Hexahydrocannabinol on the Light Cannabis Market: The Latest "New" Entry.

Introduction: Hexahydrocannabinols (HHCs), referred to as (9R)-HHC and (9S)-HHC diastereoisomers, are poorly studied cannabinoids naturally found in small concentrations in the pollen and the seeds of the hemp plants. Aim: In this study, for the first time, we describe the finding of (9R)-HHC and (9S)-HHC in two commercialized hemp derived products. Methods: The achievement of reference standards by semisynthetic or isolation approach allows us to develop and validate a gas chromatography mass spectrometry method for the identification and quantification of HHCs in hemp-derived resin. Results: The two analyzed samples showed percentage of 42.5 and 41.5 for (9R)-HHC and of 23.6 and 23.6 for (9S)-HHC. Conclusions: Despite the lack of in-depth studies about HHCs activity, potency, toxicity, and safety, these cannabinoids are emerging on the light-cannabis (hemp) market probably because legislations still do not clearly regulate them. Since analytical assay for hemp-derived products usually include only Δ9-THC, THC-A, CBD, and CBD-A, a thorough investigation could be carried out to reveal the possible addition of "new" compounds that might be a matter of safety.

Acute perchloroethylene intoxication in an elderly woman: a case report.

BACKGROUND: Perchloroethylene is a colorless, strong-smelling substance commonly used for dry cleaning. Liver and kidney toxicities and carcinogenicity are well-known occupational hazards caused by chronic perchloroethylene exposure. Acute intoxication by ingestion of nondiluted perchloroethylene is rare in the adult population owing to its strong smell and taste. Very few data are available to physicians managing patients in this situation. CASE PRESENTATION: An 89-year-old Caucasian woman accidentally drank perchloroethylene while visiting her laundry, leading to a coma within a few minutes. The poison control center provided little information about perchloroethylene toxicity after ingestion, including an estimated long biological half-life (144 hour) and detrimental effects to liver and kidneys. A long intensive care unit stay was thus expected, potentially leading to several complications. After intubation, transitory hypoxemia appeared and rapidly resolved, while mild hemodynamic instability was managed with fluid resuscitation and anti-arrhythmic drugs. Twelve hours after perchloroethylene ingestion, the patient suddenly woke up and self-extubated. Less than 24 hours after ingestion, she was discharged from the intensive care unit, and 4 days later she was discharged home. CONCLUSION: The patient drank perchloroethylene from a bottle, which prevented her from smelling it, and owing to its taste, only a small sip was likely drunk. However, a much larger intake was presumed, given her rapid and profound central nervous system depression. This case was challenging owing to the paucity of information available regarding acute perchloroethylene ingestion and the duration and magnitude of its effect. The present report will hopefully be of support for clinicians managing patients with this rare acute intoxication.

Determination of cannabinoids in hair: Indicators for illegal vs CBD-rich cannabis use.

Cannabis products rich in cannabidiol (CBD) and low in Δ9-tetrahydrocannabinol (THC) (e.g., light cannabis in Italy) are becoming widely popular and available on the market as replacements for THC preparations and tobacco for their recreational and/or therapeutic benefits. In this paper, which aims to establish alternative discrimination parameters between hair samples from CBD-rich and THC-prevalent cannabis users, cannabinoid concentrations, such as THC, CBD, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) and 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) were quantified in 127 hair samples by a GC-MS/MS technique. Initially, this analysis was able to discriminate two cohorts: cohort 1 (individuals with THC values ≥ 0.05 ng/mg and THC-COOH ≥ 0.2 pg/mg or THC-positive users, n = 60) and cohort 2 (individuals with THC values ranging between 0.01 and 0.05 ng/mg and THC-COOH or 11-OH-THC ≥ LOQs, n = 67). The evaluation of CBD/THC ratio in cohort 2 identified two further sub-cohorts 2a (CBD/THC<<1 or ~ 1, THC-prevalent cannabis users) and 2b (CBD/THC>>1, suspected CBD-rich and THC-low cannabis users). The latter showed unusual profiles for THC metabolites, in particular for 11-OH-THC. Statistical evaluation of the data of cohort 1, cohort 2a and cohort 2b yielded significant differences in CBD/THC and THC/11-OH-THC. Based on the analysis of 337 seized cannabis samples and 630 CBD-rich/light cannabis samples by GC-FID and GC-MS, respectively, we also evaluated statistical differences in the CBD/THC ratio between biological (hair) and plant-derived samples. Considering the legal implications of a positive result, the obtained findings could be relevant for the interpretation of cannabinoid concentrations in hair. Further studies are necessary to elucidate the reason behind the unusual metabolic ratios.

Endogenous 1-H-Pyrrole-2,3,5-Tricarboxylic Acid (PTCA) in Hair and Its Forensic Applications: A Pilot Study on a Wide Multi-Ethnic Population.

Over the years, several studies have shown that many factors are likely to affect the results of forensic hair analyses and complicate their interpretation. Among these factors, one of the major drawbacks in hair analysis is the affectability of deposited xenobiotics by cosmetic treatments, which could be eventually used to adulterate the sample. It is well known that some cosmetic treatments containing hydrogen peroxide, such as permanent dyeing or bleaching, lead to the formation of 1-H-pyrrole-2,3,5-tricarboxylic acid (PTCA), a melanin degradation product. Considering that PTCA is also an endogenous compound, spontaneously formed by natural oxidation of melanin, its only detection in hair is not enough to confirm a cosmetic oxidative treatment. For this reason, the aim of the present work was to develop and validate a reliable liquid-liquid extraction method in ultra-high-performance liquid chromatographic-tandem mass spectrometry for the determination of endogenous PTCA in hair from a wide multi-ethnic population (African, Arab, Asian-Pacific, Caucasian, Hispanic and Indian). According to previous studies, untreated hair samples showed a PTCA content of 8.54 ± 5.72 ng/mg (mean ± standard deviation [SD]), ranging between 0.44 and 23.7 ng/mg; after in vitro cosmetic bleaching, PTCA increased to 16.8 ± 6.95 ng/mg (range: 4.16-32.3 ng/mg). Comparing baseline PTCA levels of each subgroup with the others, we could not observe any statistically significant difference, except for Caucasians (P < 0.05), wherein the concentrations were lower. Further studies and a wider sampling are necessary to elucidate the role of PTCA as diagnostic marker of cosmetic hair treatment in forensic field.

PTCA (1-H-Pyrrole-2,3,5-Tricarboxylic Acid) as a Marker for Oxidative Hair Treatment: Distribution, Gender Aspects, Correlation with EtG and Self-Reports.

Hair analysis is an important and reliable resource for the assessment of alcohol or drug abstinence in both clinical and forensic toxicology. Recently, it has been demonstrated that hair oxidative cosmetic treatments lead to the reduction in incorporated xenobiotics in hair, such as ethyl glucuronide (EtG), a marker of alcohol abuse, and the formation of 1-H-pyrrole-2,3,5-tricarboxylic acid (PTCA), a degradation product of melanin. The aim of the present study was to investigate PTCA trends in a large number of samples in order to evaluate the reliability of this biomarker in recognizing previous cosmetic treatment in forensic analyses. Therefore, a single-step extraction followed by an high-performance liquid chromatography--tandem mass spectrometry (HPLC--MS-MS) method was established and validated for the simultaneous determination of EtG and PTCA. This method was applied to 1,219 scalp hair samples from two groups, namely self-reported untreated and in vivo treated hair, exhibiting a concentration range of 6.7 to 440.0 pg/mg for EtG (mean 26.8 pg/mg, median 14.6 pg/mg) and 0.009 to 49.8 ng/mg for PTCA (mean 0.66 ng/mg, median 0.02 ng/mg). The PTCA content was significantly different among the two experimental groups, with the in vivo treated group showing significantly higher levels of PTCA than the untreated group. Finally, an in vitro bleaching was performed and the results confirmed that a strong hair oxidative treatment may negatively affect EtG test results (false negative), whereas the mean PTCA content increased showing statistically significant differences between untreated and in vitro oxidative treated samples. The present study suggests that the determination of PTCA in routine hair analysis procedure could be useful in order to discover previous cosmetic treatment including oxidation.

Quantitative Lipidomic Analysis of Osteosarcoma Cell-Derived Products by UHPLC-MS/MS.

Changes in lipid metabolism are involved in several pathological conditions, such as cancer. Among lipids, eicosanoids are potent inflammatory mediators, synthesized from polyunsaturated fatty acids (PUFAs), which coexist with other lipid-derived ones, including endocannabinoids (ECs) and N-acylethanolamides (NAEs). In this work, a bioanalytical assay for 12 PUFAs/eicosanoids and 20 ECs/NAEs in cell culture medium and human biofluids was validated over a linear range of 0.1-2.5 ng/mL. A fast pretreatment method consisting of protein precipitation with acetonitrile followed by a double step liquid-liquid extraction was developed. The final extracts were injected onto a Kinetex ultra-high-performance liquid chromatography (UHPLC) XB-C18 column with a gradient elution of 0.1% formic acid in water and methanol/acetonitrile (5:1; v/v) mobile phase. Chromatographic separation was followed by detection with a triple-quadrupole mass spectrometer operating both in positive and negative ion-mode. A full validation was carried out in a small amount of cell culture medium and then applied to osteosarcoma cell-derived products. To the best of our knowledge, this is the first lipid profiling of bone tumor cell lines (SaOS-2 and MG-63) and their secretome. Our method was also partially validated in other biological matrices, such as serum and urine, ensuring its broad applicability as a powerful tool for lipidomic translational research.

11-OH-THC in hair as marker of active cannabis consumption: Estimating a reliable cut-off by evaluation of 672 THC-positive hair samples.

Hair analysis for the assessment of cannabis active use from passive consumption may be failed when performed by the sole detection of compounds present in plant material as well as in cannabis smoke like Δ-9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN). For this reason, the determination of 11-nor-9-carboxy-Δ-9-tetrahydrocannabinol (THC-COOH) has been proposed by the Society of Hair Testing (SoHT) in order to prove active cannabis consumption. The identification of THC-COOH in hair will continue to be complicated by its acidic nature and the critical low concentration due to the preferential incorporation of basic compounds into hair shaft. Alternatively, 11-OH-THC may be considered as a complementary marker for THC administration. Our recent study reported an accurate validated procedure for THC, CBD, CBN and 11-OH-THC in hair, based on a GC/MS-MS method in electron ionization mode. However, unlike THC-COOH, a cut-off level for 11-OH-THC in hair has not been fixed yet. For this reason, the aim of this study is to propose a concentration value for 11-OH-THC in hair analysis in order to discriminate between chronic use and external contamination. Receiver operating characteristics (ROC) analysis was applied for cut-off evaluation after 11-OH-THC quantification in a pool of 672 THC-positive hair samples. Results have shown a concentration range between 0.01-5.34 ng/mg for THC (mean 0.34 ng/mg, median 0.12), 0.00-19.2 pg/mg for THC-COOH (mean 0.72 pg/mg, median 0.19 pg/mg) and 0.01-13.33 ng/mg for 11-OH-THC (mean 1.09 ng/mg, median 0.51 ng/mg) for scalp hair and between 0.03-6.32 ng/mg for THC (mean 0.82 ng/mg, median 0.30), 0.00-42.1 pg/mg for THC-COOH (mean 2.70 pg/mg, median 1.08 pg/mg) and 0.00-7.88 ng/mg for 11-OH-THC (mean 1.70 ng/mg, median 0.89 ng/mg) for body hair. Considering these experimental data collected in our laboratory, we propose a cut-off level of 0.5 for scalp and body hair, as indicative of cannabis active consumption. The ROC curve AUCs for 11-OH-THC were 0.873 and 0.884 in 590 scalp hair and 82 body hair samples, respectively. The comparison of the results for THC-COOH (control method) and 11-OH-THC (test method) was also made by means of the Cohen's kappa statistics providing a good agreement according to both Landis & Koch and Fleiss scales. Additionally, we suggest that the detection of both THC-COOH and 11-OH-THC should be mandatory in order to prove active intake and exclude false positive results from external contamination.

Analysis of hydroxy-cocaine metabolites as evidence of cocaine consumption: Identification by parent ion search and quantitation by UHPLC-MS/MS in hair.

Over the last decade, hair analysis has become a routine procedure in most forensic laboratories and, complementary to blood and urine, hair is a unique biological matrix which gives the opportunity to establish a temporal consumption profile. Despite hair is widely used to identify drug use, environmental contamination continues to represent a challenging factor of this procedure, especially for cocaine (COC). In the last few years several strategies have been proposed in order to distinguish between actual use and external contamination, however the commonly detected COC metabolites probably are insufficient for demonstrating cocaine use through hair testing. Thus, the aim of this study is to develop an ultra high performance liquid cromatography - tandem mass spectrometry (UHPLC-MS/MS) method able to detect and quantify hydroxy-COC metabolites, as specific markers of COC abuse, in hair samples from COC consumers, thus enabling unambiguous evidence of COC consumption. At the beginning, since no commercial reference materials were available, COC-positive hair samples were tested using parent ion scan-based analysis to extract hydroxy COC metabolites target ions. Once identified, the reference materials were synthesized by our analytical laboratory allowing the development of the first UHPLC-MS/MS validated method to quantify p- and m-isomers of hydroxy COC, as well as hydroxy benzoylecgonine (BE) and hydroxy norcocaine (NCOC). The method was successfully applied to a large number of COC-positive hair samples and introduced into a routine procedure for testing drug ingestion in order to evaluate for the first-time hydroxy metabolites of COC ranges in hair and their correlation with COC and BE.

An automated sample preparation approach for routine liquid chromatography tandem-mass spectrometry measurement of the alcohol biomarkers phosphatidylethanol 16:0/18:1, 16:0/16:0 and 18:1/18:1.

BACKGROUND: Phosphatidylethanols (PEths) are currently under investigation as highly sensitive and specific direct biomarkers of long-term alcohol abuse. PEths belong to a group of aberrant phospholipids formed in erythrocyte membranes in presence of ethanol by the catalytic action of the enzyme phospholipase D on phosphatidylcholine. Compared to other alcohol biomarkers, a higher sensitivity (94.5-100%) and specificity (100%) characterizes PEth species. METHOD: Prior to detection, an important practical aspect in the work-flow of PEths analysis is the sample preparation step. To date, traditional techniques such as liquid-liquid extraction (LLE) and solid phase extraction (SPE) require multiple steps to remove blood interferences. Due to the simplicity of use and the possibility of automation, sample filtration is also a widespread technique in biomedical laboratories. In this work, a reliable sample preparation method based on an automated filtration with Phree™ Phospholipid Removal Plates (Phenomenex, California, USA) was developed to extract PEths from human whole blood. Surface characteristics of Phospholipids Removal material allow phospholipids retention on the filter and a suitable PEths recovery after elution. The blood samples were added with internal standard (IS) and purified in acetonitrile (1 mL). After centrifugation, supernatants were applied to the Phospholipids Removal Plates in an automated workstation. After washing, the phospholipids retained on the filter were eluted with 1-mL 2-propanol 1% ammonia. PEth 16:0/18:1, PEth 16:0/16:0 and PEth 18:1/18:1 were extracted using the proposed method and detected by LC-MS/MS operated in electron spray ionization (ESI). The detection of all compounds was based on multiple reaction monitoring (MRM) transitions. This method was validated for the quantitative profiling of PEth molecular species in human blood collected from heavy and social drinkers. RESULTS: The method was validated according to Food and Drug Administration (FDA) guidelines. Linearity was observed in the 25-1250 (PEth 16:0/18:1) and 5-250 (PEth 16:0/16:0 and PEth 18:1/18:1) ng/mL range with a correlation coefficient (r²) between 0.997 and 0.999 for all three compounds. Moreover, the nominal concentrations of non-zero calibrators were ±15%. Variation coefficient (%CV) was < 10% for all the analytes, while lowest limit of quantitation (LLOQ) was found to be 1.25 ng/mL for PEth 16:0/18:1, 0.50 ng/mL for PEth 16:0/16:0 and 0.50 ng/mL for PEth 18:1/18:1. Intra- and inter-day precision and accuracy were always lower than 14% and 11%, respectively. Analytical recovery was higher than 68.8% for all analytes. Sample stability at 4 °C and -20 °C showed a concentration drop lower than 20% up to 4 weeks. Extracts were stable for 7 days in the autosampler and 30 days at -20 °C and 4 °C in a closed vial. The procedure was successfully applied to blood samples collected from heavy drinkers (n = 8), social drinkers (n = 5), and teetotalers (n = 7). CONCLUSIONS: Due to the simplicity of application and the possibility of automation, sample filtration is well suited for a clinical and forensic laboratory. To monitor alcohol consumption, an analytical method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) with novel and automated sample preparation was developed and validated for the simultaneous quantification of PEth 16:0/18:1, PEth 16:0/16:0 and PEth 18:1/18:1 in whole blood samples, characterized by a fast sample preparation and lower pre-analysis costs than other extraction procedures.

A novel single-step GC-MS/MS method for cannabinoids and 11-OH-THC metabolite analysis in hair.

THC, CBD, CBN, THC-COOH and 11-OH-THC are the most popular markers of cannabis consumption and abuse. The use of this drug is a serious social problem worldwide. In this study, a method based on gas chromatography-tandem mass spectrometry (GC-MS/MS) operated in electron ionization (EI) with simple and rapid liquid-liquid extraction (LLE) and derivatization was developed and validated for the simultaneous determination of THC, CBD, CBN and 11-OH-THC in hair samples. The detection of all compounds was based on multiple reaction monitoring (MRM) transitions. The most important advantage of this method is the single-step, quick, easy and effective sample extraction procedure for THC, CBD, CBN and 11-OH-THC. The method showed a good linearity with a correlation coefficient (r2) between 0.997 and 0.999 for all substances. The variation coefficient (%CV) was <5% for THC, 11-OH-THC and CBD and <13% for CBN. The limit of detection (LOD) was 0.03 pg/mg for 11-OH-THC and it ranged from 0.3 to 1.4 pg/mg for THC, CBD and CBN. The limit of quantification was 0.1 pg/mg for 11-OH-THC and it ranged from 0.9 to 4.7 pg/mg for THC, CBD and CBN. Analytical recovery was higher than 88% for 11-OH-THC and it ranged between 68 and 97% for THC, CBD and CBN. Intra- and inter-assay precision and accuracy were always lower than 9-14% and 5-9%, respectively. In parallel, we have quantified the THC-COOH level, following the methods previously set-up by us. The whole procedure was successfully applied to more than 200 different hair samples from cannabis consumers, disclosing the presence of 11-OH-THC in a range between 0.2 pg/mg and 27 pg/mg, and the presence of THC-COOH in a range between 0.05 pg/mg and 42.05 pg/mg. These data provided a good start towards the use of 11-THC-OH as alternative hair biomarker of cannabis consumption.

LC-MS/MS method development for quantification of doxorubicin and its metabolite 13-hydroxy doxorubicin in mice biological matrices: Application to a pharmaco-delivery study.

This study describes the development of simple, rapid and sensitive liquid chromatography tandem mass spectrometry method for the simultaneous analysis of doxorubicin and its major metabolite, doxorubicinol, in mouse plasma, urine and tissues. The calibration curves were linear over the range 5-250 ng/mL for doxorubicin and 1.25-25 ng/mL for doxorubicinol in plasma and tumor, over the range 25-500 ng/mL for doxorubicin and 1.25-25 ng/mL for doxorubicinol in liver and kidney, and over the range 25-1000 ng/mL for doxorubicin and doxorubicinol in urine. The study was validated, using quality control samples prepared in all different matrices, for accuracy, precision, linearity, selectivity, lower limit of quantification and recovery in accordance with the US Food & Drug Administration guidelines. The method was successfully applied in determining the pharmaco-distribution of doxorubicin and doxorubicinol after intravenously administration in tumor-bearing mice of drug, free or nano-formulated in ferritin nanoparticles or in liposomes. Obtained results demonstrate an effective different distribution and doxorubicin protection against metabolism linked to nano-formulation. This method, thanks to its validation in plasma and urine, could be a powerful tool for pharmaceutical research and therapeutic drug monitoring, which is a clinical approach currently used in the optimization of oncologic treatments.