Screening and determination of polycyclic aromatic hydrocarbons in seafoods using QuEChERS-based extraction and high-performance liquid chromatography with fluorescence detection.

A rapid, sensitive, and accurate method for the screening and determination of polycyclic aromatic hydrocarbons (PAHs) in edible seafood is described. The method uses quick, easy, cheap, effective, rugged, and safe (QuEChERS)-based extraction and HPLC with fluorescence detection (FLD). The method was developed and validated in response to the massive Deepwater Horizon oil spill in the Gulf of Mexico. Rapid and highly sensitive PAH screening methods are critical tools needed for oil spill response; they help to assess when seafood is safe for harvesting and consumption. Sample preparation involves SPE of edible seafood portions with acetonitrile, followed by the addition of salts to induce water partitioning. After centrifugation, a portion of the acetonitrile layer is filtered prior to analysis via HPLC-FLD. The chromatographic method uses a polymeric C18 stationary phase designed for PAH analysis with gradient elution, and it resolves 15 U.S. Environmental Protection Agency priority parent PAHs in fewer than 20 min. The procedure was validated in three laboratories for the parent PAHs using spike recovery experiments at PAH fortification levels ranging from 25 to 10 000 microg/kg in oysters, shrimp, crab, and finfish, with recoveries ranging from 78 to 99%. Additional validation was conducted for a series of alkylated homologs of naphthalene, dibenzothiophene, and phenanthrene, with recoveries ranging from 87 to 128%. Method accuracy was further assessed based on analysis of National Institute of Standards and Technology Standard Reference Material 1974b. The method provides method detection limits in the sub to low ppb (microg/kg) range, and practical LOQs in the low ppb (microg/kg) range for most of the PAH compounds studied.

EVALI Vaping Liquids Part 1: GC-MS Cannabinoids Profiles and Identification of Unnatural THC Isomers.

Tetrahydrocannabinol (THC)-containing products played a major role in the 2019 US nationwide outbreak of pulmonary lung illness associated with e-cigarettes or vaping liquids (EVALI). Due to the severity of the illness which resulted in 68 deaths, a comprehensive identification of the components in the vaping liquids was required. Our laboratory received over 1000 vaping liquid products for analysis including hundreds of vaping products from EVALI patients. In this work, we present the results for the GC-MS identification of the cannabinoids from a large subset of ca. 300 Cannabis-based vaping liquids, with emphasis on the identification of a series of unnatural THC isomers. GC-MS analysis was conducted using a validated, published method in which the cannabinoids were identified as the trimethylsilyl derivatives after separation on a commercial 35% silphenylene phase. Δ9- Tetrahydrocannabinol is the naturally occurring THC isomer found in the Cannabis plant, and was found in the majority of the vaping liquids. However, we also identified the presence of one or more additional THC isomers in many of the vaping liquids including Δ8-tetrahydrocannabinol, Δ6a,10a-tetrahydrocannabinol, Δ10-tetrahydrocannabinol, and exo-tetrahydrocannabinol. Significant or major amounts of unnatural THC isomers were found in over 10% of the THC vaping liquids, with lesser amounts found in another 60% of the vaping liquids. Exposure of the Cannabis source materials (such as marijuana concentrates or converted hemp materials) to chemical and thermal treatments during manufacturing, is proposed as the primary cause for the THC isomerizations.

EVALI Vaping Liquids Part 2: Mass Spectrometric Identification of Diluents and Additives.

The vaping liquid additive vitamin E acetate (VEA) was strongly linked to the 2019 United States nationwide outbreak of pulmonary lung illness (EVALI) associated with e-cigarettes or vaping liquids. Our laboratory received over 1,000 vaping liquid products for identification of the vaping liquid additives, including hundreds of vaping products from EVALI patients. In this work, we present results obtained for the GC-MS identification of numerous vaping liquid additives in a large subset of ca. 300 Cannabis vaping liquids, including vitamin E acetate, medium chain triglycerides oil (MCT oil), polyethylene glycols, squalane, triethyl citrate, dipropylene glycol dibenzoate (DPG dibenzoate), pine rosin acids, pine rosin methyl esters, and sucrose acetate isobutyrate (SAIB). Confirmation of DPG dibenzoate and SAIB using LC-HRMS is also presented. GC-MS analysis for additives identified as the parent compounds was conducted after separation on a commercial 5% phenyl phase. GC-MS analysis for additives identified as the trimethylsilyl derivatives was conducted after separation on a commercial 35% silphenylene phase. LC-HRMS analysis was conducted using gradient elution with either C18 or phenyl-hexyl phases and determination of exact masses for the target compounds. In addition to providing rapid methods for the identification of vaping liquid additives, this work highlights the variety of Cannabis vaping liquid additives in current use.

Rapid selective screening and determination of ephedrine alkaloids using GC-MS footnote mark.

Ephedra (ma huang) has been widely used as an herb or herbal extract in both traditional Chinese medicine and Western world dietary supplements. The effects of Ephedra have been attributed to a series of six ephedrine alkaloids including ephedrine and pseudoephedrine. A GC-MS method for the ephedrine alkaloids is described which couples ammoniacal chloroform as the extraction solvent with a two-stage derivatisation scheme. This scheme produces the O-trimethylsilyl, N-trifluoracetyl derivatives (O-TMS, N-TFA) for the primary and secondary amine alkaloids, and the O-TMS derivatives for the tertiary amine alkaloids. Relatively clean extracts are obtained from complex matrices, and the six ephedrine alkaloids are effectively separated and identified. This approach was also evaluated for quantitative analysis, and was shown to provide quantitative results for ephedrine and pseudoephedrine, and good estimates for the four minor alkaloids. Figures of merit are presented for linearity, detection limits, precision and accuracy. We have applied this approach to the rapid screening and profiling of the ephedrine alkaloids in whole Ephedra plants, liquid plant extracts, dried powder plant extracts and a variety of Ephedra-containing dietary supplements.

Commercial cannabis consumer products part 2: HPLC-DAD quantitative analysis of cannabis cannabinoids.

Quantitative analysis for the cannabis cannabinoids such as cannabidiol and Δ9-tetrahydrocannabinol in commercial products is necessary for evaluating label information, and assessing dosages and exposures when the products are consumed. Herein is presented a broadly applicable HPLC-DAD method for the determination of cannabis cannabinoids in commercial consumer products and traditional plant-related substances. The current method provides chromatographic resolution of 11 cannabinoids using a commercial, mixed C18-aromatic functionality stationary phase. The method uses 95% or pure ethanol for extraction, and certain modifications which address specific matrix types are detailed herein. Extensive method validation including precision and accuracy was conducted for five cannabinoids of primary interest (CBD, Δ9-THC, CBDA, THCA, and CBN). UV detection provided excellent sensitivity with limits of quantitation (LOQs) of 10µg/g across cannabinoids. The method was applied to about 60 commercial products representing diverse product types and a broad range of cannabinoids amounts (0.01-350mg/g).

Commercial cannabis consumer products part 1: GC-MS qualitative analysis of cannabis cannabinoids.

The recent surge in the sale of cannabis-based consumer products in the US includes foods, candies, beverages, topicals, vapes/eliquids, oral supplements in various forms, recreational marijuana plants, and plant extracts or preparations. The wide variety of product and sample types has resulted in a host of new matrix interferences when conducting qualitative testing for the cannabis cannabinoids such as cannabidiol and d9-tetrahydrocannabinol. A qualitative GC-MS method is presented in this work, which uses a commercial 35% silphenylene phase to provide chromatographic resolution for 11 target cannabinoids as their trimethylsilyl derivatives (CBD, CBDA, d9THC, THCA, CBN, d8THC, CBG, CBGA, CBDV, THCV, and CBC). The method uses variants of ethanol- and acetonitrile-based extractants to successfully minimize or eliminate several types of interferents, and also provides protocols to address specific interferents such as glycerin and lactose. Method validation included spike/recovery for five cannabinoids of primary interest (spiking level 50µg/g) from a series of edible oils, foods, beverages, candies, topicals, oral OTC pharmaceuticals, glycerin, and propylene glycol. The minimum detectable concentration was established as 1.0µg/g. The method was applied to about sixty diverse commercial products, as well as to recreational marijuana plants, plant preparations, hempseed oils, and dronabinol capsules.

GHB free acid: I. Solution formation studies and spectroscopic characterization by 1HNMR and FT-IR.

In forensic evidence, gamma-hydroxybutyric acid (GHB) has frequently been encountered in one of its salt forms (gamma-hydroxybutyrate), but has also been encountered in its free acid form (GHB). Owing to the physical properties, encounters of the free acid have been largely restricted to forensic exhibits comprising aqueous solutions, such as acidic beverages that have been "spiked" or formulated with GHB salts or gamma-butyrolactone (GBL). The analysis of GHB free acid presents particular difficulties including the potential for altering the original proportions of GHB free acid, GHB carboxylate, and GBL in the course of analysis, and discrimination between GHB free acid and carboxylate forms. In this work, the formation of GHB free acid in aqueous solutions (water and/or D2O) was studied as a function of solution pH. Proton nuclear magnetic resonance (1HNMR) and Fourier-transform infrared spectrometry (FT-IR) measurements were obtained on freshly prepared mixtures of NaGHB and HCl stock solutions representing a series of points along the GHB titration curve. Both 1HNMR and FT-IR were shown to track the changing proportions of GHB free acid and carboxylate forms as a function of pH, while simultaneously monitoring for the formation of the lactone (GBL). The results were consistent with acid-base conversion behavior for a carboxylic acid. 1HNMR was shown to provide an ideal means for analysis of aqueous-based GHB/GBL forensic exhibits based on simple dilution of the neat liquid exhibit, without altering the original proportions of GHB free acid, carboxylate, and GBL in the samples.

GHB free acid: II. Isolation and spectroscopic characterization for forensic analysis.

A reference standard for gamma-hydroxybutyric acid (GHB) free acid is not commercially available, making its analysis in forensic exhibits more difficult. GHB free acid is typically encountered in aqueous solution and in the presence of the lactone, gamma-butyrolactone (GBL), presenting difficulty in Fourier transform infrared (FT-IR) analysis. The strong infrared (IR) absorptivity of the GBL carbonyl band, the shifting of the GBL carbonyl band in aqueous solutions, and the position of the O-H bend for water can mask the main carbonyl band for GHB free acid. Model solutions of beta-hydroxybutyric acid (BHB) and GBL were studied in order to further understand the masking of the GHB free acid carbonyl band in FT-IR analysis. The use of second derivative FT-IR spectroscopy was shown to provide resolution of the free acid carbonyl band, and a presumptive test for GHB free acid was developed and applied. An extension of this work included preparing, for use as a standard reference material, small amounts (< or = 10 mg) of GHB free acid. Preparation was based on the instantaneous reaction of GHB's sodium salt with a stoichiometric amount of hydrochloric acid in aqueous solution, and subsequent isolation of the free acid in neat liquid form. Both FT-IR and proton nuclear magnetic resonance spectra of the neat reference material were obtained and used to verify its identity. The isolation of GHB free acid from actual forensic exhibits is also presented, with identity confirmation using FT-IR.

The determination of nicotine and sulfate in supermarket ground beef adulterated with Black Leaf 40.

In December 2002, approximately 250 lbs. of ground beef was adulterated with nicotine sulfate by a supermarket employee and subsequently sold to the public. Soon afterward, reports of illness associated with ground beef purchased at a single store were identified. Authorities suspected the ground beef was tainted with Black Leaf 40, a banned pesticide containing approximately 40% nicotine as nicotine sulfate. Ground beef submitted to our laboratory was analyzed in concert by high performance liquid chromatography-ultraviolet detection (HPLC-UV), gas chromatography-mass spectrometry (GC-MS), and high performance anion exchange chromatography with suppressed conductivity detection. GC-MS was used to identify the samples that contained nicotine. The nicotine was confirmed and quantitated by HPLC-UV. The sulfate was identified and quantitated by high performance anion exchange chromatography with suppressed conductivity detection. Our analysis revealed that the raw tainted beef contained about 350 mg/kg nicotine free base, a potentially lethal dose of nicotine per serving for an adult. Additionally, we found elevated sulfate levels in the samples that tested positive for nicotine, providing evidence that nicotine sulfate was the probable adulterant.

Quantitation of Synthetic Cannabinoids in Plant Materials Using High Performance Liquid Chromatography with UV Detection (Validated Method).

Plant based products laced with synthetic cannabinoids have become popular substances of abuse over the last decade. Quantitative analysis for synthetic cannabinoid content in the laced materials is necessary for health hazard assessments addressing overall exposure and toxicity when the products are smoked. A validated, broadly applicable HPLC-UV method for the determination of synthetic cannabinoids in plant materials is presented, using acetonitrile extraction and separation on a commercial phenylhexyl stationary phase. UV detection provides excellent sensitivity with limits of quantitation (LOQs) less than 10 µg/g for many cannabinoids. The method was validated for several structural classes (dibenzopyrans, cyclohexylphenols, naphthoylindoles, benzoylindoles, phenylacetylindoles, tetramethylcyclopropylindoles) based on spike recovery experiments in multiple plant materials over a wide cannabinoid contents range (0.1-81 mg/g). Average recovery across 32 cannabinoids was 94% for marshmallow leaf, 95% for damiana leaf, and 92% for mullein leaf. The method was applied to a series of case-related products with determined amounts ranging from 0.2 to >100 mg/g.

Partial conversion of methanol to formaldehyde in the gas chromatography injection port and reactivity with amines.

The partial conversion of methanol (MeOH) to formaldehyde (HCHO) in the gas chromatograph (GC) injection port was studied. The presence of formaldehyde in the injection port can result in reaction with injected analytes, especially primary and secondary amines. A systematic study of this problem was undertaken using norephedrine and ephedrine as probe analytes, and experiments involving varying inlet temperature, comparisons among solvents and solvent mixtures, isotopic labeling, and formaldehyde spiking. These experiments showed a direct correlation between inlet temperature and formation of the amine-formaldehyde condensation products. The mass spectra of the norephedrine and ephedrine condensation products were consistent with addition of the methylene group derived from formaldehyde across the amine and alcohol moieties to form the corresponding oxazolidine-ring compounds. Results for the imine condensation product of didesmethylsibutramine formed in the injection port are also presented.