Laura A. Ciolino

The Chemical Interconversion of GHB and GBL: Forensic Issues and Implications

In this work, the interconversion of GHB and GBL in a variety of aqueous media was studied. The effects of solution pH and time were determined by spiking GHB or GBL into pure water and buffered aqueous solutions, and determining the GHB and GBL contents at various time intervals. The degree of GBL hydrolysis to GHB was determined for several commercial aqueous-based GBL products, and further studied as a function of time. The effects of temperature and time were also determined for five commercial beverages spiked with GHB or GBL. GHB and GBL contents were determined using high performance liquid chromatography (HPLC). GHB and/or GBL confirmations were made using gas chromatography-mass spectrometry (GC-MS) and/or infrared spectroscopy (IR). Solution pH, time, and storage temperature were determined to be important factors affecting the rate and extent of GBL hydrolysis to GHB. Under strongly alkaline conditions (pH 12.0), GBL was completely converted to GHB within minutes. In pure water, GBL reacted to form an equilibrium mixture comprising ca. 2:1 GBL:GHB over a period of months. This same equilibrium mixture was established from either GHB or GBL in strongly acidic solution (pH 2.0) within days. A substantial portion of GBL (ca. 1/3) was hydrolyzed to GHB in aqueous-based GBL products, and in spiked commercial beverages, after ambient storage for a period ranging from several weeks to several months. Heat increased and refrigeration decreased the rate of GBL hydrolysis relative to ambient conditions. These studies show that hydrolysis of GBL to GHB does occur in aqueous-based solutions, with samples and time frames that are relevant to forensic testing. Implications for forensic testing and recommendations are discussed.

Optimization study for the reversed-phase ion-pair liquid chromatographic determination of nicotine in commercial tobacco products.

The availability of published methods for the determination of nicotine in commercial tobacco products based on state-of-the-art chromatographic methods is limited. Nicotine is a diprotic base with pKas of 3.12 (pyridine ring) and 8.02 (pyrrolidine ring). Other monoprotic and diprotic bases are also present in commercial tobacco including anatabine, nornicotine, anabasine, and cotinine. In this paper, the chromatography of nicotine and the minor tobacco alkaloids under reversed-phase ion-pairing conditions is thoroughly studied. The results of this study are used to understand the retention mechanisms of the tobacco alkaloids, to examine their observed elution order with respect to fundamental analyte properties (size, functionality, and acid-base strength), and to select optimum chromatographic conditions for the determination of nicotine in commercial tobacco products.

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

ABSTRACT: In forensic evidence, γ‐hydroxybutyric acid (GHB) has frequently been encountered in one of its salt forms (γ‐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 γ‐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.

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.

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.

GHB Free Acid: II. Isolation and Spectroscopic Characterization for Forensic Analysis

ABSTRACT: A reference standard for γ‐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, γ‐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 β‐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 (≤10 mg) of GHB free acid. Preparation was based on the instantaneous reaction of GHBs 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.

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