TY - JOUR
AU - Neiswonger, Marc, A
AB - Abstract A relatively overlooked aspect of forensic science is the potential of oral cavity fluid for contributing to a forensic diagnosis. Although traditional specimens, like blood and urine, are routinely evaluated for forensic toxicology testing, fluid from the oral cavity has not been investigated as a matrix in postmortem cases. Our laboratory developed and validated qualitative and quantitative analytical methods for determining 47 medicinal and illicit drugs from oral cavity fluid. These developed methods aimed to compare results from liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses of oral cavity fluid to those of traditional matrices collected from the same postmortem subjects. Of 34 cadavers studied, 32 (including two decomposed and two drowned subjects) had detectable and quantifiable drugs in the oral cavity fluid and/or blood, urine, bile, vitreous fluid and/or liver tissue. The most significant finding was that 6-acetylmorphine (6-AM) was detected more frequently in oral cavity fluid (11 cases) than in blood and urine combined (6 cases). Compounds with a short window of detection, like the heroin metabolite, 6-AM and even heroin, could be detected more readily in oral cavity fluid than in urine. In 2017, the incidence of heroin-related overdose deaths increased to 15,958. Those data have shed light on the practicality of testing oral cavity fluid postmortem and its significance in forensic toxicology. In conclusion, this study showed that oral cavity fluid could be useful for detecting and quantifying drugs in postmortem subjects; moreover, oral cavity fluid may be particularly suitable when other matrices are limited or difficult to collect, due to body condition or putrefaction. Introduction Historically, systematic postmortem forensic toxicological analyses have relied on the analysis of multiple traditional matrices, such as blood, urine, bile (1) and liver samples (2, 3). The specific matrix used for specimen retrieval depends on the time after death and the sample consistency is determined by such factors as clotting time, fluid movement and changes in cellular components. Our laboratory has developed a method and conducted a study to determine (i) if oral cavity fluid could serve as an adjunct matrix that could be readily collected from a postmortem subject, (ii) whether we could accurately detect and quantify medicinal and illicit drugs in oral cavity fluid and (iii) to compare the results to the current testing modalities used in postmortem forensic toxicology. Our assessment of oral cavity fluid suitability as a sample matrix was based on given analytes that could be identified and quantified in postmortem subjects. The present study indicated that utilizing oral cavity fluid in postmortem toxicology was suitable and practical compared to conventional biological samples, particularly in decaying cadavers, where the number of viable samples is limited. To the best of our knowledge, no experimental studies of oral cavity fluid collected and tested from postmortem subjects have been performed. Materials and methods Sample collection Coroners and medical examiners in seven counties participated in collecting oral cavity fluid and other available matrices from 25 male and 9 female cadavers. Oral cavity fluid was collected with the Quantisal® Oral Fluid Collection Device, purchased from Immunalysis Corporation (Pomona, CA). The cellulose pad on the collector was placed sublingually in the buccal cavity, adjacent to the second and third molars. In cases of rigor mortis, the cellulose pad was placed in the buccal area between the cheek and gum, adjacent to the second and third molars, until the indicator turned a blue color or for approximately 15 min. In cases where blood, regurgitation or reflux of stomach contents was observed in the oral cavity, the collector was instructed to remove the contents utilizing a paper towel or gloved fingers to allow for proper positioning of the cellulose pad. The cellulose pad was removed and immersed into 3 mL of non-azide buffer in a transport tube. The transport tube was labeled with the date of collection and a unique identifier (e.g., PM-01) for each cadaver. When the indicator did not turn blue, it was recorded on the chain-of-custody document. Samples were shipped to the designated laboratory (based on the matrix type) for analysis. The anonymity of the cadavers was protected and approved by all participating coroners and medical examiners. Chemicals and reagents The following reagents and kits were purchased from Immunalysis Corporation (Pomona, CA): TMB chromogenic substrate, STOP Reagent, synthetic negative saliva (SNS) and oral fluid multi-analyte calibrator/control sets. In addition, the direct enzyme-linked immunosorbent assay (ELISA) kits were purchased for detecting the following drugs: amphetamine, methamphetamine, opiates, propoxyphene, phencyclidine, cocaine/benzoylecgonine, Δ9-tetrahydrocannabinol (Δ9-THC), benzodiazepines, tramadol, methadone, buprenorphine, fentanyl, oxycodone/oxymorphone, carisoprodol and meperidine. All reagent grade chemicals and certified reference standards required for analysis were purchased from Cerilliant Corporation (Round Rock, TX, USA). All solvents, including methanol (optima grade), acetonitrile (optima grade), 2-propanol (optima grade) and LC–MS grade ammonium formate (95%), were purchased from VWR (Radnor, PA, USA). Formic acid was purchased from Acros Organics (Bridgewater, NJ). The Millipore Direct-Q (Type 1) and Elix (Type 2) water purification systems were purchased from Millipore Corporation (Darmstadt, Germany). LC-MS/MS working solutions Standards Reference standards and deuterated analytes were prepared as stock standard solutions for controls in liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses. A stock standard solution (2,000 ng/mL) was prepared by diluting 50 μL of a 1-mg/mL reference standard analytes solution with methanol (MeOH), for a total volume of 25 mL. A stock internal standard solution (500 ng/mL) was prepared by diluting 750 μL of a 100-μg/mL deuterated analytes solution with MeOH, for a total volume of 150 mL. Linearity The calibrators were prepared in SNS solution at concentrations of 1, 5, 10, 50, 100, 250, 500 and 1,000 ng/mL, calculated to account for the dilution of the specimen in the transport tube buffer. Each concentration was prepared with an appropriate amount of the stock standard solution. An internal standard solution (30 μL of 500 ng/mL) was added, and the solution was diluted to the final concentration with MeOH. Finally, the solutions were mixed by vortexing at high speed for approximately 10 s. Mobile phases The aqueous mobile phase (A) was composed of 50 mL of 5 mM ammonium formate and 2 mL of 0.1% formic acid, brought to a total volume of 2 L, with Type 1 water. The organic mobile phase (B) was composed of 2 mL of 0.1% formic acid, diluted to a total volume of 2 L with acetonitrile. Sample preparation In some cases, oral cavity fluid captured on the cellulose swab did not cause the adequacy indicator to turn blue. Therefore, we weighed the collection devices prior to sample preparations (PM-16 through -34) to facilitate evaluations of samples that failed to elicit a blue signal in the adequacy indicator. Next, the cellulose pad was removed from the collector wand and placed into the buffer with a blood serum filter (16 mm × 4 in). With the collection tube in a vertical position, the rubber end of the filter sampler was inserted at a 45° angle and pushed gently down on top of the cellulose pad. Filtered oral cavity fluid/buffer mixture will rise into the barrel for analysis. An aliquot (500 μL) of filtered sample was transferred into a labeled glass culture tube for qualitative analysis. Another aliquot (500 μL) was transferred to a labeled screw-thread amber vial, which contained 120 μL MeOH and 30 μL stock internal standard solution, for quantitative analysis. The vials were capped and mixed by vortexing for approximately 10 s at high speed. Qualitative procedures Oral cavity fluid samples from cadavers PM-01 through PM-15 were screened with an in-house ELISA method that had been validated for precision and accuracy according to the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines (4). The analyzses were conducted with a Tecan® Freedom EVO® 150 (Tecan Group Ltd., Männedorf, Switzerland). Quantitative procedures All oral cavity fluid samples were analyzed on a 6460 Triple Quadrupole LC-MS/MS System coupled with a 1290 Infinity liquid chromatography system (Agilent Technologies; Santa Clara, CA, USA). HPLC was performed with a Poroshell 120 EC-C18, 3.0 × 50 mm, 2.7 μm column, maintained at 50°C. The mobile phase flow rate was 0.6 mL/min, and the gradient program is shown in Table I. At 7 min, the initial conditions were restored, and the column was allowed to equilibrate for an additional 1.5 min post-run delay, before starting the next analysis. The sample injection volume was 2.5 μL. Mass spectrometry was performed in a positive electrospray ionization mode with the following source parameters: N2 gas temperature (300°C); gas flow (12 L/min); nebulizer pressure (45 psi); sheath gas temperature (350°C); and flow rate (12 L/min). Table I. HPLC gradient parameters for analyzing oral fluid samples Time, min A (%) B (%) 0 95 5 1 95 5 3 70 30 6 30 70 6.5 5 95 7 99.5 0.5 8 99.5 0.5 Time, min A (%) B (%) 0 95 5 1 95 5 3 70 30 6 30 70 6.5 5 95 7 99.5 0.5 8 99.5 0.5 View Large Table I. HPLC gradient parameters for analyzing oral fluid samples Time, min A (%) B (%) 0 95 5 1 95 5 3 70 30 6 30 70 6.5 5 95 7 99.5 0.5 8 99.5 0.5 Time, min A (%) B (%) 0 95 5 1 95 5 3 70 30 6 30 70 6.5 5 95 7 99.5 0.5 8 99.5 0.5 View Large Results Method validation: linearity, LOD, LOQ, precision, accuracy and carryover The method for quantifying 47 analytes was validated according to SWGTOX (5). Validation results for the analytes detected in this study are shown in Table II. Analytes in oral cavity fluid showed linearity at concentrations of 5–1,000 ng/mL, with 1/× weighting, on a calibration curve (n = 7); the linear coefficient (R2) was calculated to be above 0.99. The limit of detection (LOD) for all analytes was 5 ng/mL (except fentanyl, which had a LOD of 1 ng/mL). Bias and precision were evaluated in triplicate, at three levels for five runs (n = 15); within-run and between-run precisions were <19.53% for all analytes, and accuracy was between −6.44 and 11.65%, respectively. The limit of quantitation (LOQ) was evaluated statistically with 10 × the standard error. Carryover studies were conducted by placing a blank matrix sample between each calibrator. No carryover was observed above the method’s upper LOQ. Interference studies included 12 blank matrix samples: 10 SNS; 1 internal standard; 1 standard assessed by monitoring the signal of the analytes. Interferences were below the LOD of the assay and was determined to be insignificant. No observed interferences from the matrix or from common drugs/metabolites were seen above the LOD. Two different sets of samples were prepared for ionization suppression/enhancement studies. One set of 10 neat standards were prepared in duplicate at two concentrations of 10 and 1,000 ng/mL and fortified with internal standard. A second set of matrix samples were prepared by extracting in duplicate and reconstituted/fortified with either the low or high concentration and injected 6 times. Athough ionization suppression/enhancement was observed, the other validation parameters were not affected. Evaluations were performed with Microsoft Excel. Data were analyzed with Agilent MassHunter™ Quantitative Analysis Software. Table II. LC-MS/MS acquisition parameters for drug analytes and validation results summary Analyte MRM transition (m/z) Fragmentor voltage (V) Collision energy (V) Retention time (min) Accuracy (%) Precision (RSD) LOQ (ng/mL) Δ9-THC 315→193 150 20 7.2 3.25–6.41 7.28 5.0 315→123 150 30 7.2 6.81 6-acetylmorphine 328.4→165.1 170 44 2.35 −4.81 to 4.96 3.95 5.0 328.4→152.1 170 80 6.99 Acetaminophen 152.1→110.1 100 12 1.26 8.14–11.65 7.62 50.0 152.1→65.1 100 36 4.07 Alprazolam 309.1→205.1 170 48 4.56 0.79–5.43 2.96 5.0 309.1→151.1 170 72 8.69 Amphetamine 136.2→119.1 70 4 2.20 0.86–8.82 2.43 5.0 136.2→91.0 70 20 10.19 Benzoylecgonine 290.3→168.1 135 16 2.71 −1.32 to 3.81 1.94 5.0 290.3→105.0 135 32 10.93 Buprenorphine 468.6→115.1 215 144 4.16 −0.28 to 4.62 5.72 5.0 468.6→101.1 215 44 9.46 Carisoprodol 261.3→176.1 75 4 4.52 0.54–4.39 4.95 5.0 261.3→158.1 75 4 13.17 Clonazepam 316.1→214.1 165 40 4.47 −0.86 to 3.67 7.39 5.0 316.1→151.1 165 88 17.02 Codeine 300.1→152.1 165 76 2.10 −1.39 to 7.39 2.55 10.0 300.1→115.1 165 88 10.71 Cyclobenzaprine 276.1→215.1 135 48 4.36 −3.04 to 6.03 2.55 5.0 276.1→213.1 135 88 8.13 Diazepam 285.1→193.1 160 32 5.19 −1.64 to 7.54 2.41 5.0 285.1→165.1 160 56 9.47 Fentanyl 337.5→188.1 130 20 3.86 −6.44 to 1.48 2.27 1.0 337.5→105.1 130 48 9.80 Gabapentin 172.2→154.1 80 8 2.17 2.84–9.51 2.82 5.0 172.2→55.1 80 24 2.17 4.69 Hydrocodone 300.4→171.1 155 40 2.43 0.63–9.77 4.96 10.0 300.4→128.0 155 76 9.86 Hydromorphone 286.3→157.1 180 44 1.17 0.95–8.08 4.68 10.0 286.3→128.1 180 68 15.12 Meperidine 248.3→174.1 115 16 3.32 −1.18 to 4.68 1.42 5.0 248.3→103.1 115 48 8.38 Methamphetamine 150.2→119.1 65 8 2.41 2.57–10.70 1.71 5.0 150.2→91.1 65 20 9.24 Methadone 310.4→265.2 100 8 4.53 −0.76 to 3.32 2.34 5.0 310.4→105.1 100 24 10.30 Morphine 286.3→165.1 155 48 0.82 −2.39 to 9.15 3.120 10.0 286.3→152.1 155 68 11.45 Naloxone 328.2→310.1 120 16 2.13 5.09–5.17 7.80 5.0 328.2→212.1 120 40 2.13 7.10 o-desmethyl-cis-tramadol 250.8→232.1 110 12 2.44 −3.99 to 2.65 3.17 5.0 250.8→58.2 110 12 14.10 Oxazepam 287.1→163.0 120 40 4.38 −5.43 to 3.32 5.97 5.0 287.1→104.1 120 36 19.53 Oxycodone 316.4→298.1 135 16 2.31 1.07–9.46 3.16 10.0 316.4→212.1 135 44 13.02 Oxymorphone 302.3→198.1 130 44 0.96 −0.34 to 5.18 10.0 302.3→128.1 130 88 10.78 9.47 Tramadol 264.3→58.2 90 16 3.15 0.02–6.80 1.61 5.0 264.3→42.2 90 112 3.15 1.91 Analyte MRM transition (m/z) Fragmentor voltage (V) Collision energy (V) Retention time (min) Accuracy (%) Precision (RSD) LOQ (ng/mL) Δ9-THC 315→193 150 20 7.2 3.25–6.41 7.28 5.0 315→123 150 30 7.2 6.81 6-acetylmorphine 328.4→165.1 170 44 2.35 −4.81 to 4.96 3.95 5.0 328.4→152.1 170 80 6.99 Acetaminophen 152.1→110.1 100 12 1.26 8.14–11.65 7.62 50.0 152.1→65.1 100 36 4.07 Alprazolam 309.1→205.1 170 48 4.56 0.79–5.43 2.96 5.0 309.1→151.1 170 72 8.69 Amphetamine 136.2→119.1 70 4 2.20 0.86–8.82 2.43 5.0 136.2→91.0 70 20 10.19 Benzoylecgonine 290.3→168.1 135 16 2.71 −1.32 to 3.81 1.94 5.0 290.3→105.0 135 32 10.93 Buprenorphine 468.6→115.1 215 144 4.16 −0.28 to 4.62 5.72 5.0 468.6→101.1 215 44 9.46 Carisoprodol 261.3→176.1 75 4 4.52 0.54–4.39 4.95 5.0 261.3→158.1 75 4 13.17 Clonazepam 316.1→214.1 165 40 4.47 −0.86 to 3.67 7.39 5.0 316.1→151.1 165 88 17.02 Codeine 300.1→152.1 165 76 2.10 −1.39 to 7.39 2.55 10.0 300.1→115.1 165 88 10.71 Cyclobenzaprine 276.1→215.1 135 48 4.36 −3.04 to 6.03 2.55 5.0 276.1→213.1 135 88 8.13 Diazepam 285.1→193.1 160 32 5.19 −1.64 to 7.54 2.41 5.0 285.1→165.1 160 56 9.47 Fentanyl 337.5→188.1 130 20 3.86 −6.44 to 1.48 2.27 1.0 337.5→105.1 130 48 9.80 Gabapentin 172.2→154.1 80 8 2.17 2.84–9.51 2.82 5.0 172.2→55.1 80 24 2.17 4.69 Hydrocodone 300.4→171.1 155 40 2.43 0.63–9.77 4.96 10.0 300.4→128.0 155 76 9.86 Hydromorphone 286.3→157.1 180 44 1.17 0.95–8.08 4.68 10.0 286.3→128.1 180 68 15.12 Meperidine 248.3→174.1 115 16 3.32 −1.18 to 4.68 1.42 5.0 248.3→103.1 115 48 8.38 Methamphetamine 150.2→119.1 65 8 2.41 2.57–10.70 1.71 5.0 150.2→91.1 65 20 9.24 Methadone 310.4→265.2 100 8 4.53 −0.76 to 3.32 2.34 5.0 310.4→105.1 100 24 10.30 Morphine 286.3→165.1 155 48 0.82 −2.39 to 9.15 3.120 10.0 286.3→152.1 155 68 11.45 Naloxone 328.2→310.1 120 16 2.13 5.09–5.17 7.80 5.0 328.2→212.1 120 40 2.13 7.10 o-desmethyl-cis-tramadol 250.8→232.1 110 12 2.44 −3.99 to 2.65 3.17 5.0 250.8→58.2 110 12 14.10 Oxazepam 287.1→163.0 120 40 4.38 −5.43 to 3.32 5.97 5.0 287.1→104.1 120 36 19.53 Oxycodone 316.4→298.1 135 16 2.31 1.07–9.46 3.16 10.0 316.4→212.1 135 44 13.02 Oxymorphone 302.3→198.1 130 44 0.96 −0.34 to 5.18 10.0 302.3→128.1 130 88 10.78 9.47 Tramadol 264.3→58.2 90 16 3.15 0.02–6.80 1.61 5.0 264.3→42.2 90 112 3.15 1.91 LC-MS/MS: liquid chromatography coupled with tandem mass spectrometry. MRM transition (m/z): the first number is the quantifier ion, and the arrow points to the qualifier ion; LOQ: limit of quantification. View Large Table II. LC-MS/MS acquisition parameters for drug analytes and validation results summary Analyte MRM transition (m/z) Fragmentor voltage (V) Collision energy (V) Retention time (min) Accuracy (%) Precision (RSD) LOQ (ng/mL) Δ9-THC 315→193 150 20 7.2 3.25–6.41 7.28 5.0 315→123 150 30 7.2 6.81 6-acetylmorphine 328.4→165.1 170 44 2.35 −4.81 to 4.96 3.95 5.0 328.4→152.1 170 80 6.99 Acetaminophen 152.1→110.1 100 12 1.26 8.14–11.65 7.62 50.0 152.1→65.1 100 36 4.07 Alprazolam 309.1→205.1 170 48 4.56 0.79–5.43 2.96 5.0 309.1→151.1 170 72 8.69 Amphetamine 136.2→119.1 70 4 2.20 0.86–8.82 2.43 5.0 136.2→91.0 70 20 10.19 Benzoylecgonine 290.3→168.1 135 16 2.71 −1.32 to 3.81 1.94 5.0 290.3→105.0 135 32 10.93 Buprenorphine 468.6→115.1 215 144 4.16 −0.28 to 4.62 5.72 5.0 468.6→101.1 215 44 9.46 Carisoprodol 261.3→176.1 75 4 4.52 0.54–4.39 4.95 5.0 261.3→158.1 75 4 13.17 Clonazepam 316.1→214.1 165 40 4.47 −0.86 to 3.67 7.39 5.0 316.1→151.1 165 88 17.02 Codeine 300.1→152.1 165 76 2.10 −1.39 to 7.39 2.55 10.0 300.1→115.1 165 88 10.71 Cyclobenzaprine 276.1→215.1 135 48 4.36 −3.04 to 6.03 2.55 5.0 276.1→213.1 135 88 8.13 Diazepam 285.1→193.1 160 32 5.19 −1.64 to 7.54 2.41 5.0 285.1→165.1 160 56 9.47 Fentanyl 337.5→188.1 130 20 3.86 −6.44 to 1.48 2.27 1.0 337.5→105.1 130 48 9.80 Gabapentin 172.2→154.1 80 8 2.17 2.84–9.51 2.82 5.0 172.2→55.1 80 24 2.17 4.69 Hydrocodone 300.4→171.1 155 40 2.43 0.63–9.77 4.96 10.0 300.4→128.0 155 76 9.86 Hydromorphone 286.3→157.1 180 44 1.17 0.95–8.08 4.68 10.0 286.3→128.1 180 68 15.12 Meperidine 248.3→174.1 115 16 3.32 −1.18 to 4.68 1.42 5.0 248.3→103.1 115 48 8.38 Methamphetamine 150.2→119.1 65 8 2.41 2.57–10.70 1.71 5.0 150.2→91.1 65 20 9.24 Methadone 310.4→265.2 100 8 4.53 −0.76 to 3.32 2.34 5.0 310.4→105.1 100 24 10.30 Morphine 286.3→165.1 155 48 0.82 −2.39 to 9.15 3.120 10.0 286.3→152.1 155 68 11.45 Naloxone 328.2→310.1 120 16 2.13 5.09–5.17 7.80 5.0 328.2→212.1 120 40 2.13 7.10 o-desmethyl-cis-tramadol 250.8→232.1 110 12 2.44 −3.99 to 2.65 3.17 5.0 250.8→58.2 110 12 14.10 Oxazepam 287.1→163.0 120 40 4.38 −5.43 to 3.32 5.97 5.0 287.1→104.1 120 36 19.53 Oxycodone 316.4→298.1 135 16 2.31 1.07–9.46 3.16 10.0 316.4→212.1 135 44 13.02 Oxymorphone 302.3→198.1 130 44 0.96 −0.34 to 5.18 10.0 302.3→128.1 130 88 10.78 9.47 Tramadol 264.3→58.2 90 16 3.15 0.02–6.80 1.61 5.0 264.3→42.2 90 112 3.15 1.91 Analyte MRM transition (m/z) Fragmentor voltage (V) Collision energy (V) Retention time (min) Accuracy (%) Precision (RSD) LOQ (ng/mL) Δ9-THC 315→193 150 20 7.2 3.25–6.41 7.28 5.0 315→123 150 30 7.2 6.81 6-acetylmorphine 328.4→165.1 170 44 2.35 −4.81 to 4.96 3.95 5.0 328.4→152.1 170 80 6.99 Acetaminophen 152.1→110.1 100 12 1.26 8.14–11.65 7.62 50.0 152.1→65.1 100 36 4.07 Alprazolam 309.1→205.1 170 48 4.56 0.79–5.43 2.96 5.0 309.1→151.1 170 72 8.69 Amphetamine 136.2→119.1 70 4 2.20 0.86–8.82 2.43 5.0 136.2→91.0 70 20 10.19 Benzoylecgonine 290.3→168.1 135 16 2.71 −1.32 to 3.81 1.94 5.0 290.3→105.0 135 32 10.93 Buprenorphine 468.6→115.1 215 144 4.16 −0.28 to 4.62 5.72 5.0 468.6→101.1 215 44 9.46 Carisoprodol 261.3→176.1 75 4 4.52 0.54–4.39 4.95 5.0 261.3→158.1 75 4 13.17 Clonazepam 316.1→214.1 165 40 4.47 −0.86 to 3.67 7.39 5.0 316.1→151.1 165 88 17.02 Codeine 300.1→152.1 165 76 2.10 −1.39 to 7.39 2.55 10.0 300.1→115.1 165 88 10.71 Cyclobenzaprine 276.1→215.1 135 48 4.36 −3.04 to 6.03 2.55 5.0 276.1→213.1 135 88 8.13 Diazepam 285.1→193.1 160 32 5.19 −1.64 to 7.54 2.41 5.0 285.1→165.1 160 56 9.47 Fentanyl 337.5→188.1 130 20 3.86 −6.44 to 1.48 2.27 1.0 337.5→105.1 130 48 9.80 Gabapentin 172.2→154.1 80 8 2.17 2.84–9.51 2.82 5.0 172.2→55.1 80 24 2.17 4.69 Hydrocodone 300.4→171.1 155 40 2.43 0.63–9.77 4.96 10.0 300.4→128.0 155 76 9.86 Hydromorphone 286.3→157.1 180 44 1.17 0.95–8.08 4.68 10.0 286.3→128.1 180 68 15.12 Meperidine 248.3→174.1 115 16 3.32 −1.18 to 4.68 1.42 5.0 248.3→103.1 115 48 8.38 Methamphetamine 150.2→119.1 65 8 2.41 2.57–10.70 1.71 5.0 150.2→91.1 65 20 9.24 Methadone 310.4→265.2 100 8 4.53 −0.76 to 3.32 2.34 5.0 310.4→105.1 100 24 10.30 Morphine 286.3→165.1 155 48 0.82 −2.39 to 9.15 3.120 10.0 286.3→152.1 155 68 11.45 Naloxone 328.2→310.1 120 16 2.13 5.09–5.17 7.80 5.0 328.2→212.1 120 40 2.13 7.10 o-desmethyl-cis-tramadol 250.8→232.1 110 12 2.44 −3.99 to 2.65 3.17 5.0 250.8→58.2 110 12 14.10 Oxazepam 287.1→163.0 120 40 4.38 −5.43 to 3.32 5.97 5.0 287.1→104.1 120 36 19.53 Oxycodone 316.4→298.1 135 16 2.31 1.07–9.46 3.16 10.0 316.4→212.1 135 44 13.02 Oxymorphone 302.3→198.1 130 44 0.96 −0.34 to 5.18 10.0 302.3→128.1 130 88 10.78 9.47 Tramadol 264.3→58.2 90 16 3.15 0.02–6.80 1.61 5.0 264.3→42.2 90 112 3.15 1.91 LC-MS/MS: liquid chromatography coupled with tandem mass spectrometry. MRM transition (m/z): the first number is the quantifier ion, and the arrow points to the qualifier ion; LOQ: limit of quantification. View Large Qualitative analysis of samples PM-01 through PM-15 Among the first 15 cadavers, 48 positive results for drugs were detected in the oral cavity fluids, and 43 positive results in all the other matrices combined (Figure 1). Although oral cavity fluid was screened with both ELISA and LC-MS/MS, only the LC-MS/MS data were compared qualitatively for commonalities and variances between the different matrices, due to the unknown volumes of oral cavity fluid collected from the cadavers. Of these 15 cases, two had decomposed (2 days and 10 days, respectively), and oral cavity fluid was compared to blood or bile. In these cases, we detected the following drugs: benzoylecgonine and fentanyl (oral cavity fluid and blood), morphine (only oral cavity fluid), hydrocodone (oral cavity fluid and bile) and hydromorphone (only bile). Lastly, in two separate cases, only blood samples showed positive results for clonazepam (ante mortem) and oxymorphone (reported as an interfering substance). Figure 1. View largeDownload slide Distribution of drugs found in oral fluids compared to four other matrices in 15 cadavers. Oral fluids were analyzed with LC-MS/MS. Standard assays were used for analyzing blood, urine, bile, and liver tissue. 6-AM: 6-acetyl-morphine; BE: benzoylecgonine; LC-MS/MS: liquid chromatography coupled with tandem mass spectrometry. Figure 1. View largeDownload slide Distribution of drugs found in oral fluids compared to four other matrices in 15 cadavers. Oral fluids were analyzed with LC-MS/MS. Standard assays were used for analyzing blood, urine, bile, and liver tissue. 6-AM: 6-acetyl-morphine; BE: benzoylecgonine; LC-MS/MS: liquid chromatography coupled with tandem mass spectrometry. Quantitative analysis for PM-16 through PM-34 The successful detection of drugs in the first 15 cadavers satisfied our first objective. Therefore, we analyzed the remaining samples only with LC-MS/MS (Tables III and IV). Oral cavity fluid and blood were collected from cadavers PM-33 and PM-34 after they had been submerged in a river for 5 and 10 days, respectively. In these cases, the medical examiner disclosed that the river water was drained from each decedent’s mouth prior to the collections. In these cadavers, we detected the following analytes: methamphetamine in PM-33, and amphetamine and Δ9-THC in PM-34. Figure 2 illustrates chromatograms for the analytes reported in PM-34. These findings were considered relevant and useful to forensic investigators and professionals, because they demonstrated that oral cavity fluid was a viable matrix, which could be used in addition to traditional matrices. Table III. Concentrations of analytes (ng/mL) in oral fluids vs. traditional matrices in cadavers PM-16 to PM-23 Analyte Cadaver ID PM-16 PM-17 PM-18 PM-19 PM-20 PM-21 PM-22 PM-23 6-Acetylmorphine 195.7 147.3 938.8 30.1 0.0† 250‡ 0.0† 0.0†,‡ Δ9-THC 16.5 174.4 6.0† 2.1† Acetaminophen 241.2 0.0†,‡ Alprazolam 0.0* 4.1† 221‡ Amphetamine 100.9 109.5 3433‡ 29† Benzoylecgonine 727.6 308.3 656† 313† 39616‡ 3965‡ Codeine 69.9 0.0† Cyclobenzaprine 8.3 0.0†,‡ Diazepam >1000 27.4 539† 116† Fentanyl 93.8 39.5 162.6 108.9 237.4 9.9† 3.2† 8.5† 7.4† 11.1† >100‡ >100‡ 835‡ Gabapentin >1000 10.2† Hydrocodone 24.3 302.0‡ Hydromorphone 0.0 191‡ Lorazepam 0.0 587‡ Methamphetamine 19.6 992.2 651.4 0.0†,‡ 209.0† >2000‡ 153† Morphine 967.1 149.5 >1000 10.7 1724‡ 20.4† 91† 0.0†,‡ 4,034‡ Naloxone 12.1 0.0† o-Desmethyl-cis-tramadol 21.2 1678‡ Oxazepam 0.0 52‡ Oxycodone 11.5 0.0†,‡ Tramadol 112 9750‡ Analyte Cadaver ID PM-16 PM-17 PM-18 PM-19 PM-20 PM-21 PM-22 PM-23 6-Acetylmorphine 195.7 147.3 938.8 30.1 0.0† 250‡ 0.0† 0.0†,‡ Δ9-THC 16.5 174.4 6.0† 2.1† Acetaminophen 241.2 0.0†,‡ Alprazolam 0.0* 4.1† 221‡ Amphetamine 100.9 109.5 3433‡ 29† Benzoylecgonine 727.6 308.3 656† 313† 39616‡ 3965‡ Codeine 69.9 0.0† Cyclobenzaprine 8.3 0.0†,‡ Diazepam >1000 27.4 539† 116† Fentanyl 93.8 39.5 162.6 108.9 237.4 9.9† 3.2† 8.5† 7.4† 11.1† >100‡ >100‡ 835‡ Gabapentin >1000 10.2† Hydrocodone 24.3 302.0‡ Hydromorphone 0.0 191‡ Lorazepam 0.0 587‡ Methamphetamine 19.6 992.2 651.4 0.0†,‡ 209.0† >2000‡ 153† Morphine 967.1 149.5 >1000 10.7 1724‡ 20.4† 91† 0.0†,‡ 4,034‡ Naloxone 12.1 0.0† o-Desmethyl-cis-tramadol 21.2 1678‡ Oxazepam 0.0 52‡ Oxycodone 11.5 0.0†,‡ Tramadol 112 9750‡ *LOQ: Limit of quantification. Two values are shown in each row: the first value represents the oral fluids; the second value represents †blood or ‡urine. View Large Table III. Concentrations of analytes (ng/mL) in oral fluids vs. traditional matrices in cadavers PM-16 to PM-23 Analyte Cadaver ID PM-16 PM-17 PM-18 PM-19 PM-20 PM-21 PM-22 PM-23 6-Acetylmorphine 195.7 147.3 938.8 30.1 0.0† 250‡ 0.0† 0.0†,‡ Δ9-THC 16.5 174.4 6.0† 2.1† Acetaminophen 241.2 0.0†,‡ Alprazolam 0.0* 4.1† 221‡ Amphetamine 100.9 109.5 3433‡ 29† Benzoylecgonine 727.6 308.3 656† 313† 39616‡ 3965‡ Codeine 69.9 0.0† Cyclobenzaprine 8.3 0.0†,‡ Diazepam >1000 27.4 539† 116† Fentanyl 93.8 39.5 162.6 108.9 237.4 9.9† 3.2† 8.5† 7.4† 11.1† >100‡ >100‡ 835‡ Gabapentin >1000 10.2† Hydrocodone 24.3 302.0‡ Hydromorphone 0.0 191‡ Lorazepam 0.0 587‡ Methamphetamine 19.6 992.2 651.4 0.0†,‡ 209.0† >2000‡ 153† Morphine 967.1 149.5 >1000 10.7 1724‡ 20.4† 91† 0.0†,‡ 4,034‡ Naloxone 12.1 0.0† o-Desmethyl-cis-tramadol 21.2 1678‡ Oxazepam 0.0 52‡ Oxycodone 11.5 0.0†,‡ Tramadol 112 9750‡ Analyte Cadaver ID PM-16 PM-17 PM-18 PM-19 PM-20 PM-21 PM-22 PM-23 6-Acetylmorphine 195.7 147.3 938.8 30.1 0.0† 250‡ 0.0† 0.0†,‡ Δ9-THC 16.5 174.4 6.0† 2.1† Acetaminophen 241.2 0.0†,‡ Alprazolam 0.0* 4.1† 221‡ Amphetamine 100.9 109.5 3433‡ 29† Benzoylecgonine 727.6 308.3 656† 313† 39616‡ 3965‡ Codeine 69.9 0.0† Cyclobenzaprine 8.3 0.0†,‡ Diazepam >1000 27.4 539† 116† Fentanyl 93.8 39.5 162.6 108.9 237.4 9.9† 3.2† 8.5† 7.4† 11.1† >100‡ >100‡ 835‡ Gabapentin >1000 10.2† Hydrocodone 24.3 302.0‡ Hydromorphone 0.0 191‡ Lorazepam 0.0 587‡ Methamphetamine 19.6 992.2 651.4 0.0†,‡ 209.0† >2000‡ 153† Morphine 967.1 149.5 >1000 10.7 1724‡ 20.4† 91† 0.0†,‡ 4,034‡ Naloxone 12.1 0.0† o-Desmethyl-cis-tramadol 21.2 1678‡ Oxazepam 0.0 52‡ Oxycodone 11.5 0.0†,‡ Tramadol 112 9750‡ *LOQ: Limit of quantification. Two values are shown in each row: the first value represents the oral fluids; the second value represents †blood or ‡urine. View Large Table IV. Concentration of analytes (ng/mL) in oral fluids vs. traditional matrices in cadavers (cadaver IDs indicated) Analyte Cadaver ID PM-24 PM-26 PM-27 PM-28 PM-29 PM-31 PM-32 Δ9-THC 0.0* 16.5 160.7 4.1† 6.0† 0.0† Acetaminophen >1000 >1000 0.0† 0.0‡ Amphetamine 7.0 0.0† Benzoylecgonine 6.7 276.7 0.0†,§ 360† Fentanyl 2.6 29.5 0.0† 8.2† Gabapentin >1000 >1000 10.2† 0.0‡ Hydrocodone 19.4 0.0† Meperidine 8.7 0.0‡ Methamphetamine 26.3 0.0† Morphine 0.0* 1525‡ Oxycodone >1000 682.3 87.2† 270† Analyte Cadaver ID PM-24 PM-26 PM-27 PM-28 PM-29 PM-31 PM-32 Δ9-THC 0.0* 16.5 160.7 4.1† 6.0† 0.0† Acetaminophen >1000 >1000 0.0† 0.0‡ Amphetamine 7.0 0.0† Benzoylecgonine 6.7 276.7 0.0†,§ 360† Fentanyl 2.6 29.5 0.0† 8.2† Gabapentin >1000 >1000 10.2† 0.0‡ Hydrocodone 19.4 0.0† Meperidine 8.7 0.0‡ Methamphetamine 26.3 0.0† Morphine 0.0* 1525‡ Oxycodone >1000 682.3 87.2† 270† *LOQ: Limit of quantification. Two values are shown in each row: the first value represents the oral fluids; the second value represents †blood, ‡urine or §vitreous humor. View Large Table IV. Concentration of analytes (ng/mL) in oral fluids vs. traditional matrices in cadavers (cadaver IDs indicated) Analyte Cadaver ID PM-24 PM-26 PM-27 PM-28 PM-29 PM-31 PM-32 Δ9-THC 0.0* 16.5 160.7 4.1† 6.0† 0.0† Acetaminophen >1000 >1000 0.0† 0.0‡ Amphetamine 7.0 0.0† Benzoylecgonine 6.7 276.7 0.0†,§ 360† Fentanyl 2.6 29.5 0.0† 8.2† Gabapentin >1000 >1000 10.2† 0.0‡ Hydrocodone 19.4 0.0† Meperidine 8.7 0.0‡ Methamphetamine 26.3 0.0† Morphine 0.0* 1525‡ Oxycodone >1000 682.3 87.2† 270† Analyte Cadaver ID PM-24 PM-26 PM-27 PM-28 PM-29 PM-31 PM-32 Δ9-THC 0.0* 16.5 160.7 4.1† 6.0† 0.0† Acetaminophen >1000 >1000 0.0† 0.0‡ Amphetamine 7.0 0.0† Benzoylecgonine 6.7 276.7 0.0†,§ 360† Fentanyl 2.6 29.5 0.0† 8.2† Gabapentin >1000 >1000 10.2† 0.0‡ Hydrocodone 19.4 0.0† Meperidine 8.7 0.0‡ Methamphetamine 26.3 0.0† Morphine 0.0* 1525‡ Oxycodone >1000 682.3 87.2† 270† *LOQ: Limit of quantification. Two values are shown in each row: the first value represents the oral fluids; the second value represents †blood, ‡urine or §vitreous humor. View Large Figure 2. View largeDownload slide LC-MS/MS chromatograms of oral fluid samples collected from one deceased subject (PM-34) that was drowned. Measurements are shown for (A) amphetamine (52.7 ng/mL), (B) methamphetamine (600.3 ng/mL) and (C) Δ9-THC (477 ng/mL). LC-MS/MS: liquid chromatography coupled with tandem mass spectrometry. Figure 2. View largeDownload slide LC-MS/MS chromatograms of oral fluid samples collected from one deceased subject (PM-34) that was drowned. Measurements are shown for (A) amphetamine (52.7 ng/mL), (B) methamphetamine (600.3 ng/mL) and (C) Δ9-THC (477 ng/mL). LC-MS/MS: liquid chromatography coupled with tandem mass spectrometry. In cadavers PM-17, PM-19 and PM-21, the 6-AM analyte would not have been quantified if the analyses only included blood and/or urine. Moreover, in PM-19, we quantified codeine and morphine in oral cavity fluid. Although codeine is a legal drug used in treatments for coughs, diarrhea, and moderate pain, it also is used recreationally. Knowledge of the metabolism of codeine can prevent mistaking its association with legitimate medical uses for its association with the abuse of heroin or morphine. In PM-26, benzoylecgonine was only quantified in oral cavity fluid; the blood showed a negative result for this analyte. Because the blood showed a negative result, the vitreous fluid (collected as a backup sample) was not analyzed. However, in this instance, the negative blood results may have been affected by postmortem changes, such as redistribution and hemoconcentration (5). Although vitreous fluid is considered an ideal alternative to urine and blood specimens for postmortem chemical analyses (6), for reasons unknown, it was not studied as an alternative in this case. In three instances, we did not report alprazolam, Δ9-THC, or morphine in oral cavity fluids, because the concentrations were below our established LOQ. Finally, three analytes (two benzodiazepines and one opiate) were only quantified in urine. This was not surprising, because benzodiazepines are excreted primarily in urine, and hydromorphone is a metabolite. We detected no analytes in cadavers PM-25 and PM-30. Discussion Multiple oral fluid collection devices are currently available on the market; however, many have been withdrawn or modified (7). Our laboratory chose the Quantisal® device based upon its scientific advantages, which included its efficiency, simplicity, and efficacy in recovering drugs, even at low concentrations (e.g., Δ9-THC). An additional study was conducted by our laboratory to provide evidence of drug substance stability utilizing 10 decedents from this study after being stored in a freezer at −25°C. All original positive samples remained positive after 10 months. All drugs, including THC, remained stable in this device during shipping, without additional refrigerated packaging. In the clinical setting, participants are instructed not to remove the collector until either the indicator turns blue or 10 min have passed. This device has a unique volume adequacy indicator on the stem of the wand that turns blue when 1 mL (±10%) of oral fluid has been collected. This feature ensures that sufficient volume of specimen is collected for screening, confirmation, and repeat testing. For the fluid collection in our first cadaver (PM-01), the stem of the collector did not turn blue after approximately 10 min. Therefore, the collector was removed to check for pad saturation, but it appeared to be dry, and the sample was discarded. A new collector was placed into the collection site on the opposite side of the mouth, and it remained in position for approximately 15 min. Again, the indicator did not turn blue, and it was removed to check for saturation. However, that time, the pad was visibly saturated. Based on these findings, the first collector should not have been discarded. Therefore, we decided that all collectors should remain in the buccal cavity until the indicator turned blue or for at least 15 min. Furthermore, in cadavers PM-02 through PM-15, the indicator never turned blue. Therefore, for the first 15 cadavers, we recognized that the drug concentrations in the oral cavity fluid samples could not be compared quantitatively to the concentrations in other matrices collected, due to the unknown volumes of oral cavity fluid collected. This critical observation that the indicator did not always turn blue led us to develop our correction protocol. The protocol was developed for use by this laboratory because field investigators would not have the ability to weigh each collection tube prior to use. We summed the average weights of 50 transport tubes (containing the buffer) and 50 dry collection wands. This sum gave us the average collection device weight (9.9113 g). This device weight was then subtracted from the weight of the collection device after sample collection to give the sample weight: Sample weight (g) = (sample + device) weight – average device weight. The inverse of the sample weight then was taken to obtain the correction factor (i.e., 1/sample weight). The final analyte concentrations (ng/mL) were adjusted by multiplying each analyte concentration by the correction factor. The sampling compartment is assumed to be related to the drug concentration at the site of action. However, compartmental barriers lose their integrity after death, because as time proceeds, rapid reductions in pH cause autolysis (8, 9). This process may alter the concentration of one or more drugs that were originally contained in intact compartments (8). Future investigations might include measuring amylase and/or the pH of the oral cavity fluid from cadavers. The ability to evaluate the presence of drugs in postmortem subjects demonstrated by this investigation may provide invaluable information into recent drug deaths (e.g., 6-AM), stability of drugs, and/or the contents of fluid containing saliva. During this study, our laboratory found variable, and sometimes inconsistent results between the matrices tested. This variability may have been due to varying conditions in sample matrices, collection procedures and/or analytical methods. Consequently, the sensitivity in detecting particular drugs may have been reduced, which could explain the inability to detect a given drug in some matrices. The time has come to provide an alternative matrix for key stakeholders as well as access to real-time data from the detection and quantification of medicinal and illicit drugs in apparent or suspected drug deaths (10). The major advantage of this method and collection procedure is minimal sample volume and preparation and rapid run time. The described analytical method was fully validated showing to be selective, precise, and accurate for the determination of 47 analytes in oral cavity fluid tested. We found that the quantification of drugs in oral cavity fluid generally was comparable to quantifications in traditional biological matrices. Moreover, we demonstrated that oral cavity fluid could easily and safely be collected (without the use of disposable needles). Finally, in cases where blood, vomit and maggots were present in the oral cavity, and that samples in decomposed cadavers were viable over time. Acknowledgments The authors would like to thank the professional and support teams of CWH & Pathology Associates, Inc. and the following county coroners in Ohio and Pennsylvania (Columbiana, Ashtabula, Carroll, and Montour, Fayette and Lycoming, respectively). We would also like to thank SteelFusion Clinical Toxicology Laboratory, LLC for their valuable assistance with the collection of oral cavity fluid samples and the documentation of results during examinations. Funding This work did not receive any external funding. References 1 Crouch , D.J. ( 2005 ) Oral fluid collection: the neglected variable in oral fluid testing . Forensic Science International , 150 , 165 – 173 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Sethi , S. , Goel , P. , Bhalla , S. ( 2015 ) Oral cavity: an insight to forensic diagnosis . Asia Pacific Journal of Public Health , 2 , 142 – 147 . WorldCat 3 Honey , D. , Caylor , C. , Luthi , R. , Kerrigan , S. ( 2005 ) Comparative alcohol concentrations in blood and vitreous fluid with illustrative case studies . Journal of Analytical Toxicology , 29 , 365 – 369 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Scientific Working Group for Forensic Toxicology . ( 2013 ) Scientific working group for forensic toxicology (SWGTOX) standard practices for method validation in forensic toxicology . Journal of Analytical Toxicology , 37 , 452 – 474 . Crossref Search ADS PubMed WorldCat 5 Schramm , W. , Craig , P.A. , Smith , R.H. , Berger , G.E. ( 1993 ) Cocaine and benzoylecgonine in saliva, serum, and urine . Clinical Chemistry , 39 , 481 – 487 . Google Scholar PubMed WorldCat 6 Horning , M.G. , Brown , L. , Nowlin , J. , Lertratanangkoon , K. ( 1977 ) Use of saliva in therapeutic drug monitoring . Clinical Chemistry , 23 , 157 – 164 . Google Scholar PubMed WorldCat 7 Pirro , V. , Jarmusch , A.K. , Vincenti , M. , Cooks , R.G. ( 2015 ) Direct drug analysis from oral fluid using medical swab touch spray mass spectrometry . Analytica Chimica Acta , 861 , 47 – 54 . Google Scholar Crossref Search ADS PubMed WorldCat 8 Huestis , M.A. ( 2009 ) A new ultraperformance–tandem mass spectrometry oral fluid assay for 29 illicit drugs and medications . Clinical Chemistry , 55 , 2079 – 2081 . Google Scholar Crossref Search ADS PubMed WorldCat 9 Buyuk , Y. , Eke , M. , Cagdir , S. , Karaaslan , H. ( 2009 ) Post-mortem alcohol analysis in synovial fluid: an alternative method for estimation of blood alcohol level in medico-legal autopsies? Toxicology Mechanisms and Methods , 19 , 375 – 378 . Google Scholar Crossref Search ADS PubMed WorldCat 10 Morrow , J.B. , Ropero-Miller , J.D. , Catlin , M.L. , Winokur , A.D. , Cadwallader , A.B. , Staymates , J.L. , et al. . ( 2019 ) The opioid epidemic: moving toward an integrated, holistic analytical response . Journal of Analytical Toxicology , 43 , 1 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2019. Published by Oxford University Press. All rights reserved. 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TI - Oral Cavity Fluid as an Investigative Approach for Qualitative and Quantitative Evaluations of Drugs in Postmortem Subjects
JF - Journal of Analytical Toxicology
DO - 10.1093/jat/bkz032
DA - 2019-07-24
UR - https://www.deepdyve.com/lp/oxford-university-press/oral-cavity-fluid-as-an-investigative-approach-for-qualitative-and-3ghx2K0X4s
SP - 444
VL - 43
IS - 6
DP - DeepDyve
ER -