Narcotics and Psychotropic Laboratory (NPL) Procedures
Receipt of Seized Narcotic Drugs and Psychotropic Substances
We have divided the received materials into two groups: (1) substances that can be weighed and thus reported in grams, including liquid materials (i.e. cannabis oil), powders (i.e. heroin and cocaine), vegetal materials (i.e. marijuana and damiana leaves containing synthetic cannabinoids), and solid blocks (such as cannabis and opium blocks), and (2) substances that can be counted, such as pills and capsules (i.e. tramadol, clonazepam, diazepam and alprazolam). Narcotic substances that were received by the NPL and are scheduled and documented in this report include cocaine, opium, heroin, cannabis (resin, oil and hashish), and marijuana (Figure 1). Psychotropic substances that were received by the NPL and scheduled and documented in this report include methamphetamine, synthetic cannabinoids, Khat, and psilocybin mushrooms, which can all be weighed in grams (Figure 2), and methamphetamine, amphetamine, tramadol, and benzodiazepines (such as clonazepam, diazepam, flunitrazepam, alprazolam, and bromazepam), which can all be counted as pills or capsules (Figure 3). Photographic images were taken as representative examples for some of the seized substances (Additional file 1).
Seized narcotic drugs and psychotropic substances were received by the Narcotic and Psychotropic Laboratory (NPL) of Kuwait, a division of the Forensic Laboratories, General Department of Criminal Evidence. NPL is the only accredited laboratory in Kuwait that conducts drug-related investigations and routine drug testing to provide expert opinion (reports) for the court of law. Seized materials were sent to the NPL by several governmental agencies. Additionally, drug samples brought in for analysis included details of the origin of the sample e.g. place of seizure, date, physical appearance, name of suspect. Toxicological samples data included the type of sample (blood, urine), date, name of suspect.
The current study reviews and analyses documented cases that were received by the NPL and the Forensic Toxicology Laboratory (FTL) from January 2015 to December 2018. It also highlights the protocols and the experimental procedures that were used by the laboratory specialists at the NPL to generate the reports. Only reports that were generated from cases that were positive for the presence of at least one illicit drug (i.e., those listed in the schedules of narcotic drugs or psychotropic substances) were reviewed and analyzed.
Processing Seized Materials in the NPL
All procedures were performed according to the required legal provisions and the chain of custody. Regarding the analysis of seized drugs, we followed the recommendations of the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) . The mission of SWGDRUG is to recommend minimum standards for the forensic examination of seized drugs and to seek the international acceptance of these standards .
Color (Spot) Tests
Color (spot) is the most commonly used presumptive screening test in forensic laboratories. Specific instructions and protocols described by Clarke’s Analysis of Drugs and Poisons were used for the identification of the illicit drugs . Marquis reagent was used for the detection of Opiates amphetamine, and methamphetamine, Duquenois-Levine and Fast Blue B reagent used for testing for cannabinoids in cannabis, and Scott’s reagent for the detection of cocaine.
Macroscopic and Microscopic Examination for Cannabis
Macroscopic analysis (visual characterization) was used to document different cannabis species. The microscopic examination of leafy materials was performed using a stereomicroscope (Stemi DV4, Carl Zeiss, Jena, Germany), equipped with a cold light (Zeiss KL1500 LCD, Jena, Germany). Analysis included the identification of botanical characteristics such as cytolithic hairs (bear claw appearance), elongated hairs on the underside of the leaf, and resin glands (glandular hair).
Thin Layer Chromatography (TLC)
TLC was performed on pre-coated aluminum TLC-sheets (20 × 20), with a 0.25-mm silica gel layer thickness. Solid samples were dissolved in methanol (MeOH; HiPerSolv CHROMANORM, HPLC grade, BDH prolabo) (VWR International, Fonenay-sous-Bois, France) then spotted onto a TLC plate using capillary tubes (Terumo corporation, Tokyo, Japan). The mobile phases were prepared and used according to the recommendation of Clarke’s Analysis of Drugs and Poisons . Plates visualization was acheived using a short-wave ultraviolet (UV) light source and the ultraviolet fluorescent indicator ALUGRAM® Xtra SIL G SIL UV254 (Macherey-Nagel Gmbh, Duren, Germany). Rf values were calculated for each sample, for comparison with standards.
Ultraviolet-visible Spectroscopy (UV-Vis)
Ultraviolet-visible spectroscopy technique used to identify a number of different compounds, including ketamine hydrochloride, cocaine hydrochloride, diazepam, phenobarbital, and barbital . In our laboratory, a Cary 60 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) was used for these measurements, and the spectra were recorded using the Cary WinUV Scan software (Agilent Technologies, Santa Clara, CA, USA).
Attenuated Total Reflection-Fourier Transformed Infrared (ATR-FTIR) Spectroscopy
It is used as a confirmatory method for the detection of a variety of different drugs including benzodiazepines, amphetamine, methamphetamine, MDMA, lysergic acid diethylamide (LSD), cocaine, opium, heroin, morphine, synthetic cannabinoids, and cathinones. Most of the samples that were examined using this method in our laboratory are in a solid form. IR spectra were recorded using a Bruker ALPHA spectrometer (Bruker Optics, Ettlingen, Germany).
Gas Chromatography-Mass Spectrometry (GC-MS)
The method used for GC-MS analysis in this paper was adapted from our previous study . GC-MS analysis was used exclusively for identification purposes in this study; no quantification was performed. Additionally, samples were analysed in triplicates (n=3).
GC-MS vials were analyzed in a GC 7693 Gas Chromatograph (Agilent Technologies, Santa Clara, CA, USA) with an autosampler, and mass spectroscopy was performed using a 5977B GC/MSD Mass Selective Detector (Agilent Technologies, Santa Clara, CA, USA). GC-vials, GC-vial lids, and GC-vial inserts were also purchased from Agilent Technologies (Santa Clara, CA, USA).
The GC-MS parameters were set as reported in the methodology from our previous study . The injection port temperature was set to 250 °C, the splitless injection volume was 2.0 µL under a purge flow of helium gas at 3 mL/min, and the solvent delay was set to 3 min. The wash steps were: four pre-injection washes, four post-injection washes, two sample washes, and six sample pumps. The initial temperature was set to 100 °C for 4 min. Ramp 1 was set to 10 °C/min until reaching 280 °C, where it remained for 2 min. The °C/min rate for Ramp 2 was set to 6 °C until it reached 300 °C, where it remained for 5 min. An HP-5MS UI column of 30 m length, 0.25 mm inner diameter, and 0.25 μm film thicknesses (Agilent, Waldbronn, Germany) was used with the flow rate set to 1 mL/min. The MS ionization mode was electron ionization (EI) set at −70 eV, with an ion-source temperature of 280 °C and an interface temperature of 290 °C. Ions were monitored using SCAN mode. Cayman Spectral, FORCHEM, and NIST 14 Libraries were used for comparative analysis.
Toxicology Laboratory Procedures
One of the job duties of the Forensic Toxicology Laboratory (FTL) in the General Department of Criminal Evidence is to analyze drug metabolites in biological matrices including urine and blood. Additionally, FTL is the only lab in the country authorized to analyse toxicological samples. Each toxicology analysis is then translated into an official report to confirm or deny drug abuse suspension, and to be used for subsequent legal actions.
This study only analyzes reports from specimens that yielded positive results for drug abuse. In addition, some positive cases were not reported herein, as the toxicants are irrelevant to the current study. All data were collected with permission from the Ministry of Justice and the Ministry of Interior.
Urine Sample Collection from Deceased Cases
If available, urine was syringed from the bladder of deceased individuals, using a 10 mL syringe as soon as they were admitted to the Forensic Medicine Unit (FMU). The specimen was then stored at 20 °C until analysis. Living individuals (hospitalized or apprehended) provided 10 mL of urine samples in containers, which were sealed and taped to prevent adulteration.
Blood Sample Collection
Venous blood was taken from a cubital vein by a physician or registered nurse of living or deceased individuals, using 10 mL gray-stopper evacuated glass tubes containing sodium fluoride (100 mg) and potassium oxalate (25 mg) as preservative agents.
Screening of Urine and Blood Samples
Urine samples were screened by Evidence® Drug of Abuse (DOA) Array I Urine Plus (DOA I URN P) assays (Randox, Crumlin, County Antrim, UK). Evidence® Drug of Abuse (DOA) The Ultra whole blood (DOA ULTRA WB) (Randox, Crumlin, County Antrim, UK) assay was used for semi-quantitative determination of the parent molecule and the metabolites of illicit drugs in human blood.
Liquid Chromatography with Tandem Mass Spectrometry (LC–MS/MS)
Details of the LC-MS/MS method and the materials used have been published in detail in our previous publication and are outlined below . Prior to analysis, solid phase extraction (SPE) was used to extract illicit drugs from urine and blood samples for testing. As with GC/MS analysis, samples were analysed in triplicates (n=3).
CHROMABOND® C18 columns with a volume of 3 mL containing 500 mg sorbent (Macherey-Nagel, Düren, Germany) were used for solid-phase extraction of urine samples. The pH of the urine samples (10 mL) were adjusted according to Macherey-Nagel guidelines for specific drugs of abuse and samples were then centrifuged.
The compound database used for analysis was the Forensic Database (Forensic DB). However, the identification of the unknown or examined samples by the library database in this step was only tentative. Therefore, reference standards were used following preliminary identification of the unknown sample using the library, and an LC–MS/MS procedure was developed. The LC–MS/MS procedure used in the current study was a two-step scheme that was performed after injecting 10 mg/mL of the standard in methanol and 100 ng/mL of the standard in blank urine (for urine samples) or blank blood (for blood samples). The standards were extracted from blank urine or blank blood using the SPE method and used as quality controls.
Raw data visualization, analysis, and graph creation were conducted using GraphPad Prism version 6 (Prism Software Corporation) and Microsoft Excel 2016 (Microsoft Corporation).