Concentrations of THC, CBD, and CBN in commercial hemp seeds and hempseed oil sold in Korea.

Hemp seeds and hempseed oil are marketed on- and off-line as health foods and cosmetics and have been reported to have high nutrient contents. However, because of the various side effects of cannabinoids, especially △9-tetrahydrocannabinol (THC), many countries regulate upper limits for THC in products, which creates the need for analytical techniques capable of measuring THC, cannabidiol (CBD), and cannabinol (CBN) levels in commercial hemp seeds and hempseed oil. In the present study, hemp seed and hempseed oil extracts obtained by methanol extraction, were analyzed by gas chromatography-mass spectrometry (GC/MS). Validation of the technique used was performed using calibration curves and by determining LODs, LOQs, specificities, selectivities, and intra- and inter-day precision and accuracies. In addition, matrix effects, process efficiencies, recoveries, and sample stabilities were investigated. In hemp seeds, as determined using the fully optimized method THC concentrations ranged from 0.06 to 5.91 µg/g, CBD concentrations from 0.32 to 25.55 µg/g, and CBN concentrations from 0.01 to 1.50 µg/g; CBN/THC ratios ranged from 0.1 to 1.60, and CBD/THC ratios from 0.11 to 62.56. Furthermore, the (THC + CBN)/CBD ratio of most hemp seed samples was less than one. In hempseed oil, THC concentrations ranged from 0.3 to 19.73 µg/mL, CBD concentrations from 6.66 to 63.40 µg/mL, CBN concentrations from 0.11 to 2.31 µg/mL, CBN/THC ratios from 0.12 to 0.42, and CBD/THC ratios from 3.21 to 22.50. Furthermore, (THC + CBN)/CBD ratios in all hempseed oil samples were less than one. The optimized methanol extraction-GC/MS technique was found to be satisfactory for determining THC, CBD, and CBN concentrations in hemp seeds and hempseed oil.

Analysis of cannabinoids in urine samples of short-term and long-term consumers of hemp seed products.

With the increasing consumption of hemp seed products, it is important to establish whether biological samples from consumers contain trace cannabinoids that could mistakenly be attributed to cannabis abuse. Thus, we analyzed whether the cannabinoids, 11-nor-9-carboxy-tetrahydrocannabinol, cannabidiol, and cannabinol, can be detected in the urine of short-term (1 week) and long-term (12 weeks) consumers of hemp seed products. Using three hemp seed products that have recently been highly distributed in Korea, subjects consumed 30 g of hemp seeds (Group A, 53.1 (±0.5) µg THC and Group B, 124.81 (±1.5) µg THC) or 2 capsules of hemp seed oil (Group C, 11.1 µg THC) once a day at 10am. In the short-term study, the hemp seed product was consumed for 7 days, after which a single urine sample was collected. In the long-term study, the hemp seed product was consumed for 12 weeks, and urine samples were collected at 7 day intervals. For screening of the urine samples, we used COBAS C311, and for the confirmatory analysis we used gas chromatography/mass spectrometry. In the screening, all 64 samples from the short-term study were below the detection cutoff level of 25 ng/mL. In the long-term study, out of the 480 samples, 3 samples from 2 participants were above the cutoff level, but these samples were all negative in screening. In conclusion, our study demonstrated that when hemp seed products sold in Korea are consumed in normal quantities over the short- or long-term, urine samples do not test positive for cannabinoids.

Determination of urinary metabolites of XLR-11 by liquid chromatography-quadrupole time-of-flight mass spectrometry.

Recently, use of novel synthetic cannabinoids has increased greatly despite worldwide efforts to regulate these drugs. XLR-11 ((1-[5'-fluoropentyl]indol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone), a fluorinated synthetic cannabinoid with a tetramethylcyclopropyl moiety, has been frequently abused since 2012. XLR-11 produces a number of metabolites in common with its non-fluorinated parent analogue, UR-144 ((1-pentylindol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone). Therefore, it is essential to develop effective urinary markers to distinguish between these drugs. In this study, we investigated the metabolic profile of authentic human urine specimens from suspected users of XLR-11 using liquid chromatography-quadrupole time-of-flight mass spectrometry. Furthermore, we quantified four potential XLR-11 metabolites by using commercially available reference standards. In vitro metabolism of XLR-11 and UR-144 using human liver microsomes was also investigated to compare patterns of production of hydroxypentyl metabolites. Urine samples were prepared with and without enzymatic hydrolysis, and subjected to solid-phase extraction. We identified 19 metabolites generated by oxidative defluorination, hydroxylation, carboxylation, dehydrogenation, glucuronidation, and combinations of these reactions. Among the identified metabolites, 12 were generated from a cyclopropyl ring-opened XLR-11 degradation product formed during smoking. The XLR-11 metabolite with a hydroxylated 2,4-dimethylpent-1-ene moiety was detected in most specimens after hydrolysis and could be utilized as a specific marker for XLR-11 intake. Quantitative results showed that the concentration ratio of 5- and 4-hydroxypentyl metabolites should also be considered as a useful marker for differentiating between the abuse of XLR-11 and UR-144.

A comprehensive and sensitive method for hair analysis in drug-facilitated crimes and incorporation of zolazepam and tiletamine into hair after a single exposure.

Hair is a highly relevant specimen that is used to verify drug exposure in victims of drug-facilitated crime (DFC) cases. In the present study, a new analytical method involving ultrahigh-performance liquid chromatography-tandem mass spectrometry was developed for determining the presence of model drugs, including zolazepam and tiletamine and their metabolites in hair specimens from DFCs. The incorporation of zolazepam and tiletamine into hair after a single exposure was investigated in Long-Evans rats with the ratio of the hair concentration to the area under the curve. For rapid and simple sample preparation, methanol extraction and protein precipitation were performed for hair and plasma, respectively. No interference was observed in drug-free hair or plasma, except for hair-derived diphenhydramine in blank hair. The coefficients of variance of the matrix effects were below 12%, and the recoveries of the analytes exceeded 70% in all of the matrices. The precision and accuracy results were satisfactory. The limits of quantification ranged from 20 to 50 pg in 10 mg of hair. The drug incorporation rates were 0.03 ± 0.01% for zolazepam and 2.09 ± 0.51% for tiletamine in pigmented hair. We applied the present method to real hair samples in order to determine the drug that was used in seven cases. These results suggest that this comprehensive and sensitive hair analysis method can successfully verify a drug after a single exposure in crimes and can be applied in forensic and clinical toxicology laboratories.

Simultaneous determination of five naphthoylindole-based synthetic cannabinoids and metabolites and their deposition in human and rat hair.

The continuing appearance of new synthetic cannabinoids has been a major issue in the field of forensic and clinical toxicology. In response to that, analytical methods for synthetic cannabinoids have been increasingly established in a variety of biological matrices. Since most of synthetic cannabinoids with structure similarity share some enzymatic metabolites, making the interpretation of analytical results and the discovery of the parent drug actually ingested very complicated, the investigation on metabolites of the first generation of synthetic cannabinoids with their relatively short side chains in chemical structure could be more important. Therefore, in the present study, we developed the analytical method for AM-2201, JWH-122 and MAM-2201 with JWH-018 as a precursor and their monohydroxylated metabolites in hair matrix. Also, using a rat model, AM-2201 and its monohydroxylated metabolites were identified and then the ratios of metabolite-to-parent drug were estimated to be used as criteria on external contamination. All analytes were extracted with methanol from washed and cut hair samples and the extracts were injected into LC-MS/MS with electrospray ion source in the positive ionization mode. Matrix effect and recovery were evaluated in hair matrices and no significant variations were observed. The validation results for precision and accuracy were satisfactory in both human and rat hair. The LOD and LOQ were 0.5 pg/10mg and 1.0 pg/10mg in human hair and 0.5 pg/20mg and 1.0 pg/20mg in pigmented and non-pigmented rat hair, respectively. Additionally, as a result of the animal study, there were not significant differences in the effect of pigmentation on the distribution of AM-2201 and its monohydroxylated metabolites in hair. Wide variations were observed for the concentrations of the naphthoylindole-based synthetic cannabinoids and metabolites in authentic hair samples from nine cases; those were 0.4-59.2 pg/mg for JWH-018, 0.1-0.8 pg/mg for JWH-073, 1.7-739.0 pg/mg for AM-2201, 0.1-402.0 pg/mg for JWH-122, 0.2-276.0 pg/mg for MAM-2201, 0.2-1.1 pg/mg for JWH-018 N-COOH, 0.3-37.2 pg/mg for JWH-018 N-5-OH, 0.3 pg/mg for JWH-073 N-COOH, 0.4 pg/mg for AM-2201 N-4-OH, 0.2-3.1 pg/mg for AM-2201 N-6-OHindole and 0.1-3.5 pg/mg for JWH-122 N-5-OH. This quantitative LC-MS/MS analytical method for five naphthoylindole-based synthetic cannabinoids and their metabolites was very useful to be applied to authentic hair samples, of which their analytical results suggested the incorporation of synthetic cannabinoids in the hair matrix and provided the information on ingested parent drugs.

An LC-MS/MS method for the determination of five erectile dysfunction drugs and their selected metabolites in hair.

The abuse of sildenafil and its analogous, accelerated by their inappropriate or illegal distribution, is a serious social issue globally. However, no studies have been conducted to monitor these drugs simultaneously in hair, which can provide valuable information on chronic drug use. In the present study, an LC-MS/MS method was developed for the simultaneous determination in hair of five erectile dysfunction drugs having a high risk for abuse (mirodenafil, sildenafil, tadalafil, udenafil and vardenafil) and their selected metabolites (SK3541, desmethylsildenafil, DA8164 and desethylvardenafil). The novel method was fully validated after optimizing matrix effects and extraction efficiency. The optimized sample preparation included acidic methanol extraction followed by solid phase extraction using C18 mixed mode strong cation exchange polymeric cartridges. The prepared samples were analyzed by LC-MS/MS with electrospray ion source in the positive ionization mode. The validation results proved the method to be selective, sensitive, accurate and precise, with acceptable linearity within calibration ranges. LODs ranged from 0.05 (DA8164) to 1 ng/10 mg hair (tadalafil). LOQs were 1 ng/10 mg hair except for DA8164 and vardenafil, of which they were 2.5 ng/10 mg hair. No significant variations were observed by different sources of matrices in both human and rat hair, except for tadalafil, for which a stable isotope-labeled internal standard was effective. The animal study suggested hair pigmentation was a major factor for the incorporation of the drugs and metabolites into hair. However, a wide variation of the sildenafil-to-desmethylsildenafil ratios was observed in human hair samples. The developed method will be very useful for monitoring the abuse of erectile dysfunction drugs for both legal and public health aspects.

Deaths from recreational use of propofol in Korea.

Propofol, a short-acting and sedative-hypnotic agent, induces and maintains anesthesia. Since it is known to produce mild euphoria and hallucinations, the recreational use of propofol has been a big issue in Korea. Furthermore, many deaths have occurred due to its abuse and misuse. In order to study the prevalence of abuse and deaths due to propofol, all autopsy cases conducted between 2005 and 2010 at the NFS (National Forensic Service, Korea) were monitored by checking its concentrations in the blood. Propofol was detected in 131 cases (0.88%) out of 14,673 autopsied cases within 6 years. Propofol alone was detected in 49 of 131 fatal cases, while the combination of drugs was detected with propofol in the remaining 82 cases. The concentrations of propofol from autopsied cases ranged from 0.05 to 8.83 mg/L (mean 1.66; median 0.9) and from 0.08 to 8.65 mg/L (mean 1.71; median 1.05) in the heart (n=31) and the femoral blood (n=32), respectively. The investigation of the ratio of heart to femoral blood and the difference between the concentrations in heart and femoral blood (n=15) from the same body revealed the ratio from 0.45 to 3.66 (mean 1.53; median 1.40). The autopsy resulted in accidental death after self-administration in 16 autopsied cases among 131 autopsied cases. In 16 cases, their ages ranged from 17 to 56 and 75% of them were in their 20's and 30's and 75% were female. Half of them were medical personnel including 19% of doctors and 38% of nurses. The combination of drugs was detected in 6 cases. Fluoxetine was detected in three and vecuronium was detected in two along with propofol. The cause of death in 14 cases was drug intoxication, while that in 2 cases was hanging. Due to its prevalence, Korea has become the first country that regulates propofol as a psychotropic substance.

Quantitative LC-MS/MS method in urine for the detection of drugs used to reverse the effects of chemical castration.

The chemical castration law, which targets child molesters with recidivism, was introduced in Korea in 2011. For this, leuprolide, a gonadotropin-releasing hormone agonist, is used to decrease testosterone production and suppress libido. In order to achieve efficient law enforcement, it is necessary to monitor intentional ingestion of drugs that antagonize the effect of leuprolide. Therefore, an analytical method for the simultaneous detection of mirodenafil, sildenafil, tadalafil, udenafil, vardenafil, icariin, alprostadil, and yohimbine, which are the major impotence treatment drugs, legitimately or otherwise, in Korea, as well as their selected metabolites, in human urine was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, different sample preparation methods, two solid-phase extractions with different cartridges and protein precipitation, were compared and protein precipitation was chosen for the entire study because it showed better matrix effects and recoveries. Thus, the drugs and metabolites in urine were extracted by protein precipitation and then filtered and analyzed by LC-MS/MS with polarity switching electrospray ionization. The validation results of selectivity, matrix effect, recovery, linearity, intra- and inter-assay precision and accuracy were satisfactory. The limits of detection ranged from 0.25 to 10 ng/mL, and the limits of quantification were 2.5 to 50 ng/mL. The drugs and metabolites in urine did not show any degradation under storage for 7 and 15 days at 4 and -20 °C as well as after three freeze-thaw cycles. The developed method will be very useful for monitoring the illegal use of impotence treatment drugs.

Simultaneous analysis of psychotropic phenylalkylamines in oral fluid by GC-MS with automated SPE and its application to legal cases.

Phenylalkylamine derivatives, such as methamphetamine (MA), amphetamine (AM), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), phentermine (PT), fenfluramine (FFA) and phenmetrazine (PM), and ketamine (KT) are widely abused recreational or anorectic drugs in Korea and are regulated under the Controlled Substance Act in Korea. Phenylalkylamines and ketamine analysis is normally performed using both urine and hair samples but there is no established method for the simultaneous analysis of all these phenylalkylamines and ketamine in oral fluids. Oral fluid is easy to collect/handle and can provide an indication of recent drug abuse. In this study, to confirm the presence of phenylalkylamine derivatives and ketamine in oral fluid after screening with an immunoassay, an analytical method using automated solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) was developed and fully validated according to international guidelines. The applicability of the assay was demonstrated by analyzing of authentic oral fluid samples and the results of oral fluid analysis were compared with those in urine and hair to to evaluate the feasibility of oral fluid in forensic cases. The recovery of phenylalkylamines and ketamine from oral fluid collection devices was also assessed. Oral fluid specimens from 23 drug abuse suspects submitted by the police were collected using Salivette (Sarstedt, Nümbrecht, Germany), Quantisal (Immunalysis, Pomona, CA) or direct expectoration. The samples were screened using a biochip array analyzer (Evidence Investigator, Randox, Antrim, UK). For confirmation, the samples were analyzed by GC-MS in selected-ion monitoring (SIM) mode after extraction using automated SPE (RapidTrace, Zymark, MA, USA) with a mixed-mode cation exchange cartridge (CLEAN SCREEN, 130 mg/3 ml, UCT, PA, USA) and derivatization with trifluoroacetic anhydride (TFA). The results from the immunoassay were consistent with those from GC-MS. Twenty oral fluid samples gave positive results for MA, AM, PT and/or PM among the 23 cases, which gave positive results in urine and/or hair. Although large variations in the MA, AM, PT and PM concentrations were observed in three different specimens, the oral fluid specimen was useful for demonstrating phenylalkylamines and ketamine abuse as an alternative specimen for urine.

Effects of repeated hair washing and a single hair dyeing on concentrations of methamphetamine and amphetamine in human hairs.

The effects of repeated hair washing and a single hair dyeing on concentrations of methamphetamine (MA) and amphetamine (AM) in hair samples of MA addicts were studied. Thirty-one MA positive hair samples collected from male (n = 24, 24-51 yrs) and female abusers (n = 7, 17-46 yrs) were evaluated for MA and AM concentration's changes after repeated hair washing and a single hair dyeing. Thirty-one MA positive hair samples, no additional treatment hair sample group (NAT), were treated in vitro with liquid soap or three kinds of hair dyes which were black, brown and yellow color hair dye, respectively. Quantitation of AM and MA in hair samples was utilized GC-MS using selected ion monitoring. MA and AM concentrations in NAT were 10.41 ± 8.91 ng/mg (range 1.50-30.0 ng/mg) and 2.24 ± 2.75 ng/mg (range 0.41-12.90 ng/mg). And, their concentrations were decreased about 23.3 ± 4.5% (range 16.7-32.8%) in hair repeated washing group (WAS) and 32.6 ± 4.82 (22.2-41.9) in three kinds of a single hair dyeing groups in comparison to original concentrations of MA and AM in NAT. A statistically significant difference was found between NAT and WAS or three hair dyeing groups (p < 0.01), but not between WAS and three hair dyeing groups, and not between each hair dyeing groups with each three kinds of hair dyes (p > 0.05).

Analysis of cannabis in oral fluid specimens by GC-MS with automatic SPE.

Methamphetamine (MA) is the most commonly abused drug in Korea, followed by cannabis. Traditionally, MA analysis is carried out on both urine and hair samples and cannabis analysis in urine samples only. Despite the fact that oral fluid has become increasingly popular as an alternative specimen in the field of driving under the influence of drugs (DUID) and work place drug testing, its application has not been expanded to drug analysis in Korea. Oral fluid is easy to collect and handle and can provide an indication of recent drug abuse. In this study, we present an analytical method using GC-MS to determine tetrahydrocannabinol (THC) and its main metabolite 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in oral fluid. The validated method was applied to oral fluid samples collected from drug abuse suspects and the results were compared with those in urine. The stability of THC and THC-COOH in oral fluid stored in different containers was also investigated. Oral fluid specimens from 12 drug abuse suspects, submitted by the police, were collected by direct expectoration. The samples were screened with microplate ELISA. For confirmation they were extracted using automated SPE with mixed-mode cation exchange cartridge, derivatized and analyzed by GC-MS using selective ion monitoring (SIM). The concentrations ofTHC and THC-COOH in oral fluid showed a large variation and the results from oral fluid and urine samples from cannabis abusers did not show any correlation. Thus, detailed information about time interval between drug use and sample collection is needed to interpret the oral fluid results properly. In addition, further investigation about the detection time window ofTHC and THC-COOH in oral fluid is required to substitute oral fluid for urine in drug testing.