Cannabis sativa is one of the oldest medicinal plants in the world. It was introduced into western medicine during the early 19th century. It contains a complex mixture of secondary metabolites, including cannabinoids and non-cannabinoid-type constituents. More than 500 compounds have been reported from C. sativa, of which 125 cannabinoids have been isolated and/or identified as cannabinoids. Cannabinoids are C21 terpeno-phenolic compounds specific to Cannabis. The non-cannabinoid constituents include: non-cannabinoid phenols, flavonoids, terpenes, alkaloids and others. This review discusses the chemistry of the cannabinoids and major non-cannabinoid constituents (terpenes, non-cannabinoid phenolics, and alkaloids) with special emphasis on their chemical structures, methods of isolation, and identification.

An HPLC single-laboratory validation was performed for the detection and quantification of the 11 major cannabinoids in most cannabis varieties, namely, cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabigerol (CBG), cannabidiol (CBD), tetrahydrocannabivarin (THCV), cannabinol (CBN), Δ9-trans-tetrahydrocannabinol (Δ9-THC), Δ8-trans-tetrahydrocannabinol (Δ8-THC), cannabicyclol (CBL), cannabichromene (CBC), and Δ9-tetrahydrocannabinolic acid-A (THCAA). The analysis was carried out on the biomass and extracts of these varieties. Methanol-chloroform (9:1, v/v) was used for extraction, 4-androstene-3,17-dione was used as the internal standard, and separation was achieved in 22.2 min on a C18 column using a two- step gradient elution. The method was validated for the 11 cannabinoids. The concentration-response relationship of the method indicated a linear relationship between the concentration and peak area with r2 values of >0.99 for all 11 cannabinoids. Method accuracy was determined through a spike study, and recovery ranged from 89.7 to 105.5% with an RSD of 0.19 to 6.32% for CBDA, CBD, THCV, CBN, Δ9-THC, CBL, CBC, and THCAA; recovery was 84.7, 84.2, and 67.7% for the minor constituents, CBGA, CBG, and Δ8-THC, respectively, with an RSD of 2.58 to 4.96%. The validated method is simple, sensitive, and reproducible and is therefore suitable for the detection and quantification of these cannabinoids in different types of cannabis plant materials.

Cannabis ( L.) is an annual herbaceous plant that belongs to the family Cannabaceae. -Δ-tetrahydrocannabinol (Δ-THC) and cannabidiol (CBD) are the two major phytocannabinoids accounting for over 40% of the cannabis plant extracts, depending on the variety. At the University of Mississippi, different strains of with different concentration ratios of CBD and Δ-THC, have been tissue cultured via micropropagation and cultivated. A GC-FID method has been developed and validated for the qualitative and quantitative analysis of acid and neutral cannabinoids in extracts. The method involves trimethyl silyl derivatization of the extracts. These cannabinoids include tetrahydrocannabivarian, CBD, cannabichromene,-Δ-tetrahydrocannabinol, Δ-THC, cannabigerol, cannabinol, cannabidiolic acid, cannabigerolic acid, and Δ-tetrahydrocannabinolic acid-A. The concentration-response relationship of the method indicated a linear relationship between the concentration and peak area ratio with R > 0.999 for all 10 cannabinoids. The precision and accuracy of the method were found to be ≤ 15% and ± 5%, respectively. The limit of detection range was 0.11 - 0.19 µg/mL, and the limit of quantitation was 0.34 - 0.56 µg/mL for all 10 cannabinoids. The developed method is simple, sensitive, reproducible, and suitable for the detection and quantitation of acidic and neutral cannabinoids in different extracts of cannabis varieties. The method was applied to the analysis of these cannabinoids in different parts of the micropropagated cannabis plants (buds, leaves, roots, and stems).

Continuing our previous studies analyzing drugs of abuse in municipal wastewater, a method was developed for the analysis of opiates in wastewater samples using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). Eight opiate drugs and metabolites were analyzed including codeine, hydrocodone, hydromorphone, 6-monoacetylmorphine (6-MAM, the primary urinary metabolite of heroin), morphine, norhydrocodone (the primary urinary metabolite of hydrocodone), oxycodone and oxymorphone. These drugs were chosen because of their widespread abuse. Wastewater samples were collected at both the Oxford Waste Water Treatment Plant in Oxford, Mississippi (MS) and the University Wastewater Treatment Plant in University, MS. These wastewater samples were collected on weekends in which the Ole Miss Rebel football team held home games (Vaught-Hemingway Stadium, University, MS 38677). The collected samples were analyzed using a validated method and found to contain codeine, hydrocodone, hydromorphone, morphine, norhydrocodone, oxycodone and oxymorphone. None of the samples contained 6-MAM.

A liquid chromatography-tandem mass spectrometry single-laboratory validation was performed for the detection and quantification of the 10 major cannabinoids of cannabis, namely, (-)--Δ-tetrahydrocannabinol, cannabidiol, cannabigerol, cannabichromene, tetrahydrocannabivarian, cannabinol, (-)--Δ-tetrahydrocannabinol, cannabidiolic acid, cannabigerolic acid, and Δ-tetrahydrocannabinolic acid-A, in the root extract of . Acetonitrile : methanol (80 : 20, v/v) was used for extraction; d-cannabidiol and d- tetrahydrocannabinol were used as the internal standards. All 10 cannabinoids showed a good regression relationship with > 0.99. The validated method is simple, sensitive, and reproducible and is therefore suitable for the detection and quantification of these cannabinoids in extracts of cannabis roots. To our knowledge, this is the first report for the quantification of cannabinoids in cannabis roots.

A method was developed for the analysis of stimulant drugs, opiates, synthetic opiates, PCP, and benzodiazepines in wastewater samples using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). A total of 33 compounds (stimulant-type drugs and metabolites of opiates, synthetic opiates, PCP, and benzodiazepines) were analyzed. These drugs included amphetamine (Amp) (1), methamphetamine (Meth) (2), methylenedioxyamphetamine (MDA) (3), methylenedioxymethamphetamine (MDMA) (4), methylenedioxyethylamphetamine (MDEA) (5), benzoylecgonine (BE, the major metabolite of Coc) (6), cocaine (Coc) (7), 6-monoacetylmorphine (6-MAM, the primary urinary metabolite of heroin) (8), codeine (9), hydrocodone (10), hydromorphone (11), morphine (12), norhydrocodone (the primary urinary metabolite of hydrocodone) (13), oxycodone (14), oxymorphone (15), 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrolidine (EDDP, the primary urinary metabolite of methadone) (16), fentanyl (17), meperidine (18), methadone (19), norfentanyl (the primary urinary metabolite of fentanyl) (20), normeperidine (the primary urinary metabolite of meperidine) (21), phencyclidine (PCP) (22), tramadol (23), alprazolam (24), temazepam (25), nordiazepam (26), chlordiazepoxide (27), flurazepam (28), oxazepam (29), α-OH-alprazolam (the primary urinary metabolite of alprazolam) (30), α-OH-triazolam (the primary urinary metabolite of triazolam) (31), 2-OH-ethylflurazepam (the primary urinary metabolite of flurazepam) (32), and 7-NH-flunitrazepam (the primary urinary metabolite of flunitrazepam) (33). These drugs were chosen because of their widespread abuse. Wastewater samples were collected at both the Oxford Wastewater Treatment Plant in Oxford, Mississippi (MS), and the University Wastewater Treatment Plant in University, MS. Samples were collected on weekends on which the Ole Miss Rebel football team held home games (Vaught-Hemingway Stadium, University, MS 38677). The collected samples were analyzed using a validated method and found to contain Amp, Meth, MDMA, MDA, Coc, BE, codeine, hydrocodone, hydromorphone, morphine, norhydrocodone, oxycodone, oxymorphone, tramadol, EDDP, meperidine, normeperidine, methadone, alprazolam, α-OH-alprazolam, nordiazepam, oxazepam, and temazepam. None of the samples contained MDEA, 6-MAM, fentanyl, norfentanyl, PCP, chlordiazepoxide, flurazepam, 2-OH-ethylflurazepam, 7-NH-flunitrazepam, and α-OH-triazolam.

Continuing our studies for the analyses of drugs of abuse in municipal wastewater, a method was developed for the analysis of benzodiazepines in wastewater samples using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). Ten benzodiazepines and metabolites were analyzed (structures were found), including alprazolam, α-OH-alprazolam (the primary urinary metabolite of alprazolam), chlordiazepoxide, flurazepam, 2-OH-ethylflurazepam (the primary urinary metabolite of flurazepam), 7-NH2-flunitrazepam, nordiazepam, oxazepam, temazepam and α-OH-triazolam (the primary urinary metabolite of triazolam) (representative chromatograms were found). These drugs were chosen because of their widespread abuse. Wastewater samples were collected at both the Oxford Wastewater Treatment Plant (WWTP) in Oxford, Mississippi (MS) and the University WWTP in University, MS. These wastewater samples were collected on weekends in which the Ole Miss Rebel football team held home games at the Vaught-Hemingway Stadium, University, and one weekend on which there was no game. The collected samples were analyzed using a validated method and found to contain alprazolam, α-OH-alprazolam, nordiazepam, oxazepam and temazepam. None of the samples contained chlordiazepoxide, flurazepam, 2-hydroxyethyl-flurazepam, 7-NH2-flunitrazepam and α-OH-triazolam.

Continuing our previous studies analyzing drugs of abuse in municipal wastewater, a method was developed for the analysis of miscellaneous drugs of abuse in wastewater samples using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). Eight drugs and metabolites were analyzed including 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrolidine (EDDP), fentanyl, norfentanyl, meperidine, normeperidine, methadone, phencyclidine and tramadol. These drugs were chosen because of their widespread abuse. Wastewater samples were collected at both the Oxford Waste Water Treatment Plant in Oxford, Mississippi (MS) and the University Wastewater Treatment Plant in University, MS. These wastewater samples were collected on weekends in which the University of Mississippi football team (colloquially the "Ole Miss Rebels" football team) held home games (Vaught-Hemingway Stadium, University, MS 38677). The collected samples were analyzed using a validated method and found to contain tramadol in 25 samples at quantifiable levels. EDDP, meperidine, normeperidine and methadone were also detected but were under the limit of quantitation.