 9 7 0.959 Amphetamine I 80 3 5 0.984 Aripiprazole H 50 111 8 0.951 Atenolol F 100 48 3 0.993 Baclofen E 250 11 7 0.961 Benzoylecgonine B 50 7 1 0.998 Benztropine H 10 303 6 0.972 Benzylpiperazine F 50 2 2 0.997 Brompheniramine H 25 83 13 0.877 Bupivacaine H 250 4,696 5 0.983 Buprenorphine H 10 6 4 0.986 Bupropion K 50 156 11 0.915 Buspirone H 6 69 33 0.528 Carbamazepine J 1000 5,925 9 0.940 Carbamazepine-10,11-epoxide J 500 194 2 0.995 Carisoprodol B 2000 10 7 0.961 Chlordiazepoxide K 50 120 5 0.981 Chlorpheniramine H 15 145 5 0.977 Chlorpromazine J 50 9 5 0.973 Citalopram H 10 63 10 0.926 Clomipramine J 20 35 3 0.993 Clonazepam A 30 3 4 0.984 Clozapine K 50 542 8 0.951 Cocaethylene C 50 136 2 0.997 Cocaine C 50 36 12 0.903 Codeine F 20 7 3 0.992 Cyclobenzaprine J 10 208 2 0.997 Demoxepam D 50 12 5 0.993 Desalkylflurazepam A 50 64 6 0.972 Desipramine J 20 21 4 0.991 Dextromethorphan H 10 78 8 0.952 Diazepam A 50 97 3 0.995 Diltiazem K 50 129 11 0.910 Diphenhydramine H 25 7 4 0.986 Donepezil H 45 47 4 0.989 Doxepin J 20 128 5 0.977 Doxylamine K 25 60 6 0.974 Duloxetine H 800 4 10 0.963 EDDP H 25 141 6 0.973 Ephedrine/Pseudoephedrine I 50 17 2 0.996 Etomidate A 100 9 5 0.982 Felbamate B 2500 4 12 0.889 Fentanyl C 1 10 4 0.988 Flecainide K 250 549 5 0.981 Flunitrazepam D 20 5 4 0.989 Flurazepam H 25 182 5 0.979 Fluvoxamine K 15 7 9 0.933 Gabapentin E 250 39 7 0.966 Haloperidol H 10 264 7 0.963 Hydrocodone F 20 35 2 0.998 Hydromorphone F 20 11 6 0.973 Hydroxychloroquine K 2000 336 12 0.899 Hydroxyzine K 10 82 4 0.985 Ketamine H 100 370 7 0.963 Labetalol F 45 50 5 0.983 Lamotrigine F 500 252 5 0.983 Levetiracetam G 2000 3 4 0.984 Lidocaine K 250 4303 3 0.993 Lorazepam D 25 8 6 0.972 MDA I 100 8 4 0.984 MDMA I 45 79 3 0.994 MDPV K 45 360 4 0.987 Meperidine K 25 137 5 0.980 Mephedrone I 45 64 5 0.983 Meprobamate B 1000 3 11 0.910 Mescaline F 100 3 6 0.971 Metaxalone G 1000 37 7 0.966 Methadone H 15 165 5 0.980 Methamphetamine I 45 70 2 0.995 Methocarbamol G 500 4 6 0.974 Methylone I 45 17 5 0.983 Methylphenidate K 20 464 2 0.997 Metoclopramide F 100 726 5 0.982 Metoprolol F 45 131 3 0.993 Midazolam K 45 65 4 0.987 Mirtazapine K 45 572 4 0.985 Morphine F 30 3 5 0.980 Naproxen B 14994 83 14 0.878 Norbuprenorphine H 10 3 8 0.9575 Norclomipramine J 36 276 2 0.998 Norclozapine J 45 140 3 0.993 Nordiazepam A 50 133 5 0.978 Nordoxepin J 20 65 4 0.985 Norfluoxetine H 20 7 2 0.989 Norketamine F 91 114 5 0.978 Normeperidine F 25 297 6 0.972 Norpropoxyphene (fragment) H 50 16 3 0.995 Norsertraline (fragment) H 50 113 3 0.993 Nortramadol K 2500 3 8 0.953 Nortriptyline J 20 66 3 0.994 Norvenlafaxine K 25 3 5 0.9879 o-/m-Chlorophenylpiperazine F 20 89 4 0.989 Olanzapine K 50 164 7 0.966 Oxazepam D 50 41 4 0.987 Oxycodone F 50 18 3 0.992 Oxymorphone F 15 5 5 0.981 Papaverine K 250 2,064 3 0.994 Paroxetine K 15 57 7 0.959 PCP H 250 7 12 0.901 Pentazocine H 50 425 4 0.987 Pregabalin E 250 7 7 0.958 Primidone B 750 3 11 0.910 Promethazine H 25 61 5 0.978 Propoxyphene (fragment) H 50 39 3 0.994 Propranolol K 50 194 2 0.996 Quetiapine K 50 579 5 0.983 Ranitidine F 250 231 5 0.981 Risperidone K 10 48 7 0.961 Ropinirole K 10 144 5 0.982 Sertraline H 100 18 6 0.971 Sildenafil F 100 14 3 0.994 Temazepam D 50 86 2 0.997 TFMPP K 50 386 3 0.992 Topiramate G 5000 4 14 0.889 Tramadol K 100 8 3 0.991 Trazodone K 100 539 3 0.995 Triazolam A 20 19 5 0.982 Trimipramine J 20 179 3 0.993 Vardenafil F 100 39 4 0.989 Venlafaxine F 50 3 4 0.984 Verapamil H 50 267 4 0.987 Zaleplon D 15 3 4 0.986 Ziprasidone H 40 49 9 0.944 Zolpidem K 10 169 1 0.999 Zonisamide A 750 3 13 0.875 Table II. Quantitative measurements for each of the analyte calibration curves ran concurrently with the PM samples in the PS-MS/MS drug screen Analyte ISTD Cut-off (ng/mL) S:B at cut-off Rel. error in slope (%) R2 6-Monoacetylmorphine F 20 5 3 0.993 7-Aminoclonazepam F 25 34 3 0.994 7-Aminoflunitrazepam F 20 52 3 0.992 9-Hydroxyrisperidone F 10 155 7 0.963 10,11-Dihydro-10-hydroxycarbamazepine J 500 19 6 0.976 Acetaminophen E 5000 4 9 0.942 Alfentanil K 50 257 7 0.961 Alpha-PVP K 50 214 9 0.936 Alprazolam A 5 3 2 0.996 Amitriptyline J 20 72 3 0.994 Amlodipine F 80 9 7 0.959 Amphetamine I 80 3 5 0.984 Aripiprazole H 50 111 8 0.951 Atenolol F 100 48 3 0.993 Baclofen E 250 11 7 0.961 Benzoylecgonine B 50 7 1 0.998 Benztropine H 10 303 6 0.972 Benzylpiperazine F 50 2 2 0.997 Brompheniramine H 25 83 13 0.877 Bupivacaine H 250 4,696 5 0.983 Buprenorphine H 10 6 4 0.986 Bupropion K 50 156 11 0.915 Buspirone H 6 69 33 0.528 Carbamazepine J 1000 5,925 9 0.940 Carbamazepine-10,11-epoxide J 500 194 2 0.995 Carisoprodol B 2000 10 7 0.961 Chlordiazepoxide K 50 120 5 0.981 Chlorpheniramine H 15 145 5 0.977 Chlorpromazine J 50 9 5 0.973 Citalopram H 10 63 10 0.926 Clomipramine J 20 35 3 0.993 Clonazepam A 30 3 4 0.984 Clozapine K 50 542 8 0.951 Cocaethylene C 50 136 2 0.997 Cocaine C 50 36 12 0.903 Codeine F 20 7 3 0.992 Cyclobenzaprine J 10 208 2 0.997 Demoxepam D 50 12 5 0.993 Desalkylflurazepam A 50 64 6 0.972 Desipramine J 20 21 4 0.991 Dextromethorphan H 10 78 8 0.952 Diazepam A 50 97 3 0.995 Diltiazem K 50 129 11 0.910 Diphenhydramine H 25 7 4 0.986 Donepezil H 45 47 4 0.989 Doxepin J 20 128 5 0.977 Doxylamine K 25 60 6 0.974 Duloxetine H 800 4 10 0.963 EDDP H 25 141 6 0.973 Ephedrine/Pseudoephedrine I 50 17 2 0.996 Etomidate A 100 9 5 0.982 Felbamate B 2500 4 12 0.889 Fentanyl C 1 10 4 0.988 Flecainide K 250 549 5 0.981 Flunitrazepam D 20 5 4 0.989 Flurazepam H 25 182 5 0.979 Fluvoxamine K 15 7 9 0.933 Gabapentin E 250 39 7 0.966 Haloperidol H 10 264 7 0.963 Hydrocodone F 20 35 2 0.998 Hydromorphone F 20 11 6 0.973 Hydroxychloroquine K 2000 336 12 0.899 Hydroxyzine K 10 82 4 0.985 Ketamine H 100 370 7 0.963 Labetalol F 45 50 5 0.983 Lamotrigine F 500 252 5 0.983 Levetiracetam G 2000 3 4 0.984 Lidocaine K 250 4303 3 0.993 Lorazepam D 25 8 6 0.972 MDA I 100 8 4 0.984 MDMA I 45 79 3 0.994 MDPV K 45 360 4 0.987 Meperidine K 25 137 5 0.980 Mephedrone I 45 64 5 0.983 Meprobamate B 1000 3 11 0.910 Mescaline F 100 3 6 0.971 Metaxalone G 1000 37 7 0.966 Methadone H 15 165 5 0.980 Methamphetamine I 45 70 2 0.995 Methocarbamol G 500 4 6 0.974 Methylone I 45 17 5 0.983 Methylphenidate K 20 464 2 0.997 Metoclopramide F 100 726 5 0.982 Metoprolol F 45 131 3 0.993 Midazolam K 45 65 4 0.987 Mirtazapine K 45 572 4 0.985 Morphine F 30 3 5 0.980 Naproxen B 14994 83 14 0.878 Norbuprenorphine H 10 3 8 0.9575 Norclomipramine J 36 276 2 0.998 Norclozapine J 45 140 3 0.993 Nordiazepam A 50 133 5 0.978 Nordoxepin J 20 65 4 0.985 Norfluoxetine H 20 7 2 0.989 Norketamine F 91 114 5 0.978 Normeperidine F 25 297 6 0.972 Norpropoxyphene (fragment) H 50 16 3 0.995 Norsertraline (fragment) H 50 113 3 0.993 Nortramadol K 2500 3 8 0.953 Nortriptyline J 20 66 3 0.994 Norvenlafaxine K 25 3 5 0.9879 o-/m-Chlorophenylpiperazine F 20 89 4 0.989 Olanzapine K 50 164 7 0.966 Oxazepam D 50 41 4 0.987 Oxycodone F 50 18 3 0.992 Oxymorphone F 15 5 5 0.981 Papaverine K 250 2,064 3 0.994 Paroxetine K 15 57 7 0.959 PCP H 250 7 12 0.901 Pentazocine H 50 425 4 0.987 Pregabalin E 250 7 7 0.958 Primidone B 750 3 11 0.910 Promethazine H 25 61 5 0.978 Propoxyphene (fragment) H 50 39 3 0.994 Propranolol K 50 194 2 0.996 Quetiapine K 50 579 5 0.983 Ranitidine F 250 231 5 0.981 Risperidone K 10 48 7 0.961 Ropinirole K 10 144 5 0.982 Sertraline H 100 18 6 0.971 Sildenafil F 100 14 3 0.994 Temazepam D 50 86 2 0.997 TFMPP K 50 386 3 0.992 Topiramate G 5000 4 14 0.889 Tramadol K 100 8 3 0.991 Trazodone K 100 539 3 0.995 Triazolam A 20 19 5 0.982 Trimipramine J 20 179 3 0.993 Vardenafil F 100 39 4 0.989 Venlafaxine F 50 3 4 0.984 Verapamil H 50 267 4 0.987 Zaleplon D 15 3 4 0.986 Ziprasidone H 40 49 9 0.944 Zolpidem K 10 169 1 0.999 Zonisamide A 750 3 13 0.875 Analyte ISTD Cut-off (ng/mL) S:B at cut-off Rel. error in slope (%) R2 6-Monoacetylmorphine F 20 5 3 0.993 7-Aminoclonazepam F 25 34 3 0.994 7-Aminoflunitrazepam F 20 52 3 0.992 9-Hydroxyrisperidone F 10 155 7 0.963 10,11-Dihydro-10-hydroxycarbamazepine J 500 19 6 0.976 Acetaminophen E 5000 4 9 0.942 Alfentanil K 50 257 7 0.961 Alpha-PVP K 50 214 9 0.936 Alprazolam A 5 3 2 0.996 Amitriptyline J 20 72 3 0.994 Amlodipine F 80 9 7 0.959 Amphetamine I 80 3 5 0.984 Aripiprazole H 50 111 8 0.951 Atenolol F 100 48 3 0.993 Baclofen E 250 11 7 0.961 Benzoylecgonine B 50 7 1 0.998 Benztropine H 10 303 6 0.972 Benzylpiperazine F 50 2 2 0.997 Brompheniramine H 25 83 13 0.877 Bupivacaine H 250 4,696 5 0.983 Buprenorphine H 10 6 4 0.986 Bupropion K 50 156 11 0.915 Buspirone H 6 69 33 0.528 Carbamazepine J 1000 5,925 9 0.940 Carbamazepine-10,11-epoxide J 500 194 2 0.995 Carisoprodol B 2000 10 7 0.961 Chlordiazepoxide K 50 120 5 0.981 Chlorpheniramine H 15 145 5 0.977 Chlorpromazine J 50 9 5 0.973 Citalopram H 10 63 10 0.926 Clomipramine J 20 35 3 0.993 Clonazepam A 30 3 4 0.984 Clozapine K 50 542 8 0.951 Cocaethylene C 50 136 2 0.997 Cocaine C 50 36 12 0.903 Codeine F 20 7 3 0.992 Cyclobenzaprine J 10 208 2 0.997 Demoxepam D 50 12 5 0.993 Desalkylflurazepam A 50 64 6 0.972 Desipramine J 20 21 4 0.991 Dextromethorphan H 10 78 8 0.952 Diazepam A 50 97 3 0.995 Diltiazem K 50 129 11 0.910 Diphenhydramine H 25 7 4 0.986 Donepezil H 45 47 4 0.989 Doxepin J 20 128 5 0.977 Doxylamine K 25 60 6 0.974 Duloxetine H 800 4 10 0.963 EDDP H 25 141 6 0.973 Ephedrine/Pseudoephedrine I 50 17 2 0.996 Etomidate A 100 9 5 0.982 Felbamate B 2500 4 12 0.889 Fentanyl C 1 10 4 0.988 Flecainide K 250 549 5 0.981 Flunitrazepam D 20 5 4 0.989 Flurazepam H 25 182 5 0.979 Fluvoxamine K 15 7 9 0.933 Gabapentin E 250 39 7 0.966 Haloperidol H 10 264 7 0.963 Hydrocodone F 20 35 2 0.998 Hydromorphone F 20 11 6 0.973 Hydroxychloroquine K 2000 336 12 0.899 Hydroxyzine K 10 82 4 0.985 Ketamine H 100 370 7 0.963 Labetalol F 45 50 5 0.983 Lamotrigine F 500 252 5 0.983 Levetiracetam G 2000 3 4 0.984 Lidocaine K 250 4303 3 0.993 Lorazepam D 25 8 6 0.972 MDA I 100 8 4 0.984 MDMA I 45 79 3 0.994 MDPV K 45 360 4 0.987 Meperidine K 25 137 5 0.980 Mephedrone I 45 64 5 0.983 Meprobamate B 1000 3 11 0.910 Mescaline F 100 3 6 0.971 Metaxalone G 1000 37 7 0.966 Methadone H 15 165 5 0.980 Methamphetamine I 45 70 2 0.995 Methocarbamol G 500 4 6 0.974 Methylone I 45 17 5 0.983 Methylphenidate K 20 464 2 0.997 Metoclopramide F 100 726 5 0.982 Metoprolol F 45 131 3 0.993 Midazolam K 45 65 4 0.987 Mirtazapine K 45 572 4 0.985 Morphine F 30 3 5 0.980 Naproxen B 14994 83 14 0.878 Norbuprenorphine H 10 3 8 0.9575 Norclomipramine J 36 276 2 0.998 Norclozapine J 45 140 3 0.993 Nordiazepam A 50 133 5 0.978 Nordoxepin J 20 65 4 0.985 Norfluoxetine H 20 7 2 0.989 Norketamine F 91 114 5 0.978 Normeperidine F 25 297 6 0.972 Norpropoxyphene (fragment) H 50 16 3 0.995 Norsertraline (fragment) H 50 113 3 0.993 Nortramadol K 2500 3 8 0.953 Nortriptyline J 20 66 3 0.994 Norvenlafaxine K 25 3 5 0.9879 o-/m-Chlorophenylpiperazine F 20 89 4 0.989 Olanzapine K 50 164 7 0.966 Oxazepam D 50 41 4 0.987 Oxycodone F 50 18 3 0.992 Oxymorphone F 15 5 5 0.981 Papaverine K 250 2,064 3 0.994 Paroxetine K 15 57 7 0.959 PCP H 250 7 12 0.901 Pentazocine H 50 425 4 0.987 Pregabalin E 250 7 7 0.958 Primidone B 750 3 11 0.910 Promethazine H 25 61 5 0.978 Propoxyphene (fragment) H 50 39 3 0.994 Propranolol K 50 194 2 0.996 Quetiapine K 50 579 5 0.983 Ranitidine F 250 231 5 0.981 Risperidone K 10 48 7 0.961 Ropinirole K 10 144 5 0.982 Sertraline H 100 18 6 0.971 Sildenafil F 100 14 3 0.994 Temazepam D 50 86 2 0.997 TFMPP K 50 386 3 0.992 Topiramate G 5000 4 14 0.889 Tramadol K 100 8 3 0.991 Trazodone K 100 539 3 0.995 Triazolam A 20 19 5 0.982 Trimipramine J 20 179 3 0.993 Vardenafil F 100 39 4 0.989 Venlafaxine F 50 3 4 0.984 Verapamil H 50 267 4 0.987 Zaleplon D 15 3 4 0.986 Ziprasidone H 40 49 9 0.944 Zolpidem K 10 169 1 0.999 Zonisamide A 750 3 13 0.875 Selectivity Because of the lack of chromatographic separation prior to the ionization step in PS-MS/MS, co-elution of drugs, drug metabolites and matrix components can result in interferences. Several intratarget interferences were identified and are summarized in Table III. Only intratarget interferences in which the highest calibrator of the interferent resulted in a signal above the cut-off for the target compound were included. Several of the interferences arose from structurally similar isomers that yielded the same fragment ions. In each of these cases, the intensity ratios of the major fragment ions generated by the two isomers were different; fragment ion ratios might therefore be used to distinguish these isomers if desired. In two cases, interference was observed from apparent hydrolysis of the parent drug standard in the blood calibrators. This interference was not observed in neat standards, ruling out impurities or in-source CID. Interference from non-target compounds can also occur, potentially resulting in false positives. For example, the non-target compound norcodeine could give false positives for morphine and hydromorphone (65) since all three of these compounds are structurally similar isomers. Table III. Intratarget interferences Target compound Interferent Extent of Interferencea Possible cause of interference Tramadol Norvenlafaxine 45% Isomers that generate the same fragment ions Hydromorphone Morphine 20% Isomers that generate the same fragment ions Morphine Hydromorphone 5% Isomers that generate the same fragment ions Morphine 6-Monoacetylmorphine 25% Partial hydrolysis of 6-MAM to morphine in blood Codeine Hydrocodone 5% Isomers that generate the same fragment ions Hydrocodone Codeine 20% Isomers that generate the same fragment ions Benzoylecgonine Cocaine 35% Partial hydrolysis of cocaine to benzoylecgonine in blood Target compound Interferent Extent of Interferencea Possible cause of interference Tramadol Norvenlafaxine 45% Isomers that generate the same fragment ions Hydromorphone Morphine 20% Isomers that generate the same fragment ions Morphine Hydromorphone 5% Isomers that generate the same fragment ions Morphine 6-Monoacetylmorphine 25% Partial hydrolysis of 6-MAM to morphine in blood Codeine Hydrocodone 5% Isomers that generate the same fragment ions Hydrocodone Codeine 20% Isomers that generate the same fragment ions Benzoylecgonine Cocaine 35% Partial hydrolysis of cocaine to benzoylecgonine in blood aThe extent of interference is expressed as (apparent concentration of target compound)/(actual concentration of interferent)*100%. Table III. Intratarget interferences Target compound Interferent Extent of Interferencea Possible cause of interference Tramadol Norvenlafaxine 45% Isomers that generate the same fragment ions Hydromorphone Morphine 20% Isomers that generate the same fragment ions Morphine Hydromorphone 5% Isomers that generate the same fragment ions Morphine 6-Monoacetylmorphine 25% Partial hydrolysis of 6-MAM to morphine in blood Codeine Hydrocodone 5% Isomers that generate the same fragment ions Hydrocodone Codeine 20% Isomers that generate the same fragment ions Benzoylecgonine Cocaine 35% Partial hydrolysis of cocaine to benzoylecgonine in blood Target compound Interferent Extent of Interferencea Possible cause of interference Tramadol Norvenlafaxine 45% Isomers that generate the same fragment ions Hydromorphone Morphine 20% Isomers that generate the same fragment ions Morphine Hydromorphone 5% Isomers that generate the same fragment ions Morphine 6-Monoacetylmorphine 25% Partial hydrolysis of 6-MAM to morphine in blood Codeine Hydrocodone 5% Isomers that generate the same fragment ions Hydrocodone Codeine 20% Isomers that generate the same fragment ions Benzoylecgonine Cocaine 35% Partial hydrolysis of cocaine to benzoylecgonine in blood aThe extent of interference is expressed as (apparent concentration of target compound)/(actual concentration of interferent)*100%. Evaluating the PS-MS/MS drug screen on postmortem samples Thirty blinded postmortem blood samples were obtained from a toxicology laboratory and analyzed using PS-MS/MS. The toxicology lab performed their typical screen and confirm workflow, which consisted of screening by a combination of immunoassay and LC–MS-MS followed by a separate quantitative LC–MS-MS assay for confirmation. A complete list of the drugs detected by the two methods and the determined concentrations can be found in Supplementary Table III. PS-MS/MS detected a total of 97 drugs across all samples, whereas the LC–MS-MS screen and confirm detected 89. Of the 89 drug detections from the LC–MS-MS screen/confirm, 7 were not detected by the PS-MS/MS method (false negatives—FNs). Of the 7 false negatives, 5 were detected by the LC–MS-MS assay at concentrations below the PS-MS/MS detection limit. For the other two results, quantitative analysis was not performed by the toxicology lab, so the drug concentrations are unknown. In total, some 4,000 negative results were determined by both screening methods. Of the 97 PS-MS/MS screening detections, 7 instances were not detected by the LC–MS-MS screen/confirm and were treated as false positives (FPs). Of these 7 FP, 2 were opiates detected in the presence of other opiates. For example, the PS-MS/MS assay screened positive for both hydromorphone and morphine, whereas only morphine was confirmed positive by LC–MS-MS. Hydromorphone and morphine are isomers that yield many of the same fragment ions, albeit in different ratios. Of the remaining 5 FP, the source of interference is not known; 4 of them were at low levels near the cut-off (within a factor of 2). Finally, of the 97 drugs screened positive by PS-MS/MS, 7 were not screened for by the toxicology lab because they were not included in the panel ordered by the customer; these detection events were excluded from the data analysis. Qualitatively, the rates of occurrence for positive and negative results can be used in measuring a method’s true positive rate, false positive rate, positive predictive value (PPV) and negative predictive value (NPV). These values are defined as (66): truepositiverate=TPTP+FN. (1) truenegativerate=TNTN+FP. (2) PPV=TPTP+FP. (3) NPV=TNTN+FN. (4) As summarized in Table IV, the true positive rate of the PS-MS/MS screen was 92.1% while the true negative rate was 99.8%. The calculated true positive rate for the PS-MS/MS assay included drugs at concentrations below the PS-MS/MS detection limits; the true positive rate for drugs within the reporting range was 100%. No drug targets above the assay cut-off were detected by LC–MS-MS but not by paper spray. These results compare favorably with immunoassay drug screens; a recent study showed that the true positive rate was about 85% across all drug classes, with some drugs (MDMA and PCP) having a 0% true positive rate (67). About 92.1% of all positive PS hits were genuinely present in the samples (positive predictive value) and 99.8% of all undetected drugs were confirmed to be absent in the samples (negative predictive value). While a significantly larger number of samples are needed to confidently assign the true positive and negative rates of paper spray MS/MS, these results indicate that the method shows good promise as a drug screening method. Table IV. Qualitative results of the PS-MS/MS-based drug screen relative to LC–MS-MS-based confirmatory assays Parameter Result True Positive (TP) 82 False Positive (FP) 7 True Negative (TN) ~4,000 False Negative (FN) 7 True positive rate (eqn 1) 92.1% True negative rate (eqn 2) 99.8% Positive Predictive Value (eqn 3) 92.1% Negative Predictive Value (eqn 4) 99.8% Parameter Result True Positive (TP) 82 False Positive (FP) 7 True Negative (TN) ~4,000 False Negative (FN) 7 True positive rate (eqn 1) 92.1% True negative rate (eqn 2) 99.8% Positive Predictive Value (eqn 3) 92.1% Negative Predictive Value (eqn 4) 99.8% Table IV. Qualitative results of the PS-MS/MS-based drug screen relative to LC–MS-MS-based confirmatory assays Parameter Result True Positive (TP) 82 False Positive (FP) 7 True Negative (TN) ~4,000 False Negative (FN) 7 True positive rate (eqn 1) 92.1% True negative rate (eqn 2) 99.8% Positive Predictive Value (eqn 3) 92.1% Negative Predictive Value (eqn 4) 99.8% Parameter Result True Positive (TP) 82 False Positive (FP) 7 True Negative (TN) ~4,000 False Negative (FN) 7 True positive rate (eqn 1) 92.1% True negative rate (eqn 2) 99.8% Positive Predictive Value (eqn 3) 92.1% Negative Predictive Value (eqn 4) 99.8% The quantitative results obtained by the PS-MS/MS screening method were compared to the LC–MS-MS quantitative confirmations using Passing-Bablok regression (Figure 2). The two methods were highly correlated, with a Pearson’s correlation coefficient (r) of 0.996. The concentrations determined by PS-MS were, on average, slightly higher than the LC–MS-MS values as reflected by the slope value of 1.17. A complete comparison of the quantitative results can be found in Supplementary Table III. It is important to note that the PS-MS/MS method was developed as a rapid screening method, whereas the HPLC method was developed for quantitative confirmation. The quantitative performance of PS-MS can be improved by decreasing the number of targets and by using isotopically labeled internal standards for each analyte. Figure 2. View largeDownload slide Comparison of concentrations for the drugs detected and quantitated from the PM samples by both HPLC- and PS-based screening methods using a log-log plot. The Passing-Bablok regression is shown as a solid line; perfect correlation is shown as a dashed line. Figure 2. View largeDownload slide Comparison of concentrations for the drugs detected and quantitated from the PM samples by both HPLC- and PS-based screening methods using a log-log plot. The Passing-Bablok regression is shown as a solid line; perfect correlation is shown as a dashed line. Screening for acidic drugs The screening method described thus far was carried out via a single paper spray analysis in the positive ion mode. Some acidic drugs, such as barbiturates, did not form abundant positive ions and were suited for the negative ion mode. Using a different solvent system optimized for negative ionization, we tested the PS-MS/MS screening method for barbiturates in the negative ion mode. Phenytoin, which like the barbiturates has an acidic cyclic imide moiety, was also included. The limits of detection and calibration performances in the whole blood calibrators are shown in Table V. The LODs for each target lie below or within their therapeutic ranges, which are all >1 μg/mL (64). The calibration curves for each target showed relative slope errors of 3–7% with R2 of 0.95 or higher. Table V. Limits of detection and calibration summary for acidic drug targets Analyte LOD (ng/mL) Rel. error in slope (%) R2 Butabarbital 229 3 0.99 Butalbital 263 4 0.98 Amobarbital 321 5 0.97 Phenobarbital 502 4 0.98 Secobarbital 286 4 0.98 Thiopental 1,100 4 0.98 Phenytoin 919 7 0.95 Analyte LOD (ng/mL) Rel. error in slope (%) R2 Butabarbital 229 3 0.99 Butalbital 263 4 0.98 Amobarbital 321 5 0.97 Phenobarbital 502 4 0.98 Secobarbital 286 4 0.98 Thiopental 1,100 4 0.98 Phenytoin 919 7 0.95 Table V. Limits of detection and calibration summary for acidic drug targets Analyte LOD (ng/mL) Rel. error in slope (%) R2 Butabarbital 229 3 0.99 Butalbital 263 4 0.98 Amobarbital 321 5 0.97 Phenobarbital 502 4 0.98 Secobarbital 286 4 0.98 Thiopental 1,100 4 0.98 Phenytoin 919 7 0.95 Analyte LOD (ng/mL) Rel. error in slope (%) R2 Butabarbital 229 3 0.99 Butalbital 263 4 0.98 Amobarbital 321 5 0.97 Phenobarbital 502 4 0.98 Secobarbital 286 4 0.98 Thiopental 1,100 4 0.98 Phenytoin 919 7 0.95 Conclusion Paper spray MS/MS drug screening methods were developed for the rapid screening of over 140 drugs and drug metabolites commonly encountered in toxicological analyses. Separate methods were developed in the positive and negative ion modes, each of which could be completed in a few minutes without sample preparation. Semi-quantitative analysis was carried out by generating calibration curves for each of the analytes. The positive ion drug screen, which included >95% of the target drugs, was tested on 30 postmortem blood specimens. Comparison with independent LC–MS-MS screening and confirmation testing showed good agreement. The sensitivity of PS-MS/MS coupled to a quadrupole-orbitrap mass spectrometer was adequate for postmortem drug screening of all of the drugs investigated here. Screening for a large panel of drugs in a single run made it impossible to optimize the experimental conditions for each drug class; better sensitivity could be expected by reducing the number of target compounds. Also, some potent emerging drugs of abuse such as carfentanil require screening cut-offs well below 1 ng/mL. Direct analysis approaches that employ analyte preconcentration are one way to achieve such low detection limits (21, 68, 69). It is also important to realize the selectivity limitations of PS-MS. Because there is no chromatographic separation, co-elution of compounds can result in interferences. Use of fragment ion ratios or gas-phase separations via differential mobility (65) or ion mobility are potential solutions to this problem. Additional study on a more exhaustive list of potential interferences is needed. Finally, the results presented here represent the results of a single run; investigation into method robustness and precision will be needed. Nevertheless, this study showed promising results that demonstrate the capability of PS-MS/MS to perform rapid, sensitive and selective drug screening from blood samples. Supplementary data Supplementary material is available at Journal of Analytical Toxicology online. Acknowledgments This project was supported by Award No. 2014-R2-CX-K007 awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect those of the Department of Justice. 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TI - Toxicological Drug Screening using Paper Spray High-Resolution Tandem Mass Spectrometry (HR-MS/MS)
JF - Journal of Analytical Toxicology
DO - 10.1093/jat/bky001
DA - 2018-01-25
UR - https://www.deepdyve.com/lp/oxford-university-press/toxicological-drug-screening-using-paper-spray-high-resolution-tandem-r2LIhR4ST9
SP - 1
EP - 310
VL - Advance Article
IS - 5
DP - DeepDyve
ER -