Development and validation of a liquid chromatography tandem mass spectrometry method for the determination of cannabinoids and phase I and II metabolites in meconium

1 DEVELOPMENT AND VALIDATION OF A LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY METHOD FOR THE DETERMINATION OF CANNABINOIDS AND PHASE I AND II METABOLITES IN MECONIUM Pablo Prego-Meleiroa, Elena Lendoiroa, Marta Concheirob, Angelines Cruza, Manuel López-Rivadullaa, Ana de Castroa aToxicology Service, Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain bDepartment of Sciences, John Jay College of Criminal Justice, City University of New York, New York, NY, USA Authors’ emails: Pablo Prego-Meleiro: [email protected] Elena Lendoiro: elena.lendoi[email protected] Marta Concheiro: mconcheiro[email protected] Angelines Cruz: [email protected] Manuel López-Rivadulla: [email protected] Ana de Castro: [email protected] Corresponding author: Dra. Ana de Castro Toxicology Service, Institute of Forensic Sciences, Faculty of Medicine C/San Francisco s/n, 15782 Santiago de Compostela, Spain Phone: +34 881812446; Fax: +34 881812459 E-mail: [email protected] 2 Abstract A liquid chromatography–tandem mass spectrometry (LC–MSMS) method was developed and fully validated for the determination of ∆9-tetrahydrocannabinol (THC), 11hydroxyTHC (OHTHC), 11-nor-9-carboxyTHC (THCCOOH), 8-β-11-dihydroxyTHC (diOHTHC), cannabinol, cannabidiol, and THC and THCCOOH glucuronides in 0.25±0.02 g meconium. Samples were homogenized in methanol and subjected to cation exchange solid-phase extraction. Chromatographic separation was performed on a Kinetex C18 column (50 mm × 2.1 mm, 2.6 μm) at 35 ºC, with a gradient of 0.1% formic acid in water and acetonitrile at a flow rate of 0.3 mL/min; total run time was 10 min. Two transitions per analyte were monitored in MRM mode. The method was specific and sensitive; LOD was from 1 to 2ng/g, and LOQ from 4 to 10 ng/g; linearity ranged from 4 to 400 ng/g for all the analytes, except for THC glucuronide (10 to 400 ng/g); intra-assay, inter-assay and total imprecision were <11.2%, <13.45% and <15.6%, respectively; accuracy ranged from 93.9% to 109.0% of the target concentration; matrix effect, extraction and process efficiency ranged from -26.4% to -71.4%, 49.9% to 69.5% and 14.3% to 45.0%, respectively. The inclusion of THC and THCCOOH glucuronides avoided the need for the hydrolysis process, thus facilitating sample pretreatment. Application of the method to 19 authentic meconium specimens from uncontrolled pregnancies or women suspicious of drug consumption revealed fetal cannabis exposure in 4 newborns. THCCOOH (24.1-288.8 ng/g), diOHTHC (53.2-332.4 ng/g), THC (4.2-7.7 ng/g), CBN (30.7-93.3 ng/g) and CBD (7.1-251.5 ng/g) were detected in all cases; THCCOOH glucuronide (190.2-306.8 ng/g) in 3 cases; and OHTHC (11.9 ng/g) in the remaining one; however, THC glucuronide was not identified in any specimen. Keywords: cannabis, metabolite, glucuronide, meconium, LC-MS/MS 3 Highlights • A LC-MS/MS method was developed for the determination of cannabinoids in meconium. • Main THC metabolites, including CarboxyTHC and THC glucuronides are quantified. • Detection of THCCOOH glucuronide avoids hidrolysis to increase method sensitivity. • The method was applied to 19 meconium specimens from uncontrolled pregnancies. 4 1. Introduction According to the 2014 World Drug Report, 5.2% of the world population aged 15-64 had used an illicit drug in 2012. Among them, cannabis is the most widely consumed worldwide, with estimated consumption prevalence between 2.7% and 4.9% in this age range [1]. Drug use during pregnancy is related to a higher rate of fetal and medical obstetric complications [2-4]. There is still no definite consensus on the effects of prenatal exposure to cannabis, mainly due to the common association to other drugs. However, some studies reported that prenatal exposure to marijuana might have developmental consequences, shorter gestation length, decreased birth weight and deficit in other growth measures [5-8]. Other authors described sleep disturbances or high-pitched cry [9, 10], among other detrimental effects. Data on drug use during pregnancy are very scarce. Prevalence of drug use among pregnant women is usually made by indirect estimation according to the information available on drug use surveys for women on childbearing age (15 to 44 years). The National Survey on Drug Use and Health is the only report available about prevalence of illicit drug users among pregnant women [11]. A direct method to obtain data about drug consumption during pregnancy is the maternal interview; however, drug use is usually underestimated due to maternal fear of legal repercussions and/or social stigmatization [12]. The analysis of biological matrices from the mother, the newborn, or tissues developed during pregnancy (placenta, umbilical cord, amniotic fluid) provides an objective determination of drug use during pregnancy, as reported by several authors [13-16]. Lendoiro et al. [13] determined by the analysis of hair segments that 15.4% of randomly selected pregnant women had consumed illicit drugs (12.4% cocaine, 3.8% cannabis, 1% opiates and 1% ketamine), 22.5% medicines (3.3% methadone, 11% benzodiazepines, 9.1% antidepressants, 1% zopiclone and 1.4% fentanyl) and 3.9% alcohol; these results were much higher than those provided by the maternal interview in that study (1.4% cocaine, 2.9% cannabis, 1% opiates, 1.9% methadone, 1.9% benzodiazepines, 0.5% antidepressants, and 13.7% alcohol) [13]. Falcon et al. [14], identified in serum and hair specimens illicit drug consumption in 30% pregnant women who voluntarily interrupted their pregnancy during the first trimester (20.4% were positive for cannabis, 14.1% for cocaine and 4.2% for opiates). García-Algar et al. [15] analyzed meconium specimens from a low socioeconomic population and detected 10.9% positivity for drugs of abuse (4.7% heroin, 2.6% cocaine and 5.3% cannabis); maternal interview from the same population identified heroin, cocaine and cannabis consumption in 5 0.3%, 1.2% and 1,5% of the pregnant women, respectively. Finally, Lozano et al. [16] detected the prenatal exposure to cannabis in 5.3% newborns. Among the different matrices available, meconium, the first fecal matter from the newborn, is currently considered the sample of choice to detect prenatal exposure to drugs, as this matrix provides more complete information than other neonatal matrices such as urine or cord blood. Meconium starts its formation between the 12-16th week of pregnancy, and accumulates until birth [17,18]; therefore, it provides direct information about fetal drug exposure from the second or, more likely, the third trimester of pregnancy [19]. In addition, meconium is easily and noninvasively obtained directly from the diaper between 1 and 5 days after delivery. Moreover, meconium specimens remain stable when storage at ≤-20 °C and, under these conditions, cannabinoids remain unchanged for a period of at least six months [20]. Several methods for the determination of few cannabinoids in meconium have been published to date using GC or LC-MS [20-26]. Although identification of cannabinoides in meconium might be possible without hydrolyzing the sample in some cases, this process was performed in all reported methods as it was proved that hydrolysis increases the positivity identification rate. This process allows glucuronides cleavage, with the subsequent increase of the free analytes concentration and, therefore, the sensitivity of the method [21, 25]. A way to avoid sample hydrolysis without a negative impact on fetal cannabinoids exposure detection through meconium analysis could be the identification of the main glucuronides present in this matrix with their direct inclusion in the analytical method. The aim of this work was the development and validation of a LC-MS/MS method for the determination of CBN, cannabidiol (CBD), and ∆9-tetrahydrocannabinol (THC) and their main metabolites in meconium, including THC and 11-nor-9-carboxyTHC (THCCOOH) glucuronides, thus avoiding the hydrolysis step needed for the determination of the free analytes. 2. Materials and methods 2.1. Chemicals THC, 11-hydroxyTHC (OHTHC), THCCOOH, CBN and CBD standars at 1 mg/mL, and THCCOOH glucuronide, and the deuterated internal standards (IStd) THC-d3, OHTHC-d3, THCCOOH-d3, CBN-d3 and CBD-d3 at 0.1 mg/mL in methanol were purchased from Cerilliant (Round Rock, TX, USA). 8-β-11-dihydroxyTHC (diOHTHC) and diOHTHCd6 standards at 0.1 mg/mL in methanol, and THC glucuronide standard at 0.01 mg/mL in 6 methanol were from ElSohly Laboratories (Oxford, MS, USA). Water was purified with a Milli-Q water system (Millipore, Le-Mont-sur-Lausanne, Switzerland). Chromasolv® gradient grade methanol and reagent grade dichloromethane were from Sigma-Aldrich (Steinheim, Germany). Chromasolv® LC-MS grade 2-propanol was from Fluka. Reagent grade formic acid 98-100%, ammonium hydroxide 32% and hydrochloric acid 37%, and LCMS grade acetonitrile were from Scharlau Chemie (Sentmenat, Spain). Solid phase extraction Oasis MCX cartridges (3 cc, 60 mg) were purchased from Waters Corp. (Milford, MA, USA). 2.2. Blank meconium specimens For the preparation of the calibration curves and quality control (QC) samples we employed meconium specimens sent to the laboratory for toxicological analysis in which cannabinoids absence had been previously confirmed. 2.3. Preparation of calibration and QC solutions For the preparation of the calibration curve, a working solution containing all the compounds, except diOHTHC and the glucuronides, at 10 µg/mL was generated in methanol from the individual ampoules. This solution was diluted to obtain 0.1 µg/mL working solution, to which diOHTHC and the glucuronides were added at the same concentration. Finally, this solution was further diluted to obtained 0.01 µg/mL working solution. Six to seven calibrators at 4, 10, 20, 40, 100, 200 and 400 ng/g were elaborated by addition of the appropriate volume of the described working solutions to blank meconium samples. Different working solutions at 0.05, 0.1 and 1 µg/mL in methanol were prepared for the generation of low, medium and high QC samples (6, 30 and 300 ng/g, respectively). A working solution containing all the IStds at 1 µg/mL was prepared by dilution of the original individual ampoules in methanol. 2.4. Sample pretreatment 0.25±0.01 g meconium were weighed into Pyrex® glass tubes. The tubes were centrifuged for a few seconds to push the sample to the bottom of the tube, and 25 μL of the IStd at 1 μg/mL and 1 mL of methanol were subsequently added for sample homogenization. After mechanical shaking for 30 min, the sample was centrifuged for 10 minutes at 5000 rpm. The supernatant was subsequently evaporated in a water bath at 40 °C with a stream of nitrogen gas in order to remove the methanol and reconditioned the sample at acid pH to performed the solid phase extraction (SPE). The extract was reconstituted in 200 μL methanol to increase cannabinoids solubility, vortexed, and 2 mL of 1% formic acid in water were added. 2.5. SPE procedure 7 Oasis MCX cartridges (3cc, 60 mg) were employed for cation exchange solid-phase extraction. The samples were directly loaded without a preconditioning step, and the cartridges was subsequently washed with 2 mL of acetonitrile:water (15:85, v/v). After 5 min cartridge drying, the analytes of interest were eluted using 2 mL of dichloromethane:2propanol (50:50, v/v). The eluates were evaporated using a TurboVap LV evaporator (Zymark, Hopkinton, MA, USA) and reconstituted in 50 μL 0.1% formic acid in water:acetonitrile (60:40, v/v). The reconstituted extracts were transferred to Eppendorf tubes inside glass insert vials for centrifugation 10 minutes at 14000 rpm. Finally, the glass inserts were transferred into injection vials for LC-MS/MS analysis. 2.6. LC-MS/MS The HPLC system was an Alliance 2795 Separation Module with an Alliance series column heater/cooler (Waters Corp.). Chromatographic separation was performed with a Kinetex C18 (50 mm x 2.1 mm, 2.6 μm) reversed-phase analytical column (Phenomenex, Torrance, CA, USA), maintained at 35 ºC. A C18 security guard column for 2.1 mm internal diameter analytical columns (Phenomenex) was employed. Formic acid 0.1% in water (A) and acetonitrile (B) were used as mobile phase at a flow rate of 0.3 mL/min. Gradient was programmed as follow: 40% B from 0 to 0.2 min, linearly increased to 100% until min 6, to return to initial conditions at min 6.8. A divert valve was set to direct the flow to the MS from 1.5 to 7.5 min, and to waste the remaining time. The autosampler was maintained at 6 ºC. The mass spectrometer was a Quattro MicroTM API ESCI triple quadrupole (Waters Corp.). The instrument was operated in electrospray in the positive mode (ESI+) to produce protonated molecules of the analytes with the following optimized settings: capillary voltage 3.0 kV; source block and desolvation gas (nitrogen) temperature 150 ºC and 400 ºC, respectively; desolvation and cone gas (nitrogen) flow rate 800 L/h and 80 L/h, respectively. Data were recorded on multiple reaction monitoring (MRM) mode. A post-column infusion of each individual analyte (1 or 10 µg/mL, depending on the analyte sensitivity) at 10 μL/min connected with a “T” valve to the chromatographic effluent (formic acid 0.1% in water:ACN, 50:50, v/v) was employed to select MRM transitions, cone voltages and collision energies for the target analytes and IStds. Data acquisition was controlled with MassLynx 4.0 software and processed with QuanLynx 4.1 software (Waters Corp.). 2.7. Method validation The following parameters were studied for method validation: linearity, selectivity, limits of detection (LOD) and quantification (LOQ), intra-assay, inter-assay and total imprecision, 8 accuracy, potential glucuronide hydrolysis, matrix effect, extraction and process efficiency [27, 28]. Linearity was evaluated by the analysis of calibration curves from 4 or 10 to 400 ng/g on four different days, using 6-7 calibration levels. The straight-line fit was performed by linear regression, and a weighting factor of 1/x was applied. Acceptable linearity was achieved if the coefficient of determination (r2) was ≥0.99, and residuals were <20% at the LOQ, and <15% at the remaining concentrations. Selectivity was evaluated by assessment of endogenous and exogenous interferences. Potential endogenous interferences were assessed by the analysis of blank meconium samples collected from 10 different sources, and fortified with the IStd. Exogenous interferences were assessed by the analysis of blank meconium samples fortified with 43 common drugs of abuse and medicines at 0.5 µg/g The following drugs were tested: morphine, codeine, 6-acetylmorphine, methadone, 2-ethylidene-1,5-dimethyl-3,3diphenylpyrrolidine (EDDP), amphetamine, methamphetamine, 3,4methylendioxyamphetamine (MDA), 3,4-methylendioxymethamphetamine (MDMA), 3,4methylendioxyethylamphetamine (MDEA), cocaine, benzoylecgonine, ecgonine methylester, cocaethylene, lysergic acid diethylamide (LSD), ketamine, norketamine, gammahydroxybutyric acid (GHB), nicotine, cotinine, fentanyl, amitriptyline, paroxetine, zolpidem, zopiclone, ibuprofen, omeprazole, acetaminophen, diclofenac, naproxen, alprazolam, temazepam, lormetazepam, lorazepam, flunitrazepam, 7-aminoflunitrazepam, clonazepam, diazepam, nordiazepam, oxazepam, triazolam, nitrazepam, bromazepam. The LOD was defined as the lowest concentration at which the two MRM transitions monitored for each analyte could be identified with a signal-to-noise >3 and appropriate ion ratio, and within ±0.2 min of the mean calibrators retention time. LOD was determined by the analysis of fortified blank mecomium samples at decreasing concentrations. LOQ was defined as the lowest concentration that could be quantified with adequate precision (%CV <20%) and accuracy (% of target concentration ±20%), and with a signalto-noise >10. The LOQ was calculated by the analysis of five replicates at the lowest concentration of the calibration curve. Imprecision and accuracy were assessed at low, medium and high QC concentrations for THC, OHTHC, THCCOOH, diOHTHC, CBN, CBD and THCCOOH glucuronide, and at medium and high QC concentrations for THC glucuronide. These parameters were evaluated by the analysis of five replicates for each concentration on four different days (n=20). Intra and inter-assay, and total imprecision was determined by calculation of the coefficient of 9 variation (%CV) following Krouwer and Rabinowitz’ recommendations [29], and using SPSS v. 20.0 statistical software. %CV was required to be less than 15%. Accuracy was expressed as the percentage of the nominal concentration, and was required to be within 85115%. Possible hydrolysis of THC and THCCOOH glucuronides was evaluated by the analysis of meconium fortified only with THC glucuronide at 300 ng/g and the IStd (n=3), and meconium fortified only with THCCOOH glucuronide at 300 ng/g and the IStd (n=3). Matrix effect, extraction and process efficiency were calculated at 30 and 300 ng/g following Matuszewski et al. recommendations [30]. Matrix effect was assessed by comparing average analyte peak area in blank meconium samples from 10 different sources fortified after extraction, with average peak area when the analytes were prepared at the same concentration in formic acid 0.1% in water:ACN (60:40, v/v) (n=5). Extraction efficiency was calculated by comparing average analyte peak area in samples fortified with the analytes before extraction (n=5) with average peak area obtained in blank samples fortified after extraction (n=5). Process efficiency was evaluated by comparing average analyte peak area in samples fortified with the analytes before extraction (n=5) with average peak area when the analytes were prepared at the same concentration in formic acid 0.1% in water:ACN (60:40, v/v) (n=5). 2.8. Application to authentic cases As a proof of the method, 19 authentic meconium specimens received in our laboratory for toxicological analysis during 2015 were analyzed using the described LC-MS/MS method. 3. Results 3.1. Method development and validation Chromatographic elution of all the analytes was achieved in 6.5 min, and the total chromatographic run was 10 min. Quantification was based on the most prominent MRM transition. A second transition was monitored for qualitative purposes to fulfill the European Commission Decision 2002/657/EC identification criteria using mass spectrometric techniques [31]. Table 1 shows quantification and qualification transitions, cone voltages, collision energies, retention time and selected IStd for each analyte. For sample extraction, we assayed liquid-liquid extraction in acid conditions employing hexane as extraction solvent, and SPE using Strata Drug, Strata X (Phenomenex) and Oasis MAX cartridges (Waters Corp.). For Strata Drug we employed a similar protocol to that proposed by Phenomenex for the extraction of cannabis and metabolites in different matrices [32-34]. In our case, after meconium homogenization, the sample was acidified, and washing 16 [17] J. 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Matuszewski, M.L. Constanzer, C.M Chavez-Eng, Strategies for the assessment of matrix effect in quantitative bioanalytical, Anal. Chem. 75 (2003) 3019–3030. DOI: 10.1021/ac020361s. [31] European Union Decision 2002/657/EC (17/8/2002), Commision decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off. J. Eur. Commun. 221 (2002) 8-36. [32] https://www.phenomenex.com/Application/Detail/19947?returnURL=/Search (Accessed on 25.12.17). [33] https://az621941.vo.msecnd.net/documents/c627fd54-a483-46b8-be605212a8d5d51f.pdf (Accessed on 25.12.17). [34] http://separations.co.za/wp-content/uploads/2014/08/Strata-Drug-B.pdf (Accessed on 25.12.17). 18 Tables Table 1. MRM transitions, cone voltage (CV), collision energy (CE), retention time (Rt) and internal standard (IStd) selected for each analyte. Analyte MRM transition CV (V) CE (eV) Rt (min) IStd THC 315.3>193.4 315.3>135.2 27 24 6.40 THC-d3 OHTHC 331.3>313.5 331.3>193.4 25 14 4.70 OH-THC-d3 diOHTHC 347.3>329.4 347.3>311.5 25 12 2.85 diOHTHC-d6 THCCOOH 345.2>327.4 345.2>299.4 40 16 4.82 THCCOOH-d3 CBN 311.3>223.4 311.3>293.4 30 22 6.28 CBN-d3 CBD 315.3>193.4 315.3>135.2 27 24 5.74 CBD-d3 THC glucuronide 315.3>193.4 315.3>123.1 50 24 3.97 diOHTHC-d6 THCCOOH glucuronide 345.2>327.4 345.2>299.4 40 20 3.20 diOHTHC-d6 Underlined transitions were used for quantification. THC: tetrahydrocannabinol; OHTHC: hydroxy-tetrahydrocannabinol; diOH-THC: di-hydroxy-tetrahydrocannabinol; THCCOOH: carboxy-tetrahydrocannabinol; CBN: cannabidiol; CBD: cannabidiol 19 Table 2. Calibration parameters, limit of detection (LOD) and quantification (LOQ) for all the analytes. Analyte LOD (ng/g) LOQ (ng/g) Linearity (ng/g) Intercept ± SD (n = 4) Slope 1 (x) ± SD (n = 4) Slope 2 (x2) ± SD (n = 4) r2 ± SD (n=4) THC 1 4 4-400 0.5777 ± 0.4031 0.5405 ± 0.0055 - 0.9985 ± 0.0012 OHTHC 1 4 4-400 0.0686 ± 0.0274 0.0640 ± 0.0059 - 0.9981 ± 0.0004 diOHTHC 2 4 4-400 0.2786 ± 0.2709 0.3305 ± 0.0082 - 0.9984 ± 0.0005 THCCOOH 1 4 4-400 0.2442 ± 0.3144 0.6628 ± 0.0312 - 0.9980 ± 0.0005 CBN 1 4 4-400 0.1939 ± 0.0350 0.4509 ± 0.0796 - 0.9990 ± 0.0007 CBD 2 4 4-400 0.3194 ± 0.3198 0.4902 ± 0.0312 - 0.9986 ± 0.0011 THC glucuronide 2 10 10-400 16.7870 ± 12.0963 2.5879 ± 1.0326 - 0.9919 ± 0.0044 THCCOOH glucuronide 2 4 4-400 2.3998 ± 1.2693 1.9935 ± 1.6865 -0.0015 ± 0.0024 0.9971 ± 0.0010 THC: tetrahydrocannabinol; OH-THC: hydroxy-tetrahydrocannabinol; diOH-THC: di-hydroxytetrahydrocannabinol; THCCOOH: carboxy-tetrahydrocannabinol; CBN: cannabidiol; CBD: cannabidiol 20 Table 3. Results for imprecision and accuracy in meconium at low, medium and high QC concentrations. Analyte Intra-assay imprecision (n =20; %CV) Inter-assay imprecision (n =20; %CV) Total imprecision (n = 0; %CV) Accuracy (n =20; % target concentration) 6 ng/g 30 ng/g 300 ng/g 6 ng/g 30 ng/g 300 ng/g 6 ng/g 30 ng/g 300 ng/g 6 ng/g 30 ng/g 300 ng/g THC 5.7 4.0 1.7 7.7 4.8 4.1 9.6 6.3 4.4 97.8 106.7 106.8 OHTHC 7.9 4.4 3.2 6.2 6.8 5.1 10.0 8.1 6.0 99.7 107.5 107.4 diOHTHC 0.1 6.3 5.0 0.1 0.0 5.1 0.2 6.3 7.2 96.6 99.0 96.6 THCCOOH 6.3 2.4 2.9 3.3 4.3 2.5 7.1 4.9 3.8 101.8 108.4 109.0 CBN 4.2 2.9 2.2 0.0 3.3 2.6 4.2 4.4 3.4 93.9 105.2 108.8 CBD 6.3 3.6 2.8 7.4 2.6 1.1 9.7 4.4 3.0 100.8 108.7 110.6 THC glucuronide - 7.0 11.2 - 13.5 10.9 - 15.2 15.6 - 100.6 99.2 THCCOOH glucuronide 7.8 7.4 8.1 0.0 3.2 3.8 7.8 8.1 9.0 105.1 96.5 99.4 THC: tetrahydrocannabinol; OH-THC: hydroxy-tetrahydrocannabinol; diOH-THC: di-hydroxytetrahydrocannabinol; THCCOOH: carboxy-tetrahydrocannabinol; CBN: cannabidiol; CBD: cannabidiol 21 Table 4. Matrix effect, extraction and process efficiency results at medium (30 ng/g) and high QC (300 ng/g) concentrations. Analyte Matrix effect (%CV) (n=10) Extraction efficiency (%) (%CV) (n=5) Process efficiency (%) (n=5) 30 ng/g 300 ng/g 30 ng/g 300 ng/g 30 ng/g 300 ng/g THC -35.5 (33.1) -26.4 (35.6) 50.2 (19.1) 61.1 (29.0) 32.4 45.0 THC-d3 -38.8 (32.1) -30.4 (35.5) 51.5 (15.7) 60.9 (27.8) 30.0 42.4 OHTHC -51.1 (42.0) -59.4 (48.0) 55.2 (37.9) 63.1 (41.5) 27.1 25.6 OHTHC-d3 -53.5 (46.5) 61.4 (49.6) 51.4 (41.4) 62.3 (42.9) 29.3 24.0 diOHTHC -53.2 (26.9) -61.5 (24.5) 67.9 (7.6) 69.5 (7.5) 30.7 26.7 diOHTHC-d6 -54.5 (21.7) -54.1 (20.4) 62.3 (11.3) 68.4 (7.7) 28.3 31.4 THCCOOH -62.4 (34.6) -61.6 (43.3) 51.8 (26.1) 56.7 (31.2) 19.5 21.8 THCCOOH-d3 -62.5 (35.5) -60.4 (42.5) 62.3 (11.3) 68.4 (7.7) 28.3 31.4 CBN -57.6 (36.8) -52.5 (36.6) 50.0 (10.7) 63.5 (22.8) 21.2 30.0 CBN-d3 -58.4 (35.6) -54.0 (36.4) 49.1 (10.0) 62.5 (21.7) 20.4 28.8 CBD -52.3 (31.6) -57.5 (38.0) 49.9 (16.2) 67.2 (17.1) 23.8 28.5 CBD-d3 -53.7 (32.1) -58.6 (39.1) 48.2 (14.4) 66.8 (17.3) 22.3 27.7 THC glucuronide -71.3 (25.7) -67.8 (26.5) 61.7 (18.8) 66.8 (17.7) 17.7 21.5 THCCOOH glucuronide -71.4 (30.8) -71.3 (20.9) 50.1 (18.0) 64.3 (12.3) 14.3 18.5 THC: tetrahydrocannabinol; OH-THC: hydroxy-tetrahydrocannabinol; diOH-THC: di-hydroxy-tetrahydrocannabinol; THCCOOH: carboxy-tetrahydrocannabinol; CBN: cannabidiol; CBD: cannabidiol 22 Table 5. Coefficient of determination (%CV) for the ratio analyte peak area/IStd peak area obtained in the specimens employed to evaluate the matrix effect (n=10), at 30 and 300 ng/g. Analyte %CV at 30 ng/g %CV at 300 ng/g THC 3.7 2.0 OHTHC 7.2 5.4 diOHTHC 13.2 7.2 THCCOOH 2.2 2.4 CBN 1.8 1.7 CBD 1.2 2.4 THC glucuronide 9.7 14.9 THCCOOH glucuronide 15.9 12.2 THC: tetrahydrocannabinol; OH-THC: hydroxytetrahydrocannabinol; diOH-THC: di-hydroxytetrahydrocannabinol; THCCOOH: carboxy-tetrahydrocannabinol; CBN: cannabidiol; CBD: cannabidiol 23 Table 6. Cannabinoids concentrations in the 4 positive meconium specimens. Case/year Analyte concentrations (ng/g) THC OHTHC diOHTHC THCCOOH CBN CBD THCCOOH glucuronide THC glucuronide 1910/2015 5.6 0.0 53.2 45.0 70.5 112.1 190.2 0.0 1944/2015 7.4 0.0 53.2 288.8 92.2 251.5 291.4 0.0 2177/2015 4.2 11.9 236.2 24.1 30.7 7.1 0.0 0.0 2497/2015 7.7 0.0 332.4 53.8 93.3 76.4 306.8 0.0 THC: tetrahydrocannabinol; OH-THC: hydroxy-tetrahydrocannabinol; diOH-THC: di-hydroxytetrahydrocannabinol; THCCOOH: carboxy-tetrahydrocannabinol; CBN: cannabidiol; CBD: cannabidiol 24 Figures Fig. 1. MRM chromatograms of the quantifier transition of the analytes in a blank meconium sample (A) and a blank meconium sample fortified at the LOQ (B). THC: tetrahydrocannabinol; OHTHC: 11-hydroxyTHC; diOHTHC: 8-β-11-dihydroxyTHC; THCCOOH: 11-nor-9-carboxyTHC; CBN: cannabidiol; CBD: cannabidiol. diOHTHC 347.3>329.4 THCCOOH glucuronide 345.2>327.4 OHTHC 331.3>313.5 THC glucuronide 315.3>193.4 THCCOOH 345.2>327.4 CBD 315.3>193.4 THC 315.3>193.4 CBN 311.3>223.4 A diOHTHC 347.3>329.4 THCCOOH glucuronide 345.2>327.4 OHTHC 331.3>313.5 THC glucuronide 315.3>193.4 THCCOOH 345.2>327.4 CBD 315.3>193.4 THC 315.3>193.4 CBN 311.3>223.4 B