Research article Received: 15 August 2013
Revised: 21 July 2014
Accepted: 7 August 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3464
Determination of drugs of abuse in bovine dentin using liquid chromatography– electrospray ionization tandem mass spectrometry J. Spinner,a,b M. Klima,a J. Kempf,a L. M. Huppertz,a V. Auwärter,a M. J. Altenburgerc and M. A. Neukamma* Drugs deposited in human teeth are well preserved; the spectrum of toxicological investigations may therefore be supplemented by an analysis method for drugs in teeth. A liquid chromatography–electrospray ionization tandem mass spectrometry assay for the detection and quantification of basic drugs of abuse in bovine dentin samples was developed and validated. The drugs and metabolites amphetamine, methamphetamine, methylenedioxymethylamphetamine, methylenedioxyethylamphetamine, codeine, morphine, cocaine and benzoylecgonine were extracted from 50 mg ground dentin powder by ultrasonication for 60 min in methanol 3 times. The extracts were analyzed on a triple-quadrupole mass-spectrometer in multiple reaction monitoring mode. The method was validated and proved to be accurate, precise, selective, specific and stable with good linearity within the calibration range and a lower limit of quantification of 10 to 20 pg/mg. To artificially load bovine dentin samples with drugs, the natural process of de- and remineralization in the oral cavity was mimicked by a pH-cycling experiment. The artificially drug-loaded dentin samples showed drug concentrations of 20 to 80 pg/mg. The method can be applied in further in vitro experiments as well as in post-mortem cases, especially where limited sample tissue is available. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: LC-ESI-MS/MS; dentin; dental hard tissue; drugs of abuse; alternative matrix
Introduction
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To investigate fatal poisonings with drugs, it is crucial to gain information about the drug intake of a person. However, in cases of severely putrefied bodies – e.g. after longer post mortem intervals or exhumation – no suitable samples of body fluids and sometimes also no organ tissues can be obtained for chemical–toxicological analysis. In teeth, however, deposited drugs or poisons are likely to be protected from biological or chemical degradation.[1,2] Morphine, for example, can be detected in teeth of persons who died of heroine overdose at least for 2 years after death.[3] Therefore, the analysis of dental hard tissue (dentin, enamel) may allow a retrospective view on the drug intake of a person. The whole tooth consists of the hard tissues (enamel and dentin), the pulp tissue in the inside of the tooth and the overlaying cementum with the periodontal ligament (see Fig. 1). Consumed substances can have contact with the dental tissue in two ways. First, from the oral cavity, the oral fluid (and its content) has contact with the enamel or exposed dentin. Second, from the inside: the blood vessels in the pulp have contact with the inner surface of the dentin. In the oral cavity the pH declines due to exogenous acidic compounds (e.g. from food) and endogenous acidic compounds secreted by the cariogenic biofilm. Subsequently, calcium and phosphate dissolve from enamel and dentin. When the biofilm
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and other acidic compounds are removed, the pH increases and remineralization takes place. Saliva, which is a supersaturated solution of calcium and phosphate, leads then to a reuptake of minerals into dental hard tissues. During this remineralization phase also other substances present in oral fluid like pigments from food, beverages (e.g. black tea or red wine), tobacco or spices are visibly adsorbed on the surface of teeth or deposited in teeth.[4,5] Haustein et al. studied the transport of substances through the enamel and dentin into intact and carious teeth in vitro.[6] The authors investigated some low aliphatic alcohols, organic acids, barbital, procaine and some sugar derivatives. All substances penetrated the enamel, which is regarded to behave like a porous and ion-selective membrane. The penetration rates depended on the pH of the incubation medium and on the lipophilicity of the substance. Schüssl et al. found that after a single
* Correspondence to: M. A. Neukamm, Institute of Forensic Medicine, University Medical Center Freiburg, Freiburg, Germany. E-mail: [email protected] a Institute of Forensic Medicine, University Medical Center Freiburg, Freiburg, Germany b Institute of Forensic Medicine, University of Bern, Bern, Switzerland c Department of Operative Dentistry and Periodontology, University Medical Center Freiburg, Freiburg, Germany
Copyright © 2014 John Wiley & Sons, Ltd.
Drugs of abuse in dentin using LC-ESI-MS/MS methylenedioxyethylamphetamine (MDEA), methylenedioxymethylamphetamine (MDMA), codeine, morphine, cocaine, benzoylecgonine) in dentin was developed. The method was applied to drug-positive bovine dentin samples, produced by a pH-cycling experiment, which mimicked the natural process of de- and remineralization in the oral cavity in a standardized way. The pH-cycling mimics exogenous diffusion of substances into dentin via drug-containing saliva. Without the influences of, e.g. oral fluid or biofilm on the tooth surface, the incorporation characteristics of different substances into dentin can be compared directly.
Materials and methods Chemicals and reference standards
Figure 1. Anatomy of the tooth and the periodont. Contact with drugs takes place via the blood stream and via the oral fluid (figure modified, original: Jordi March i Nogué [CC-BY-SA-3.0 (http://creativecommons.org/ licenses/by-sa/3.0) or GFDL (http://www.gnu.org/copyleft/fdl.html)] via Wikimedia Commons).
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Preparation of the stock solution of standards To prepare the stock solution of standards, appropriate volumes of reference solutions were mixed and diluted with methanol to a final concentration of 1.0 μg/ml. The stability of the stock solution for 5 months was proven.
Preparation of working solutions To prepare the working solutions I and II, the stock solution of standards was diluted with methanol to contain 50 ng/ml (I) or 100 ng/ml (II) of each analyte. To prepare the internal standard working solution, appropriate volumes of reference solutions of deuterated standards were mixed and diluted with methanol to a final concentration of 100 ng/ml of each compound.
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dose of the antibiotics amoxicillin and clindamycin, the concentration in the root of a tooth correlated with the serum concentration. The antibiotics were determined by a quantitative liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method.[7] In toxicology literature, there are not many publications focusing on the use of teeth as analytical specimen. Besides a study on the assessment of passive smoke exposure of children by analysis of nicotine and cotinine in deciduous teeth,[8,9] there is a forensic study on the determination of alcohol intake behaviour by analysis of ethyl glucuronide in dental hard tissue.[10] For drugs of abuse, to our knowledge there are only studies on the determination of opiates (pilot study[3]) and cocaine.[11] In these studies[3,8,9,11] at least 1 g of human dental material was used. Also the dental pulp – which is interspersed by blood vessels – was apparently not removed. Hence, it can not be determined if the detected substances were a deposit from blood circulation in the pulp chamber or were incorporated due to remineralization processes. For the use of teeth as a forensic-toxicological matrix it is necessary to investigate the quantitative relationship between drug exposure and determined concentrations, as well as to basically understand the mechanisms of deposition. Only if this is understood, valid conclusions can be drawn on the drug intake history of a person. As a basis for the study on the general and substancespecific mechanisms of deposition, an LC-ESI-MS/MS method for the quantitative determination of different substances relevant in forensic toxicology (amphetamine, methamphetamine,
All solvents and substances were at least of analytical or HPLC grade. Formic acid, 2-(4-(2-hydroxy ethyl)-1-piperazinyl) ethanesulfonic acid, isopropanol and potassium chloride were purchased from Carl Roth GmbH (Karlsruhe, Germany). Ammonium formate, hydrochloric acid, methylhydroxydiphosphonate (MHDP) and lactic acid were obtained from Sigma-Aldrich (Steinheim, Germany). Potassium dihydrogen phosphate, calcium chloride, calcium chloride dihydrate and acetic acid were from Merck (Darmstadt, Germany). Purified water (Aqua bidest) was obtained from Fresenius Kabi AG, Bad Homburg auf der Höhe, Germany. Methanol was purchased from Chem solute (Th. Geyer, Renningen, Germany). Technovit 4071 Liquid and Technovit 4071 chemically setting resin powder was from Heraeus Kulzer (Hanau, Germany), and thymol was from AppliChem (Darmstadt, Germany). Deionized water for preparation of the LC mobile phase was prepared with a cartridge deionizer from Memtech (Moorenweis, Germany). Amphetamine, amphetamine-d5, benzoylecgonine, benzoylecgonine-d3, cocaine, cocaine-d3, codeine, codeine-d3, MDEA, MDEA-d5, MDMA, MDMA-d5, methamphetamine, methamphetamine-d5, morphine and morphine-d3 were purchased from LGCStandards (Wesel, Germany). All non deuterated standards except cocaine were obtained as certified solutions with the concentration 1.0 mg/ml in methanol; cocaine was obtained as certified solution with the concentration 1.0 mg/ml in acetonitrile. All deuterated standards except cocaine-d3 were obtained as certified solutions with the concentration of 100 μg/ml in methanol; cocaine-d3 was obtained as certified solution with the concentration 100 μg/ml in acetonitrile.
J. Spinner et al. Calibration standards and controls The dentin calibration standards (10, 30, 50, 70 and 90 pg/mg for methamphetamine, MDMA, MDEA benzoylecgonine, morphine and codeine; 30, 50, 70 and 90 pg/mg for amphetamine and cocaine) were prepared by adding adequate amounts of the working solution I or II and 10 μl of the internal standard working solution to 50 mg of drug-free dentin powder prior to extraction. The quality control samples with concentrations of 20 pg/mg and 80 pg/mg were prepared in the same manner using separate working solutions. The calibration range was chosen to cover the expected concentrations in artificial drug-positive samples. Drug-free bovine dentin powder was prepared as follows: dentin pellets were cooled with liquid nitrogen and ground using a ball mill (MM2, Retsch, Haan, Germany) for 15 min. Matrix free solvent calibration standards (10, 30, 50, 70 and 90 pg/mg, 30, 50, 70 and 90 pg/mg for amphetamine and cocaine) were prepared by adding adequate amounts of the working solution I or II and 10 μl of the internal standard working solution to adequate amounts of the liquid chromatography solvent A/B 95/5 (v/v) to yield 100 μl. Solvent A consisted of water with 2% formic acid and 2.0 mmol/l ammonium formate and solvent B was methanol with 0.1% formic acid.
incubated for 9 days with four cycles of demineralization (0.5 h each) and remineralization (2.5 h each). During night time, the pellets were stored in remineralization solution for 12 h. The pellets were rinsed with purified water after each step. After 36 h, the demineralization and remineralization solutions were renewed. After pH-cycling, the pellets were rinsed again with purified water and then dried in the oven at 37 °C for approx. 20 h and stored in closed tubes at 20 °C until analysis. The stability of all analytes in the drugcontaining remineralization solution was checked. All analytes were stable for at least 10 days, so the stability of the substances during the pH-cycling experiment is assured. Method for stability testing: A volume of 10 ml of the drugcontaining remineralization solution was stored in a clear glass bottle at room temperature. Aliquots of 400 μl were sampled every 24 h for 10 days. Another 400 μl was sampled after another approximately 10 and 20 days, respectively. Stability was presumed if the percental decline of the peak area of the analyte was below the imprecision of the method. All analytes were stable for at least 10 days. Sample extraction Preparation of dentin powder
Preparation of dentin pellets Bovine dentin can be used as an alternative for human dental material.[12] Dentin was obtained from freshly extracted bovine incisors of the second dentition tested negative for Bovine Spongiform Encephalopathy. The teeth were stored in 0.1% thymol at 8 °C before further processing. Round specimens, 3.5 mm in diameter, were drilled out of each tooth’s root using a custom-made trephine bur (Gebr. Brasseler, Lembo, Germany). After removing tissue residues the samples were embedded in chemically setting resin (Technovit 4071). The pellets were subsequently polished with the grinding machine and wet sand paper to a grit of 4000 to remove root cement and to create uniform dentin pellets. After removing the resin, the pellets were ultrasonicated for 10 min at 20 °C in purified water to clean the pellets from grinding sludge. Preparation of artificial drug-positive dentin samples
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To create uniform initial carious-like lesions, the dentin pellets were demineralized at room temperature for 7 days in a demineralization solution according to Buskes et al.[13] containing 3 mM calcium chloride dihydrate, 3 mM potassium dihydrogen phosphate, 60 μM MHDP, 10% lactic acid (40%) and traces of thymol. A pH of 5 was set by adding the adequate amount of potassium hydroxide (5 M). Lesion formation was verified by light microscopy and transverse microradiography (TMR, Inspector Research, Amsterdam, The Netherlands). After demineralization, the samples were rinsed with purified water and dried at 37 °C for 20 h. To simulate the natural process of de- and remineralization in the oral cavity, pH-cycling was performed according to ten Cate et al.[14] as follows: 25 pre-demineralized dentin pellets of bovine teeth were incubated alternately with a demineralization solution without analytes (pH = 4.8) and a remineralization solution containing the analytes (pH = 7). The remineralization solution contained 1.0 mg/l of the following drugs: amphetamine, methamphetamine, MDMA, MDEA, codeine, morphine, cocaine and benzoylecgonine. The pellets were
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Dentin pellets were cooled with liquid nitrogen and ground using a ball mill (MM2, Retsch, Haan, Germany) for 15 min. To 50 mg of dentin powder 10 μl of the internal standard solution was added. Extraction The following steps were executed three times: The powder was suspended with 500 μl of methanol. The sample was vortexed and extracted in an ultrasonic bath for 60 min at room temperature (the temperature of the bath rose during the ultrasonication from about 22 °C to maximal 45 °C). The extraction time was optimized (see section method optimization). After centrifugation, the supernatant was collected. To complete the extraction cycle, the residue was resuspended in 100 μl of methanol and vortexed. After centrifugation, the supernatants were combined. The combined supernatants of three extraction cycles were reduced under a gentle stream of nitrogen at 40 °C to a small residual volume (approximately 2 μl) (the number of extraction cycles was optimized, see section method optimization). After addition of 100 μl of a mixture of isopropanol:HCl 3/1 (v/v) to prevent the evaporation of amphetamines, the solution was evaporated to dryness. The dry residue was reconstituted in 100 μl of liquid chromatography solvents A/B 95/5 (v/v). Method optimization To 50 mg of artificial drug-positive dentin powder 500 μl of methanol and 10 μl of the internal standard working solution were added. To evaluate the optimal extraction procedure, each sample was extracted in 1 to 5 cycles in an ultrasonic bath for the extraction time of 10 min, 30 min or 60 min, respectively. The experiment was carried out with three powder samples for each extraction time and cycle number. The samples were analyzed and the peak area ratios of analyte to internal standard of each extraction time and cycle number were compared.
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Drugs of abuse in dentin using LC-ESI-MS/MS Liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) The LC-ESI-MS/MS system included an API 5000 triple quadrupole mass spectrometer (AB Sciex, Darmstadt, Germany) fitted with a Shimadzu HPLC system consisting of 3 LC-10ADVP binary pumps, a CTO-10 AC column oven, a HTC PAL autosampler and SCL-10AVP controller. Chromatographic separation of all substances was achieved using a Luna PFP column (150 mm × 2 mm, 5 μm) with a guard column with the same stationary phase (4 mm × 2 mm) (Phenomenex, Aschaffenburg, Germany) and gradient elution. The gradient was as follows: initially, 5% of solvent B at a flow rate of 0.4 ml/min. During 10 min solvent B increased up to 98% at a flow rate of 0.6 ml/min which was kept for 2.5 min. The flow rate was decreased to 0.4 ml/min again within 0.5 min at 98% B. Initial conditions were restored and held for 2 min to re-equilibrate the system. Post column addition of isopropanol at a flow rate of 0.1 ml/min was applied to improve sensitivity. The injection volume was 20 μl. The respective multiple reaction monitoring (MRM) transitions and the corresponding potentials for all analytes and internal standards are shown in Table 1. The mass spectrometer was operated in positive ionization mode. The ion spray voltage was set to 1500 V. The gas settings were as follows: curtain gas: 20 psi; collision gas: 9 psi, ion source gas 1: 60 psi; ion source gas 2: 70 psi. The ion source temperature was set to 500 °C. Data acquisition and analysis For data acquisition, the software Analyst 1.5.1 (AB Sciex, Darmstadt, Germany) was applied. For data analysis, Analyst 1.5.1 and Valistat 2.0 (Arvecon GmbH, Walldorf, Germany) were used.
Method validation The LC-ESI-MS/MS method was validated for the quantification of the chosen drugs in dentin according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh).[15] Therefore, selectivity, linearity, limit of detection, limit of quantification, accuracy and precision, autosampler stability, stability at 2–8 °C, matrix effects,[16] extraction recovery, overall process efficiency and application of a solvent calibration were examined. Selectivity To assess potential interferences with common drugs of abuse, endogenous compounds as well as reagents and materials used for sample preparation the following samples were analyzed: six drug-free dentin samples without the addition of standards, two drug-free dentin samples fortified with internal standards as well as one drug-free dentin sample fortified with the following drugs or metabolites in the concentration range of 20 pg/mg to 16 ng/mg: cannabinoids: Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9tetrahydrocannabinol, 11-nor-Δ9-carboxytetrahydrocannabinol; benzodiazepines and structurally unrelated agonists of the GABA receptor complex: 7-aminoclonazepam, 7-aminoflunitrazepam, alprazolam, α-hydroxyalprazolam, bromazepam, brotizolam, camazepam, chlordiazepoxide, clobazam, clonazepam, clotiazepam, delorazepam, diazepam, estazolam, flunitrazepam, flurazepam, lorazepam, lormetazepam, medazepam, midazolam, nitrazepam, nordazepam, norflunitrazepam, oxazepam, temazepam, tetrazepam, triazolam, α-hydroxytriazolam, zaleplon, zolpidem, zopiclone; synthetic cannabinoids: JWH-007, JWH-015, JWH-018, JWH-018-adamantyl derivative,
Table 1. MRM transitions, internal standards, retention times and corresponding voltages for the liquid chromatographic–mass spectrometric analysis. (Q1) mass-to-charge ratio of precursor; (amu) atomic mass unit; (Q3) mass-to-charge ratio of fragment; (DP) declustering potential; (EP) entrance potential; (CE) collision energy; (CXP) collision cell exit potential; (tR) retention time. The transitions used for quantitation are marked with an asterisk Analyte
Q1 [amu]
Q3 [amu]
DP [V]
EP [V]
CE [V]
CXP [V]
tR [min]
Amphetamine
136.1 150.1
MDMA
194.1
MDEA
208.1
Cocaine
304.2
Benzoylecgonine
290.2
Morphine
286.2
Codeine
300.2
53 53 57 57 60 60 55 55 65 65 65 65 90 90 85 85
5.0 5.0 5.0 5.0 6.0 6.0 4.0 4.0 4.0 5.0 5.0 5.0 6.0 6.0 5.5 5.5
11 26 30 13 14 30 16 34 26 73 25 75 57 80 58 33
3.0 3.5 3.5 4.0 4.5 3.0 4.0 3.5 3.5 2.5 4.0 3.0 4.0 4.0 4.0 5.0
3.5
Methamphetamine
119.1* 91.0 91.0* 119.1 163.1* 105.1 163.1* 105.1 182.1* 77.0 168.2* 77.0 153.1* 128.1 165.1* 215.2
Internal standard Ampethamine-d5 Methampethamine-d5 MDMA-d5 MDEA-d5 Cocaine-d3 Benzoylecgonine-d3 Morphine-d3 Codeine-d3
141.1 155.1 199.1 213.1 307.2 293.2 289.2 303.2
93.0 92.0 165.1 163.1 185.1 171.1 152.1 165.1
53 57 60 55 65 65 90 85
5.0 5.0 6.0 4.0 5.0 5.0 6.0 5.5
26 30 14 16 26 25 57 58
3.0 3.5 4.5 4.0 3.5 4.0 4.0 4.0
Copyright © 2014 John Wiley & Sons, Ltd.
3.9 4.5 5.3 5.6 2.0 3.6
3.5 3.8 3.9 4.5 5.3 5.5 2.0 3.6
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3.9
J. Spinner et al. JWH-019, JWH-020, JWH-073, JWH-081, JWH-122, JWH-200, JWH-203, JWH-210, JWH-250, JWH-251, JWH-307, JWH-387, JWH-398, JWH-412, AM-694, AM-1220, AM-2201, AM-2233, CRA-13, methanandamide, RCS-4, RCS-4 ortho isomer, RCS-8, WIN 48,098, WIN 55,212-2. All drug-free dentin samples were from different sources. Analytical limits For evaluation of the calibration model five calibration curves were analyzed. The limit of detection (LOD) and lower limit of quantification (LLOQ) were determined by analyzing five samples at concentrations of 10, 20, 30, 50, 70 and 90 pg/mg. For estimation of LOD and LLOQ the software Valistat 2.0 was used. The LOD was estimated as the lowest concentration level at which the signal to noise (S/N) ratio of the quantifier and qualifier transition was ≥3. The LLOQ was the lowest concentration at which bias and precision (RSD) of 5 consecutive samples were ≤20%. Accuracy and precision To assess the accuracy and precision of the method, high and low level quality control samples were prepared, extracted and analyzed. Two samples of each quality control concentration level were analyzed daily on five consecutive days. For statistical evaluation and calculation of the accuracy (bias) as well as the intraday and interday precision, the software Valistat 2.0 was used. Autosampler stability and stability at 2–8 °C The stability of the analytes and internal standards in processed samples was studied at the low and high quality control concentration levels. Two samples of each concentration level were extracted, evaporated to dryness, reconstituted in mobile Table 2. Peak area ratios and standard deviation of analyte to internal standard at 10 min, 30 min or 60 min of extraction by ultrasonication in methanol (n = 3) Analyte Amphetamine Methamphetamine MDMA MDEA Cocaine Benzoylecgonine Morphine Codeine
10 min
30 min
60 min
4.45 ± 0.45 3.65 ± 0.12 2.00 ± 0.02 1.92 ± 0.02 0.29 ± 0.02 1.12 ± 0.05 3.89 ± 0.05 1.29 ± 0.08
5.32 ± 0.20 4.09 ± 0.15 2.26 ± 0.07 2.05 ± 0.03 0.33 ± 0.03 1.31 ± 0.04 4.04 ± 0.43 1.44 ± 0.07
5.94 ± 0.54 4.62 ± 0.15 2.69 ± 0.23 2.48 ± 0.20 0.39 ± 0.03 1.56 ± 0.03 5.35 ± 0.42 1.70 ± 0.02
phase and pooled before analysis. Both concentration levels were injected every 3 h over a total period of 12 h. To test the stability of the analytes and internal standards in prepared samples, reconstituted samples were stored in an autosampler vial at 2–8 °C and analyzed at 24 h and 72 h. The stabilities were determined by comparing the absolute peak areas. The maximum accepted difference between highest and lowest peak area was 25%.
Matrix effects, extraction recovery, overall process efficiency Matrix effects, extraction recovery and overall process efficiency were determined according to the procedure suggested by Matuszewski et al.[16] Three sample sets were prepared: set 1 consisted of dilutions of analytes and internal standards in liquid chromatography solvents A/B 95/5 (v/v). Set 2 consisted of extracts of five drug-free dentin samples fortified with analytes and internal standards. Set 3 consisted of five different drug-free dentin samples fortified with analytes and internal standards prior to extraction. All experiments regarding matrix effects, recovery and process efficiency were carried out at the low concentration of 20 pg/mg and the high concentration of 80 pg/mg.
Evaluation of solvent calibration Solvent calibration standards (10, 30, 50, 70 and 90 pg/mg) were prepared by adding adequate amounts of the working solution I or II and 10 μl of the internal standard working solution to liquid chromatography solvents A/B 95/5 (v/v). Five solvent calibration curves were analyzed. The F-Test (Funk, 99% significance) was applied to test for variance homogeneity of the dentin and the solvent calibration curves. In addition, quality control dentin samples were analyzed using the solvent calibration.
Results and discussion Extraction time optimization The extraction method was optimized by comparing the analyte to internal standard peak area ratios at different extraction times (10 min, 30 min and 60 min; see Table 2) and extraction cycles (1 to 5 cycles, see Table 3). Ultrasonication of the dentin powder in methanol for 60 min resulted in the highest analyte to internal standard peak area ratio for all analytes compared to ultrasonication for 10 min and 30 min.
Table 3. Peak area ratios and standard deviation of analyte to internal standard after 1 to 5 ultrasonication cycles of 60 min, each Analyte
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Amphetamine Methamphetamine MDMA MDEA Cocaine Benzoylecgonine Morphine Codeine
Cycle 1
Cycle 2
Cycle 3
Cycle 4
Cycle 5
5.94 ± 0.54 4.62 ± 0.15 2.69 ± 0.23 2.49 ± 0.20 0.39 ± 0.03 1.57 ± 0.03 5.38 ± 0.46 1.70 ± 0.02
0.61 ± 0.22 1.06 ± 0.26 0.67 ± 0.18 0.61 ± 0.14 0.09 ± 0.02 0.43 ± 0.14 1.21 ± 0.41 0.42 ± 0.11
0.19 ± 0.06 0.42 ± 0.13 0.31 ± 0.12 0.26 ± 0.08 0.05 ± 0.01 0.27 ± 0.11 0.74 ± 0.30 0.42 ± 0.06
0.05 ± 0.01 0.07 ± 0.06 0.07 ± 0.01 0.06 ± 0.01 0.01 ± 0.00 0.08 ± 0.01 0.19 ± 0.04 0.05 ± 0.01
0.08 ± 0.03 0.14 ± 0.05 0.09 ± 0.03 0.08 ± 0.03 0.02 ± 0.00 0.11 ± 0.04 0.32 ± 0.11 0.08 ± 0.03
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J. Mass Spectrom. 2014, 49, 1306–1313
Drugs of abuse in dentin using LC-ESI-MS/MS Optimization of ultrasonication cycle numbers
Matrix effects, extraction recovery and overall process efficiency
The extracted amount of all analytes decreased from the first to the third 60 min ultrasonic cycle (see Table 3). The fourth and fifth cycles yielded equal to or less than 7% of the first cycle. Therefore, the dentin powder was extracted three times for 60 min for all samples in this paper.
At the low concentration (20 pg/mg) matrix effects ranged from 28% to 63% (RSD ≤ 18%); at the high concentration (80 pg/mg) matrix effects ranged from 52% to 81% (RSD ≤ 12%) (see Table 4). Recoveries ranged from 88% to 100% (RSD ≤ 13%) at the low concentration and from 86% to 102% (RSD ≤ 10%) at the high concentration. Process efficiencies ranged from 26% to 62% (RSD 24%) at the low concentration and from 47% to 80% (RSD ≤ 9.6%) at the high concentration. The matrix effects are compensated by the deuterated analyte used as internal standard.
Method validation Selectivity and specificity Blank and zero samples as well as the sample fortified with other drugs and metabolites (cannabinoids, benzodiazepines and related hypnotics and synthetic cannabinoids) did not reveal any relevant interference on the MRM transitions of the analytes or the internal standards. Calibration model For all compounds, a linear relationship between the area ratio of the analyte to deuterated standard and the concentration in the range of LLOQ to the highest calibrator was confirmed by Mandel’s test (99% significance). The coefficients of correlation (R) of all calibrations were higher than or equal to 0.992 (see Table 4). To compensate for heteroscedasticity, a weighted least squares model with a weighting factor 1/x was applied for all compounds. No outliers were detected in all calibrations (Grubbs test). Precision and accuracy Precision and accuracy data are shown in Table 4. The intraday and interday precision for each compound at both concentration levels was below or equal than 6.6% (RSD ≤ 6.6%). Bias was below or equal to 7.4%. Therefore, each analyte fulfilled the acceptance criteria for accuracy (bias within ≤15% of the accepted reference value) and precision (≤15% RSD) at both concentration levels. Stability All analytes were stable in the autosampler for 9 h (low concentration level: interval between lowest and highest value ≤20%) and for 12 h, respectively (high concentration level: interval between lowest and highest value ≤24%. Cocaine showed only 9 h stability in the high concentration level). Since no degradation occurs during 9 h, batch times of up to 9 h for all analytes in a broad range of concentrations are possible, e.g. overnight measurement. During storage in the refrigerator at 2–8 °C, all analytes were stable in the worked up samples for at least 24 h, allowing a repeated or delayed analysis from the same extract. Analytical limits
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Homogeneity of variance of the dentin and the solvent calibration curves was given for all analytes. For the low dentin concentration level (20 pg/mg) processed with the solvent calibration, the calculated concentrations deviated about ±30%, which is an acceptable range. For the high concentration level (80 pg/mg) the calculated concentrations deviated about ±50–70%, which is not acceptable. Using the solvent calibration for processing of dentin samples is therefore only applicable/reliable in the 20 pg/mg concentration range. For all results presented in this work, a dentin matrix calibration was applied. Application of the method to drug-positive dentin samples Three artificially loaded drug-positive dentin samples were analyzed with the presented method. The applied artificial loading conditions were chosen to approach the approximate maximal drug concentration[17] and daily pH course expected in the oral fluid of a drug consumer (1 mg/l). In the presented study, dentin was used as model material. Parts of the odontoblasts reach into the dentinal tubules and might have an effect on the diffusion of the drugs into the dentin. However, ions and small molecules are able to diffuse through the dentinal tubules into the pulp.[18] This effect can be observed even though there is an outstream of dentin liquor and the mentioned organic matrix from the odontoblasts. The clinical situation for every tooth, regarding the above mentioned effects, can not be evaluated, as they can be influenced by, e.g. endodontic treatment or dentin alteration. Enamel, which has an inorganic crystal structure is only influenced through the exogenous pathway i. e. by drugs that are consumed orally or nasally. In contrast, drug findings in coronal dentin, which is covered by enamel, would be a result of the deposition of drugs in dentinal tubules by odontoblasts in the pulp tissue. In real cases, dentin is often not completely covered by enamel and thus exposed to the oral cavity. To interpret findings in real cases, both routes of deposition should be investigated. So, initially, dentin was used as model matrix for this proof-ofconcept study. The measured concentrations of all analytes ranged from 20 pg/mg to 80 pg/mg dentin (see Fig. 2). Amphetamine, methamphetamine and MDMA were detected in the highest concentrations: 74 to 85 pg/mg. The lowest concentration was found for benzoylecgonine, being 22 pg/mg. The different concentrations of the drugs in dentin can be explained by different deposition rates of the substances having different physico-chemical properties like pKa, lipophilicity, H-bonds, molecular weight, molecule size and protein binding. Small lipophilic molecules are prone to deposition in dental hard tissue,[6] which could be the reason for the
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The lower limit of quantification was 10 pg/mg for methamphetamine, MDMA, MDEA, benzoylecgonine, morphine and codeine. The lower limit of quantification for amphetamine and cocaine was 20 pg/mg (see Table 4). Even though 20 pg/mg is below the calibration range for amphetamine and cocaine, the RSD for precision and accuracy at 20 pg/mg is clearly below 20% (RSD less or equal 4.6%). For all analytes, the limit of detection was set to the level of the lower limit of quantification. The upper limit of quantification was set to the highest calibration level (90 pg/mg).
Solvent calibration
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wileyonlinelibrary.com/journal/jms (RSD) [%] (RSD) [%] (Bias) [%] [%] [%] [%] [%] [%] [%] (RSD) [%] (RSD) [%] (Bias) [%] [%] [%] [%] [%] [%] [%]
High concentration (80 pg/mg) Intraday imprecision (n = 10) Interday imprecision (n = 5) Accuracy (n = 10) Matrix effect RSD (n = 5) Recovery RSD (n = 5) Process efficiency RSD (n = 5)
[pg/mg] [pg/mg]
Low concentration (20 pg/mg) Intraday imprecision (n = 10) Interday imprecision (n = 5) Accuracy (n = 10) Matrix effect RSD (n = 5) Recovery RSD (n = 5) Process efficiency RSD (n = 5)
LOD Calibration range Lowest correlation coefficient (R) (n = 5)
Unit
1.4 3.1 4.9 55 7.6 86 9.0 47 4.2
2.3 3.6 1.2 50 5.9 100 5.1 50 4.5
20 30–90 1.000
Amphetamine
1.5 3.3 4.6 58 5.6 89 6.0 52 5.8
2.6 2.8 6.0 37 8.2 95 4.1 35 6.5
10 10–90 0.993
Methamphetamine
5.6 5.7 5.0 57 5.7 95 6.0 54 5.3
4.4 5.6 7.4 42 8.7 94 3.2 40 10
10 10–90 0.999
MDMA
6.6 6.6 1.9 57 4.6 96 4.6 54 5.5
3.5 6.4 3.0 39 7.5 92 2.9 36 9.0
10 10–90 0.997
MDEA
1.1 2.8 3.1 81 1.4 99 4.1 80 3.9
3.2 4.6 0.0 56 8.3 95 7.1 53 4.8
20 30–90 0.999
Cocaine
2.1 2.6 1.1 49 4.7 98 10 48 8.4
1.0 4.1 2.1 28 15 96 13 26 5.3
10 10–90 1.000
Benzoylecgonine
2.1 4.1 2.7 53 12 96 7.8 50 9.6
2.9 3.4 2.6 60 18 88 8.0 53 22
10 10–90 0.998
Morphine
1.9 3.5 1.1 52 9.1 102 1.2 53 9.1
4.2 5.5 1.6 63 18 98 6.2 62 24
10 10–90 0.994
Codeine
Table 4. Calibration and validation data: limit of detection (LOD), calibration range, lowest correlation coefficient of 5 calibrations, intraday and interday precision, accuracy and respective relative standard deviation (RSD), matrix effect, recovery and process efficiency and RSD at 20 pg/mg and at 80 pg/mg, respectively
J. Spinner et al.
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Drugs of abuse in dentin using LC-ESI-MS/MS concentrations can then be compared to the drug intake history of the deceased to further evaluate relationships between measured concentration and patterns of drug use. With the presented method, the spectrum of toxicological investigations will be reasonably supplemented especially in post-mortem cases with limited sample tissue available.
References
Figure 2. Mean concentrations (±RSD) of all drugs in artificially drugpositive dentin samples (n = 3). For the preparation of artificially drugpositive samples see Material and methods section.
amphetamines (amphetamine, methamphetamine, MDMA and MDEA) appearing in higher concentrations than the other drugs. On the other hand, just as in hair analysis, influences of extraction efficiency or irreversible binding of substances to the dental matrix causing low recoveries cannot be determined quantitatively in real samples. Development and application of a suitable in vitro model can help to interpret these effects. In our study, the concentrations are lower by a factor of around 1.000 compared to those found by Cattaneo et al.[3] and Pellegrini et al.[11] In the study with post mortem material,[3] the high measured drug concentrations could derive mainly from high post mortem drug concentrations in the blood and therefore also in the dental pulp, which was apparently not removed prior to analysis. The teeth of drug addicts[11] were probably exposed to drugs repeatedly over several months or years, whereas in our study, the dental material was exposed to a drug solution for only nine days during the artificial loading phase. Longer exposure suggests the deposition of higher drug amounts which in turn would result in higher measured concentrations. To our knowledge, this is the first time, substances were determined in dentin without potential influences of residual dental pulp.
Conclusion
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The presented liquid chromatography–tandem mass spectrometry method proved to be suitable for the detection and quantification of the common drugs of abuse amphetamine, methamphetamine, MDMA, MDEA, cocaine, benzoylecgonine, morphine and codeine in dentin samples using 50 mg of dental hard tissue. The method demonstrated to be accurate, precise, selective and specific with satisfying linearity within the calibrated range. Lower limits of quantification were 10 – 20 pg/mg. Evaluation of the analytes’ stability in processed samples suggests to analyze samples within 9 h after extraction. In artificially drug-loaded dentin samples, slightly higher concentrations of amphetamines (amphetamine, methamphetamine, MDMA, MDEA) compared to cocaine and benzoylecgonine might be explained by different physico-chemical properties of the drugs. The method can be applied in further experiments with different artificial drug-exposure conditions. In further experiments, the method should be used to quantitatively analyze drug positive tooth samples, e.g. in post-mortem cases. The measured
[1] M. J. Altenburger, J. F. Schirrmeister, A. Lussi, M. Klasser, E. Hellwig. In situ fluoride retention and remineralization of incipient carious lesions after the application of different concentrations of fluoride. Eur. J. Oral Sci. 2009, 117, 58. [2] G. Skopp. Preanalytic aspects in postmortem toxicology. Forensic Sci. Int. 2004, 142, 75. [3] C. Cattaneo, F. Gigli, F. Lodi, M. Grandi. The detection of morphine and codeine in human teeth: an aid in the identification and study of human skeletal remains. J. Forensic Odontostomatol. 2003, 21, 1. [4] N. Conforti, S. Mankodi, Y. P. Zhang, P. Chaknis, M. E. Petrone, W. DeVizio, A. R. Volpe. Clinical study to compare extrinsic stain formation in subjects using three dentifrice formulations. Compend. Contin. Educ. Dent. Suppl. 2000, 27, 23. [5] A. H. Schuurs, L. Abraham-Inpijn, J. P. van Straalen, S. H. Sastrowijoto. An unusual case of black teeth. Oral Surg. Oral Med. Oral Pathol. 1987, 64, 427. [6] K. O. Haustein, G. Thiele, U. Stangel. Transport of various substances through human enamel and dentine. Int. J. Clin. Pharmacol. Ther. 1994, 32, 483. [7] Y. Schüssl, K. Pelz, J. Kempf, J. E. Otten. Concentrations of amoxicillin and clindamycin in teeth following a single dose of oral medication. Clin. Oral Investig. 2013, 18, 35–40. [8] J. A. Pascual, D. Diaz, J. Segura, O. Garcia-Algar, O. Vall, P. Zuccaro, R. Pacifici, S. Pichini. A simple and reliable method for the determination of nicotine and cotinine in teeth by gas chromatography/mass spectrometry. Rapid Commun. Mass Spectrom. 2003, 17, 2853. [9] O. Garcia-Algar, O. Vall, J. Segura, J. A. Pascual, D. Diaz, L. Mutnoz, P. Zuccaro, R. Pacifici, S. Pichini. Nicotine concentrations in deciduous teeth and cumulative exposure to tobacco smoke during childhood. J. Am. Med. Assoc. 2003, 290, 196. [10] C. Zeren, A. Keten, S. Celik, I. Damlar, N. Daglioglu, A. Celiker, B. Karaarslan. Demonstration of ethyl glucuronide in dental tissue samples by liquid chromatography/electro-spray tandem mass spectrometry. J. Forensic Leg. Med. 2013, 20, 706. [11] M. Pellegrini, A. Casa, E. Marchei, R. Pacifici, R. Mayne, V. Barbero, O. Garcia-Algar, S. Pichini. Development and validation of a gas chromatography-mass spectrometry assay for opiates and cocaine in human teeth. J. Pharm. Biomed. Anal. 2006, 40, 662. [12] M. J. Altenburger, J. F. Schirrmeister, K. T. Wrbas, E. Hellwig. Remineralization of artificial interproximal carious lesions using a fluoride mouthrinse. Am. J. Dent. 2007, 20, 385. [13] J. A. Buskes, J. Christoffersen, J. Arends. Lesion formation and lesion remineralization in enamel under constant composition conditions. A new technique with applications. Caries Res. 1985, 19, 490. [14] J. M. Ten Cate, B. Nyvad, Y. M. Van de Plassche-Simons, O. Fejerskov. A quantitative analysis of mineral loss and shrinkage of in vitro demineralized human root surfaces. J. Dent. Res. 1991, 70, 1371. [15] F. T. Peters, M. Hartung, M. Herbold, G. Schmitt, T. Daldrup, F. Musshoff. Anhang B zu den Richtlinien der GTFCh zur Qualitätssicherung bei forensisch-toxikologischen Untersuchungen; Anforderungen an die Validierung von Analysenmethoden. Toxichem. Krimtech. 2009, 76, 185. [16] B. K. Matuszewski, M. L. Constanzer, C. M. Chavez-Eng. Strategies for the Assessment of Matrix Effect in Quantitative Bioanalytical Methods Based on HPLC-MS/MS. Anal. Chem. 2003, 75, 3019. [17] O. H. Drummer. Review: Pharmacokinetics of illicit drugs in oral fluid. Forensic Sci. Int. 2005, 150, 133. [18] D. Sharma, J. A. McGuire, J. T. Gallob, P. Amini. Randomised clinical efficacy trial of potassium oxalate mouthrinse in relieving dentinal sensitivity. J. Dent. 2013, 41(Suppl 4), S40.
Revised: 21 July 2014
Accepted: 7 August 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3464
Determination of drugs of abuse in bovine dentin using liquid chromatography– electrospray ionization tandem mass spectrometry J. Spinner,a,b M. Klima,a J. Kempf,a L. M. Huppertz,a V. Auwärter,a M. J. Altenburgerc and M. A. Neukamma* Drugs deposited in human teeth are well preserved; the spectrum of toxicological investigations may therefore be supplemented by an analysis method for drugs in teeth. A liquid chromatography–electrospray ionization tandem mass spectrometry assay for the detection and quantification of basic drugs of abuse in bovine dentin samples was developed and validated. The drugs and metabolites amphetamine, methamphetamine, methylenedioxymethylamphetamine, methylenedioxyethylamphetamine, codeine, morphine, cocaine and benzoylecgonine were extracted from 50 mg ground dentin powder by ultrasonication for 60 min in methanol 3 times. The extracts were analyzed on a triple-quadrupole mass-spectrometer in multiple reaction monitoring mode. The method was validated and proved to be accurate, precise, selective, specific and stable with good linearity within the calibration range and a lower limit of quantification of 10 to 20 pg/mg. To artificially load bovine dentin samples with drugs, the natural process of de- and remineralization in the oral cavity was mimicked by a pH-cycling experiment. The artificially drug-loaded dentin samples showed drug concentrations of 20 to 80 pg/mg. The method can be applied in further in vitro experiments as well as in post-mortem cases, especially where limited sample tissue is available. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: LC-ESI-MS/MS; dentin; dental hard tissue; drugs of abuse; alternative matrix
Introduction
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To investigate fatal poisonings with drugs, it is crucial to gain information about the drug intake of a person. However, in cases of severely putrefied bodies – e.g. after longer post mortem intervals or exhumation – no suitable samples of body fluids and sometimes also no organ tissues can be obtained for chemical–toxicological analysis. In teeth, however, deposited drugs or poisons are likely to be protected from biological or chemical degradation.[1,2] Morphine, for example, can be detected in teeth of persons who died of heroine overdose at least for 2 years after death.[3] Therefore, the analysis of dental hard tissue (dentin, enamel) may allow a retrospective view on the drug intake of a person. The whole tooth consists of the hard tissues (enamel and dentin), the pulp tissue in the inside of the tooth and the overlaying cementum with the periodontal ligament (see Fig. 1). Consumed substances can have contact with the dental tissue in two ways. First, from the oral cavity, the oral fluid (and its content) has contact with the enamel or exposed dentin. Second, from the inside: the blood vessels in the pulp have contact with the inner surface of the dentin. In the oral cavity the pH declines due to exogenous acidic compounds (e.g. from food) and endogenous acidic compounds secreted by the cariogenic biofilm. Subsequently, calcium and phosphate dissolve from enamel and dentin. When the biofilm
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and other acidic compounds are removed, the pH increases and remineralization takes place. Saliva, which is a supersaturated solution of calcium and phosphate, leads then to a reuptake of minerals into dental hard tissues. During this remineralization phase also other substances present in oral fluid like pigments from food, beverages (e.g. black tea or red wine), tobacco or spices are visibly adsorbed on the surface of teeth or deposited in teeth.[4,5] Haustein et al. studied the transport of substances through the enamel and dentin into intact and carious teeth in vitro.[6] The authors investigated some low aliphatic alcohols, organic acids, barbital, procaine and some sugar derivatives. All substances penetrated the enamel, which is regarded to behave like a porous and ion-selective membrane. The penetration rates depended on the pH of the incubation medium and on the lipophilicity of the substance. Schüssl et al. found that after a single
* Correspondence to: M. A. Neukamm, Institute of Forensic Medicine, University Medical Center Freiburg, Freiburg, Germany. E-mail: [email protected] a Institute of Forensic Medicine, University Medical Center Freiburg, Freiburg, Germany b Institute of Forensic Medicine, University of Bern, Bern, Switzerland c Department of Operative Dentistry and Periodontology, University Medical Center Freiburg, Freiburg, Germany
Copyright © 2014 John Wiley & Sons, Ltd.
Drugs of abuse in dentin using LC-ESI-MS/MS methylenedioxyethylamphetamine (MDEA), methylenedioxymethylamphetamine (MDMA), codeine, morphine, cocaine, benzoylecgonine) in dentin was developed. The method was applied to drug-positive bovine dentin samples, produced by a pH-cycling experiment, which mimicked the natural process of de- and remineralization in the oral cavity in a standardized way. The pH-cycling mimics exogenous diffusion of substances into dentin via drug-containing saliva. Without the influences of, e.g. oral fluid or biofilm on the tooth surface, the incorporation characteristics of different substances into dentin can be compared directly.
Materials and methods Chemicals and reference standards
Figure 1. Anatomy of the tooth and the periodont. Contact with drugs takes place via the blood stream and via the oral fluid (figure modified, original: Jordi March i Nogué [CC-BY-SA-3.0 (http://creativecommons.org/ licenses/by-sa/3.0) or GFDL (http://www.gnu.org/copyleft/fdl.html)] via Wikimedia Commons).
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Preparation of the stock solution of standards To prepare the stock solution of standards, appropriate volumes of reference solutions were mixed and diluted with methanol to a final concentration of 1.0 μg/ml. The stability of the stock solution for 5 months was proven.
Preparation of working solutions To prepare the working solutions I and II, the stock solution of standards was diluted with methanol to contain 50 ng/ml (I) or 100 ng/ml (II) of each analyte. To prepare the internal standard working solution, appropriate volumes of reference solutions of deuterated standards were mixed and diluted with methanol to a final concentration of 100 ng/ml of each compound.
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dose of the antibiotics amoxicillin and clindamycin, the concentration in the root of a tooth correlated with the serum concentration. The antibiotics were determined by a quantitative liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method.[7] In toxicology literature, there are not many publications focusing on the use of teeth as analytical specimen. Besides a study on the assessment of passive smoke exposure of children by analysis of nicotine and cotinine in deciduous teeth,[8,9] there is a forensic study on the determination of alcohol intake behaviour by analysis of ethyl glucuronide in dental hard tissue.[10] For drugs of abuse, to our knowledge there are only studies on the determination of opiates (pilot study[3]) and cocaine.[11] In these studies[3,8,9,11] at least 1 g of human dental material was used. Also the dental pulp – which is interspersed by blood vessels – was apparently not removed. Hence, it can not be determined if the detected substances were a deposit from blood circulation in the pulp chamber or were incorporated due to remineralization processes. For the use of teeth as a forensic-toxicological matrix it is necessary to investigate the quantitative relationship between drug exposure and determined concentrations, as well as to basically understand the mechanisms of deposition. Only if this is understood, valid conclusions can be drawn on the drug intake history of a person. As a basis for the study on the general and substancespecific mechanisms of deposition, an LC-ESI-MS/MS method for the quantitative determination of different substances relevant in forensic toxicology (amphetamine, methamphetamine,
All solvents and substances were at least of analytical or HPLC grade. Formic acid, 2-(4-(2-hydroxy ethyl)-1-piperazinyl) ethanesulfonic acid, isopropanol and potassium chloride were purchased from Carl Roth GmbH (Karlsruhe, Germany). Ammonium formate, hydrochloric acid, methylhydroxydiphosphonate (MHDP) and lactic acid were obtained from Sigma-Aldrich (Steinheim, Germany). Potassium dihydrogen phosphate, calcium chloride, calcium chloride dihydrate and acetic acid were from Merck (Darmstadt, Germany). Purified water (Aqua bidest) was obtained from Fresenius Kabi AG, Bad Homburg auf der Höhe, Germany. Methanol was purchased from Chem solute (Th. Geyer, Renningen, Germany). Technovit 4071 Liquid and Technovit 4071 chemically setting resin powder was from Heraeus Kulzer (Hanau, Germany), and thymol was from AppliChem (Darmstadt, Germany). Deionized water for preparation of the LC mobile phase was prepared with a cartridge deionizer from Memtech (Moorenweis, Germany). Amphetamine, amphetamine-d5, benzoylecgonine, benzoylecgonine-d3, cocaine, cocaine-d3, codeine, codeine-d3, MDEA, MDEA-d5, MDMA, MDMA-d5, methamphetamine, methamphetamine-d5, morphine and morphine-d3 were purchased from LGCStandards (Wesel, Germany). All non deuterated standards except cocaine were obtained as certified solutions with the concentration 1.0 mg/ml in methanol; cocaine was obtained as certified solution with the concentration 1.0 mg/ml in acetonitrile. All deuterated standards except cocaine-d3 were obtained as certified solutions with the concentration of 100 μg/ml in methanol; cocaine-d3 was obtained as certified solution with the concentration 100 μg/ml in acetonitrile.
J. Spinner et al. Calibration standards and controls The dentin calibration standards (10, 30, 50, 70 and 90 pg/mg for methamphetamine, MDMA, MDEA benzoylecgonine, morphine and codeine; 30, 50, 70 and 90 pg/mg for amphetamine and cocaine) were prepared by adding adequate amounts of the working solution I or II and 10 μl of the internal standard working solution to 50 mg of drug-free dentin powder prior to extraction. The quality control samples with concentrations of 20 pg/mg and 80 pg/mg were prepared in the same manner using separate working solutions. The calibration range was chosen to cover the expected concentrations in artificial drug-positive samples. Drug-free bovine dentin powder was prepared as follows: dentin pellets were cooled with liquid nitrogen and ground using a ball mill (MM2, Retsch, Haan, Germany) for 15 min. Matrix free solvent calibration standards (10, 30, 50, 70 and 90 pg/mg, 30, 50, 70 and 90 pg/mg for amphetamine and cocaine) were prepared by adding adequate amounts of the working solution I or II and 10 μl of the internal standard working solution to adequate amounts of the liquid chromatography solvent A/B 95/5 (v/v) to yield 100 μl. Solvent A consisted of water with 2% formic acid and 2.0 mmol/l ammonium formate and solvent B was methanol with 0.1% formic acid.
incubated for 9 days with four cycles of demineralization (0.5 h each) and remineralization (2.5 h each). During night time, the pellets were stored in remineralization solution for 12 h. The pellets were rinsed with purified water after each step. After 36 h, the demineralization and remineralization solutions were renewed. After pH-cycling, the pellets were rinsed again with purified water and then dried in the oven at 37 °C for approx. 20 h and stored in closed tubes at 20 °C until analysis. The stability of all analytes in the drugcontaining remineralization solution was checked. All analytes were stable for at least 10 days, so the stability of the substances during the pH-cycling experiment is assured. Method for stability testing: A volume of 10 ml of the drugcontaining remineralization solution was stored in a clear glass bottle at room temperature. Aliquots of 400 μl were sampled every 24 h for 10 days. Another 400 μl was sampled after another approximately 10 and 20 days, respectively. Stability was presumed if the percental decline of the peak area of the analyte was below the imprecision of the method. All analytes were stable for at least 10 days. Sample extraction Preparation of dentin powder
Preparation of dentin pellets Bovine dentin can be used as an alternative for human dental material.[12] Dentin was obtained from freshly extracted bovine incisors of the second dentition tested negative for Bovine Spongiform Encephalopathy. The teeth were stored in 0.1% thymol at 8 °C before further processing. Round specimens, 3.5 mm in diameter, were drilled out of each tooth’s root using a custom-made trephine bur (Gebr. Brasseler, Lembo, Germany). After removing tissue residues the samples were embedded in chemically setting resin (Technovit 4071). The pellets were subsequently polished with the grinding machine and wet sand paper to a grit of 4000 to remove root cement and to create uniform dentin pellets. After removing the resin, the pellets were ultrasonicated for 10 min at 20 °C in purified water to clean the pellets from grinding sludge. Preparation of artificial drug-positive dentin samples
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To create uniform initial carious-like lesions, the dentin pellets were demineralized at room temperature for 7 days in a demineralization solution according to Buskes et al.[13] containing 3 mM calcium chloride dihydrate, 3 mM potassium dihydrogen phosphate, 60 μM MHDP, 10% lactic acid (40%) and traces of thymol. A pH of 5 was set by adding the adequate amount of potassium hydroxide (5 M). Lesion formation was verified by light microscopy and transverse microradiography (TMR, Inspector Research, Amsterdam, The Netherlands). After demineralization, the samples were rinsed with purified water and dried at 37 °C for 20 h. To simulate the natural process of de- and remineralization in the oral cavity, pH-cycling was performed according to ten Cate et al.[14] as follows: 25 pre-demineralized dentin pellets of bovine teeth were incubated alternately with a demineralization solution without analytes (pH = 4.8) and a remineralization solution containing the analytes (pH = 7). The remineralization solution contained 1.0 mg/l of the following drugs: amphetamine, methamphetamine, MDMA, MDEA, codeine, morphine, cocaine and benzoylecgonine. The pellets were
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Dentin pellets were cooled with liquid nitrogen and ground using a ball mill (MM2, Retsch, Haan, Germany) for 15 min. To 50 mg of dentin powder 10 μl of the internal standard solution was added. Extraction The following steps were executed three times: The powder was suspended with 500 μl of methanol. The sample was vortexed and extracted in an ultrasonic bath for 60 min at room temperature (the temperature of the bath rose during the ultrasonication from about 22 °C to maximal 45 °C). The extraction time was optimized (see section method optimization). After centrifugation, the supernatant was collected. To complete the extraction cycle, the residue was resuspended in 100 μl of methanol and vortexed. After centrifugation, the supernatants were combined. The combined supernatants of three extraction cycles were reduced under a gentle stream of nitrogen at 40 °C to a small residual volume (approximately 2 μl) (the number of extraction cycles was optimized, see section method optimization). After addition of 100 μl of a mixture of isopropanol:HCl 3/1 (v/v) to prevent the evaporation of amphetamines, the solution was evaporated to dryness. The dry residue was reconstituted in 100 μl of liquid chromatography solvents A/B 95/5 (v/v). Method optimization To 50 mg of artificial drug-positive dentin powder 500 μl of methanol and 10 μl of the internal standard working solution were added. To evaluate the optimal extraction procedure, each sample was extracted in 1 to 5 cycles in an ultrasonic bath for the extraction time of 10 min, 30 min or 60 min, respectively. The experiment was carried out with three powder samples for each extraction time and cycle number. The samples were analyzed and the peak area ratios of analyte to internal standard of each extraction time and cycle number were compared.
Copyright © 2014 John Wiley & Sons, Ltd.
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Drugs of abuse in dentin using LC-ESI-MS/MS Liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) The LC-ESI-MS/MS system included an API 5000 triple quadrupole mass spectrometer (AB Sciex, Darmstadt, Germany) fitted with a Shimadzu HPLC system consisting of 3 LC-10ADVP binary pumps, a CTO-10 AC column oven, a HTC PAL autosampler and SCL-10AVP controller. Chromatographic separation of all substances was achieved using a Luna PFP column (150 mm × 2 mm, 5 μm) with a guard column with the same stationary phase (4 mm × 2 mm) (Phenomenex, Aschaffenburg, Germany) and gradient elution. The gradient was as follows: initially, 5% of solvent B at a flow rate of 0.4 ml/min. During 10 min solvent B increased up to 98% at a flow rate of 0.6 ml/min which was kept for 2.5 min. The flow rate was decreased to 0.4 ml/min again within 0.5 min at 98% B. Initial conditions were restored and held for 2 min to re-equilibrate the system. Post column addition of isopropanol at a flow rate of 0.1 ml/min was applied to improve sensitivity. The injection volume was 20 μl. The respective multiple reaction monitoring (MRM) transitions and the corresponding potentials for all analytes and internal standards are shown in Table 1. The mass spectrometer was operated in positive ionization mode. The ion spray voltage was set to 1500 V. The gas settings were as follows: curtain gas: 20 psi; collision gas: 9 psi, ion source gas 1: 60 psi; ion source gas 2: 70 psi. The ion source temperature was set to 500 °C. Data acquisition and analysis For data acquisition, the software Analyst 1.5.1 (AB Sciex, Darmstadt, Germany) was applied. For data analysis, Analyst 1.5.1 and Valistat 2.0 (Arvecon GmbH, Walldorf, Germany) were used.
Method validation The LC-ESI-MS/MS method was validated for the quantification of the chosen drugs in dentin according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh).[15] Therefore, selectivity, linearity, limit of detection, limit of quantification, accuracy and precision, autosampler stability, stability at 2–8 °C, matrix effects,[16] extraction recovery, overall process efficiency and application of a solvent calibration were examined. Selectivity To assess potential interferences with common drugs of abuse, endogenous compounds as well as reagents and materials used for sample preparation the following samples were analyzed: six drug-free dentin samples without the addition of standards, two drug-free dentin samples fortified with internal standards as well as one drug-free dentin sample fortified with the following drugs or metabolites in the concentration range of 20 pg/mg to 16 ng/mg: cannabinoids: Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9tetrahydrocannabinol, 11-nor-Δ9-carboxytetrahydrocannabinol; benzodiazepines and structurally unrelated agonists of the GABA receptor complex: 7-aminoclonazepam, 7-aminoflunitrazepam, alprazolam, α-hydroxyalprazolam, bromazepam, brotizolam, camazepam, chlordiazepoxide, clobazam, clonazepam, clotiazepam, delorazepam, diazepam, estazolam, flunitrazepam, flurazepam, lorazepam, lormetazepam, medazepam, midazolam, nitrazepam, nordazepam, norflunitrazepam, oxazepam, temazepam, tetrazepam, triazolam, α-hydroxytriazolam, zaleplon, zolpidem, zopiclone; synthetic cannabinoids: JWH-007, JWH-015, JWH-018, JWH-018-adamantyl derivative,
Table 1. MRM transitions, internal standards, retention times and corresponding voltages for the liquid chromatographic–mass spectrometric analysis. (Q1) mass-to-charge ratio of precursor; (amu) atomic mass unit; (Q3) mass-to-charge ratio of fragment; (DP) declustering potential; (EP) entrance potential; (CE) collision energy; (CXP) collision cell exit potential; (tR) retention time. The transitions used for quantitation are marked with an asterisk Analyte
Q1 [amu]
Q3 [amu]
DP [V]
EP [V]
CE [V]
CXP [V]
tR [min]
Amphetamine
136.1 150.1
MDMA
194.1
MDEA
208.1
Cocaine
304.2
Benzoylecgonine
290.2
Morphine
286.2
Codeine
300.2
53 53 57 57 60 60 55 55 65 65 65 65 90 90 85 85
5.0 5.0 5.0 5.0 6.0 6.0 4.0 4.0 4.0 5.0 5.0 5.0 6.0 6.0 5.5 5.5
11 26 30 13 14 30 16 34 26 73 25 75 57 80 58 33
3.0 3.5 3.5 4.0 4.5 3.0 4.0 3.5 3.5 2.5 4.0 3.0 4.0 4.0 4.0 5.0
3.5
Methamphetamine
119.1* 91.0 91.0* 119.1 163.1* 105.1 163.1* 105.1 182.1* 77.0 168.2* 77.0 153.1* 128.1 165.1* 215.2
Internal standard Ampethamine-d5 Methampethamine-d5 MDMA-d5 MDEA-d5 Cocaine-d3 Benzoylecgonine-d3 Morphine-d3 Codeine-d3
141.1 155.1 199.1 213.1 307.2 293.2 289.2 303.2
93.0 92.0 165.1 163.1 185.1 171.1 152.1 165.1
53 57 60 55 65 65 90 85
5.0 5.0 6.0 4.0 5.0 5.0 6.0 5.5
26 30 14 16 26 25 57 58
3.0 3.5 4.5 4.0 3.5 4.0 4.0 4.0
Copyright © 2014 John Wiley & Sons, Ltd.
3.9 4.5 5.3 5.6 2.0 3.6
3.5 3.8 3.9 4.5 5.3 5.5 2.0 3.6
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3.9
J. Spinner et al. JWH-019, JWH-020, JWH-073, JWH-081, JWH-122, JWH-200, JWH-203, JWH-210, JWH-250, JWH-251, JWH-307, JWH-387, JWH-398, JWH-412, AM-694, AM-1220, AM-2201, AM-2233, CRA-13, methanandamide, RCS-4, RCS-4 ortho isomer, RCS-8, WIN 48,098, WIN 55,212-2. All drug-free dentin samples were from different sources. Analytical limits For evaluation of the calibration model five calibration curves were analyzed. The limit of detection (LOD) and lower limit of quantification (LLOQ) were determined by analyzing five samples at concentrations of 10, 20, 30, 50, 70 and 90 pg/mg. For estimation of LOD and LLOQ the software Valistat 2.0 was used. The LOD was estimated as the lowest concentration level at which the signal to noise (S/N) ratio of the quantifier and qualifier transition was ≥3. The LLOQ was the lowest concentration at which bias and precision (RSD) of 5 consecutive samples were ≤20%. Accuracy and precision To assess the accuracy and precision of the method, high and low level quality control samples were prepared, extracted and analyzed. Two samples of each quality control concentration level were analyzed daily on five consecutive days. For statistical evaluation and calculation of the accuracy (bias) as well as the intraday and interday precision, the software Valistat 2.0 was used. Autosampler stability and stability at 2–8 °C The stability of the analytes and internal standards in processed samples was studied at the low and high quality control concentration levels. Two samples of each concentration level were extracted, evaporated to dryness, reconstituted in mobile Table 2. Peak area ratios and standard deviation of analyte to internal standard at 10 min, 30 min or 60 min of extraction by ultrasonication in methanol (n = 3) Analyte Amphetamine Methamphetamine MDMA MDEA Cocaine Benzoylecgonine Morphine Codeine
10 min
30 min
60 min
4.45 ± 0.45 3.65 ± 0.12 2.00 ± 0.02 1.92 ± 0.02 0.29 ± 0.02 1.12 ± 0.05 3.89 ± 0.05 1.29 ± 0.08
5.32 ± 0.20 4.09 ± 0.15 2.26 ± 0.07 2.05 ± 0.03 0.33 ± 0.03 1.31 ± 0.04 4.04 ± 0.43 1.44 ± 0.07
5.94 ± 0.54 4.62 ± 0.15 2.69 ± 0.23 2.48 ± 0.20 0.39 ± 0.03 1.56 ± 0.03 5.35 ± 0.42 1.70 ± 0.02
phase and pooled before analysis. Both concentration levels were injected every 3 h over a total period of 12 h. To test the stability of the analytes and internal standards in prepared samples, reconstituted samples were stored in an autosampler vial at 2–8 °C and analyzed at 24 h and 72 h. The stabilities were determined by comparing the absolute peak areas. The maximum accepted difference between highest and lowest peak area was 25%.
Matrix effects, extraction recovery, overall process efficiency Matrix effects, extraction recovery and overall process efficiency were determined according to the procedure suggested by Matuszewski et al.[16] Three sample sets were prepared: set 1 consisted of dilutions of analytes and internal standards in liquid chromatography solvents A/B 95/5 (v/v). Set 2 consisted of extracts of five drug-free dentin samples fortified with analytes and internal standards. Set 3 consisted of five different drug-free dentin samples fortified with analytes and internal standards prior to extraction. All experiments regarding matrix effects, recovery and process efficiency were carried out at the low concentration of 20 pg/mg and the high concentration of 80 pg/mg.
Evaluation of solvent calibration Solvent calibration standards (10, 30, 50, 70 and 90 pg/mg) were prepared by adding adequate amounts of the working solution I or II and 10 μl of the internal standard working solution to liquid chromatography solvents A/B 95/5 (v/v). Five solvent calibration curves were analyzed. The F-Test (Funk, 99% significance) was applied to test for variance homogeneity of the dentin and the solvent calibration curves. In addition, quality control dentin samples were analyzed using the solvent calibration.
Results and discussion Extraction time optimization The extraction method was optimized by comparing the analyte to internal standard peak area ratios at different extraction times (10 min, 30 min and 60 min; see Table 2) and extraction cycles (1 to 5 cycles, see Table 3). Ultrasonication of the dentin powder in methanol for 60 min resulted in the highest analyte to internal standard peak area ratio for all analytes compared to ultrasonication for 10 min and 30 min.
Table 3. Peak area ratios and standard deviation of analyte to internal standard after 1 to 5 ultrasonication cycles of 60 min, each Analyte
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Amphetamine Methamphetamine MDMA MDEA Cocaine Benzoylecgonine Morphine Codeine
Cycle 1
Cycle 2
Cycle 3
Cycle 4
Cycle 5
5.94 ± 0.54 4.62 ± 0.15 2.69 ± 0.23 2.49 ± 0.20 0.39 ± 0.03 1.57 ± 0.03 5.38 ± 0.46 1.70 ± 0.02
0.61 ± 0.22 1.06 ± 0.26 0.67 ± 0.18 0.61 ± 0.14 0.09 ± 0.02 0.43 ± 0.14 1.21 ± 0.41 0.42 ± 0.11
0.19 ± 0.06 0.42 ± 0.13 0.31 ± 0.12 0.26 ± 0.08 0.05 ± 0.01 0.27 ± 0.11 0.74 ± 0.30 0.42 ± 0.06
0.05 ± 0.01 0.07 ± 0.06 0.07 ± 0.01 0.06 ± 0.01 0.01 ± 0.00 0.08 ± 0.01 0.19 ± 0.04 0.05 ± 0.01
0.08 ± 0.03 0.14 ± 0.05 0.09 ± 0.03 0.08 ± 0.03 0.02 ± 0.00 0.11 ± 0.04 0.32 ± 0.11 0.08 ± 0.03
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J. Mass Spectrom. 2014, 49, 1306–1313
Drugs of abuse in dentin using LC-ESI-MS/MS Optimization of ultrasonication cycle numbers
Matrix effects, extraction recovery and overall process efficiency
The extracted amount of all analytes decreased from the first to the third 60 min ultrasonic cycle (see Table 3). The fourth and fifth cycles yielded equal to or less than 7% of the first cycle. Therefore, the dentin powder was extracted three times for 60 min for all samples in this paper.
At the low concentration (20 pg/mg) matrix effects ranged from 28% to 63% (RSD ≤ 18%); at the high concentration (80 pg/mg) matrix effects ranged from 52% to 81% (RSD ≤ 12%) (see Table 4). Recoveries ranged from 88% to 100% (RSD ≤ 13%) at the low concentration and from 86% to 102% (RSD ≤ 10%) at the high concentration. Process efficiencies ranged from 26% to 62% (RSD 24%) at the low concentration and from 47% to 80% (RSD ≤ 9.6%) at the high concentration. The matrix effects are compensated by the deuterated analyte used as internal standard.
Method validation Selectivity and specificity Blank and zero samples as well as the sample fortified with other drugs and metabolites (cannabinoids, benzodiazepines and related hypnotics and synthetic cannabinoids) did not reveal any relevant interference on the MRM transitions of the analytes or the internal standards. Calibration model For all compounds, a linear relationship between the area ratio of the analyte to deuterated standard and the concentration in the range of LLOQ to the highest calibrator was confirmed by Mandel’s test (99% significance). The coefficients of correlation (R) of all calibrations were higher than or equal to 0.992 (see Table 4). To compensate for heteroscedasticity, a weighted least squares model with a weighting factor 1/x was applied for all compounds. No outliers were detected in all calibrations (Grubbs test). Precision and accuracy Precision and accuracy data are shown in Table 4. The intraday and interday precision for each compound at both concentration levels was below or equal than 6.6% (RSD ≤ 6.6%). Bias was below or equal to 7.4%. Therefore, each analyte fulfilled the acceptance criteria for accuracy (bias within ≤15% of the accepted reference value) and precision (≤15% RSD) at both concentration levels. Stability All analytes were stable in the autosampler for 9 h (low concentration level: interval between lowest and highest value ≤20%) and for 12 h, respectively (high concentration level: interval between lowest and highest value ≤24%. Cocaine showed only 9 h stability in the high concentration level). Since no degradation occurs during 9 h, batch times of up to 9 h for all analytes in a broad range of concentrations are possible, e.g. overnight measurement. During storage in the refrigerator at 2–8 °C, all analytes were stable in the worked up samples for at least 24 h, allowing a repeated or delayed analysis from the same extract. Analytical limits
J. Mass Spectrom. 2014, 49, 1306–1313
Homogeneity of variance of the dentin and the solvent calibration curves was given for all analytes. For the low dentin concentration level (20 pg/mg) processed with the solvent calibration, the calculated concentrations deviated about ±30%, which is an acceptable range. For the high concentration level (80 pg/mg) the calculated concentrations deviated about ±50–70%, which is not acceptable. Using the solvent calibration for processing of dentin samples is therefore only applicable/reliable in the 20 pg/mg concentration range. For all results presented in this work, a dentin matrix calibration was applied. Application of the method to drug-positive dentin samples Three artificially loaded drug-positive dentin samples were analyzed with the presented method. The applied artificial loading conditions were chosen to approach the approximate maximal drug concentration[17] and daily pH course expected in the oral fluid of a drug consumer (1 mg/l). In the presented study, dentin was used as model material. Parts of the odontoblasts reach into the dentinal tubules and might have an effect on the diffusion of the drugs into the dentin. However, ions and small molecules are able to diffuse through the dentinal tubules into the pulp.[18] This effect can be observed even though there is an outstream of dentin liquor and the mentioned organic matrix from the odontoblasts. The clinical situation for every tooth, regarding the above mentioned effects, can not be evaluated, as they can be influenced by, e.g. endodontic treatment or dentin alteration. Enamel, which has an inorganic crystal structure is only influenced through the exogenous pathway i. e. by drugs that are consumed orally or nasally. In contrast, drug findings in coronal dentin, which is covered by enamel, would be a result of the deposition of drugs in dentinal tubules by odontoblasts in the pulp tissue. In real cases, dentin is often not completely covered by enamel and thus exposed to the oral cavity. To interpret findings in real cases, both routes of deposition should be investigated. So, initially, dentin was used as model matrix for this proof-ofconcept study. The measured concentrations of all analytes ranged from 20 pg/mg to 80 pg/mg dentin (see Fig. 2). Amphetamine, methamphetamine and MDMA were detected in the highest concentrations: 74 to 85 pg/mg. The lowest concentration was found for benzoylecgonine, being 22 pg/mg. The different concentrations of the drugs in dentin can be explained by different deposition rates of the substances having different physico-chemical properties like pKa, lipophilicity, H-bonds, molecular weight, molecule size and protein binding. Small lipophilic molecules are prone to deposition in dental hard tissue,[6] which could be the reason for the
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The lower limit of quantification was 10 pg/mg for methamphetamine, MDMA, MDEA, benzoylecgonine, morphine and codeine. The lower limit of quantification for amphetamine and cocaine was 20 pg/mg (see Table 4). Even though 20 pg/mg is below the calibration range for amphetamine and cocaine, the RSD for precision and accuracy at 20 pg/mg is clearly below 20% (RSD less or equal 4.6%). For all analytes, the limit of detection was set to the level of the lower limit of quantification. The upper limit of quantification was set to the highest calibration level (90 pg/mg).
Solvent calibration
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wileyonlinelibrary.com/journal/jms (RSD) [%] (RSD) [%] (Bias) [%] [%] [%] [%] [%] [%] [%] (RSD) [%] (RSD) [%] (Bias) [%] [%] [%] [%] [%] [%] [%]
High concentration (80 pg/mg) Intraday imprecision (n = 10) Interday imprecision (n = 5) Accuracy (n = 10) Matrix effect RSD (n = 5) Recovery RSD (n = 5) Process efficiency RSD (n = 5)
[pg/mg] [pg/mg]
Low concentration (20 pg/mg) Intraday imprecision (n = 10) Interday imprecision (n = 5) Accuracy (n = 10) Matrix effect RSD (n = 5) Recovery RSD (n = 5) Process efficiency RSD (n = 5)
LOD Calibration range Lowest correlation coefficient (R) (n = 5)
Unit
1.4 3.1 4.9 55 7.6 86 9.0 47 4.2
2.3 3.6 1.2 50 5.9 100 5.1 50 4.5
20 30–90 1.000
Amphetamine
1.5 3.3 4.6 58 5.6 89 6.0 52 5.8
2.6 2.8 6.0 37 8.2 95 4.1 35 6.5
10 10–90 0.993
Methamphetamine
5.6 5.7 5.0 57 5.7 95 6.0 54 5.3
4.4 5.6 7.4 42 8.7 94 3.2 40 10
10 10–90 0.999
MDMA
6.6 6.6 1.9 57 4.6 96 4.6 54 5.5
3.5 6.4 3.0 39 7.5 92 2.9 36 9.0
10 10–90 0.997
MDEA
1.1 2.8 3.1 81 1.4 99 4.1 80 3.9
3.2 4.6 0.0 56 8.3 95 7.1 53 4.8
20 30–90 0.999
Cocaine
2.1 2.6 1.1 49 4.7 98 10 48 8.4
1.0 4.1 2.1 28 15 96 13 26 5.3
10 10–90 1.000
Benzoylecgonine
2.1 4.1 2.7 53 12 96 7.8 50 9.6
2.9 3.4 2.6 60 18 88 8.0 53 22
10 10–90 0.998
Morphine
1.9 3.5 1.1 52 9.1 102 1.2 53 9.1
4.2 5.5 1.6 63 18 98 6.2 62 24
10 10–90 0.994
Codeine
Table 4. Calibration and validation data: limit of detection (LOD), calibration range, lowest correlation coefficient of 5 calibrations, intraday and interday precision, accuracy and respective relative standard deviation (RSD), matrix effect, recovery and process efficiency and RSD at 20 pg/mg and at 80 pg/mg, respectively
J. Spinner et al.
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J. Mass Spectrom. 2014, 49, 1306–1313
Drugs of abuse in dentin using LC-ESI-MS/MS concentrations can then be compared to the drug intake history of the deceased to further evaluate relationships between measured concentration and patterns of drug use. With the presented method, the spectrum of toxicological investigations will be reasonably supplemented especially in post-mortem cases with limited sample tissue available.
References
Figure 2. Mean concentrations (±RSD) of all drugs in artificially drugpositive dentin samples (n = 3). For the preparation of artificially drugpositive samples see Material and methods section.
amphetamines (amphetamine, methamphetamine, MDMA and MDEA) appearing in higher concentrations than the other drugs. On the other hand, just as in hair analysis, influences of extraction efficiency or irreversible binding of substances to the dental matrix causing low recoveries cannot be determined quantitatively in real samples. Development and application of a suitable in vitro model can help to interpret these effects. In our study, the concentrations are lower by a factor of around 1.000 compared to those found by Cattaneo et al.[3] and Pellegrini et al.[11] In the study with post mortem material,[3] the high measured drug concentrations could derive mainly from high post mortem drug concentrations in the blood and therefore also in the dental pulp, which was apparently not removed prior to analysis. The teeth of drug addicts[11] were probably exposed to drugs repeatedly over several months or years, whereas in our study, the dental material was exposed to a drug solution for only nine days during the artificial loading phase. Longer exposure suggests the deposition of higher drug amounts which in turn would result in higher measured concentrations. To our knowledge, this is the first time, substances were determined in dentin without potential influences of residual dental pulp.
Conclusion
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The presented liquid chromatography–tandem mass spectrometry method proved to be suitable for the detection and quantification of the common drugs of abuse amphetamine, methamphetamine, MDMA, MDEA, cocaine, benzoylecgonine, morphine and codeine in dentin samples using 50 mg of dental hard tissue. The method demonstrated to be accurate, precise, selective and specific with satisfying linearity within the calibrated range. Lower limits of quantification were 10 – 20 pg/mg. Evaluation of the analytes’ stability in processed samples suggests to analyze samples within 9 h after extraction. In artificially drug-loaded dentin samples, slightly higher concentrations of amphetamines (amphetamine, methamphetamine, MDMA, MDEA) compared to cocaine and benzoylecgonine might be explained by different physico-chemical properties of the drugs. The method can be applied in further experiments with different artificial drug-exposure conditions. In further experiments, the method should be used to quantitatively analyze drug positive tooth samples, e.g. in post-mortem cases. The measured
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