Easy and convenient millimole‐scale synthesis of new, potential biomarkers for gamma‐hydroxybutyric acid (GHB) intake: Feasible for analytical laboratories

New biomarkers indicating the abuse of drugs and alcohol are still of major interest for clinical and forensic sciences. The endogenous neurotransmitter and approved drug, gamma-hydroxybutyric acid (GHB), is often illegally used for drug-facilitated crimes by spiking GHB into alcoholic beverages. Analytical detection windows of only 6 h in blood and 12 h in urine are often too short to provide reliable proof of GHB ingestion. Therefore, new biomarkers are needed to prove exogenous GHB administration. Previously, amino acid GHB conjugates were discovered in an untargeted metabolomics screening and fatty acid esters with GHB were recently discussed as promising biomarkers to enlarge the analytical detection time windows. However, the development of analytical methods is still slowed down since reference compounds for targeted screenings are still missing. In this paper, we describe simple procedures for the rapid synthesis and purification of amino acid GHB conjugates as well as fatty acid esters, which can be adopted in analytical and clinical/forensic laboratories. Structural characterization data, together with IR, $^1$H-nuclear magnetic resonance (NMR), $^{13}$C-NMR, high-resolution mass spectra (MS), and MS/MS spectra in positive and negative ionization mode are reported for all obtained GHB conjugates and GHB conjugate precursors

Exploring new dimensions: Single and multi-block analysis of essential oils using DBDI-MS and FT-IR for enhanced authenticity control

Background: Essential oils (EOs) are complex mixtures of volatile hydrocarbons with a wide range of applications in the pharmaceutical, fragrance and food industry. The composition of EOs is highly variable and can affect their quality and pharmaceutical efficacy. Moreover, the high economic value of EOs, such as those obtained from Rosa damascena, make falsification and misclassification a lucrative business. Consequently, adulterations can lead to serious health consequences for consumers. While current quality control methods for EOs involve analysing their chromatographic profile or comparing their Fourier transform infrared (FT-IR) spectra, these methods can be time-consuming or lack sensitivity. To address these issues, we compared state-of-the-art quality control methods, including gas chromatography flame ionization detection (GC-FID) quantification and enantiomeric ratio determination, FT-IR spectrometry with dielectric barrier discharge ionization coupled to triple quadrupole mass spectrometer (DBDI-MS), in a chemometric single- and multi-block approach. Results: Our results show that the best classification accuracy of 94.7% for R. damascena samples was obtained using GC-FID combined with partial least square discriminant analysis (PLS-DA). Comparatively, the enantiomeric ratios did not improve classification accuracy. In contrast, fragmentation data from DBDI-MS (Q3), which was acquired in a fraction of the analysis time and without extensive sample preparation, achieved a classification accuracy of 84.2%. We also found that combining FT-IR with parent ion DBDI-MS (Q1) data in a multi-block sequentially orthogonalized partial least squares linear discriminant analysis (SO-PLS-LDA) model improved classification accuracy, compared to their respective single-block PLS-DA models. Significance: Overall, our study demonstrates that DBDI, as an ambient ionization method, has significant potential for high-throughput screening. When combined with MS, it can produce comparable classification accuracies to conventional methods, while offering the added benefits of speed and convenience. As such, DBDI-MS is a promising tool for EO quality control.ISSN:0003-2670ISSN:1873-432

Determination of Major, Minor and Chiral Components as Quality and Authenticity Markers of Rosa damascena Oil by GC-FID

Rose oil is traditionally produced by the water distillation of Rosa damascena and is of high economic value due to the low essential oil yield. It is therefore a common target for adulteration, which can cause harm to consumers. Current standards for authenticity control only consider the analysis of major components and overlook minor quality markers as well as the enantiomeric ratio of terpenes, which have proven useful in originality determination. The aim of this study was the development of two analytical GC-FID methods for the analysis of 21 and 29 rose oil analytes including major, minor and chiral components on a DB-wax and BGB 178 30% CD (chiral) capillary column, respectively. The total run time for both methods was within 60 min. For all target analytes, the % bias at the lower and upper calibration range varied from −7.8 to 13.2% and −13.1 to 5.2% analysed on the DB-wax column and 0.5 to 13.3% and −6.9 to 7.0% analysed on the chiral column. The chiral analysis successfully separated the enantiomers (+/−)-camphene, (+/−)-rose oxide, (+/−)-linalool, (+/−)-citronellol and (+/−)-citronellyl acetate, as well as the diastereomers of citral and β-damascenone. Both methods were applied to the analysis of 10 authentic rose oil samples and the enantiomeric/diastereomeric ratios, as well as the content of major and minor components, were determined. The identity of the analysed components in the authentic samples was further confirmed by GC-MS.ISSN:2223-774

Easy and convenient millimole-scale synthesis of new, potential biomarkers for gamma-hydroxybutyric acid (GHB) intake: Feasible for analytical laboratories

New biomarkers indicating the abuse of drugs and alcohol are still of major interest for clinical and forensic sciences. The endogenous neurotransmitter and approved drug, gamma-hydroxybutyric acid (GHB), is often illegally used for drug-facilitated crimes by spiking GHB into alcoholic beverages. Analytical detection windows of only 6 h in blood and 12 h in urine are often too short to provide reliable proof of GHB ingestion. Therefore, new biomarkers are needed to prove exogenous GHB administration. Previously, amino acid GHB conjugates were discovered in an untargeted metabolomics screening and fatty acid esters with GHB were recently discussed as promising biomarkers to enlarge the analytical detection time windows. However, the development of analytical methods is still slowed down since reference compounds for targeted screenings are still missing. In this paper, we describe simple procedures for the rapid synthesis and purification of amino acid GHB conjugates as well as fatty acid esters, which can be adopted in analytical and clinical/forensic laboratories. Structural characterization data, together with IR, H-1-nuclear magnetic resonance (NMR), C-13-NMR, high-resolution mass spectra (MS), and MS/MS spectra in positive and negative ionization mode are reported for all obtained GHB conjugates and GHB conjugate precursors.ISSN:1942-7611ISSN:1942-760

Towards extending the detection window of gamma-hydroxybutyric acid—An untargeted metabolomics study in serum and urine following controlled administration in healthy men

In forensic toxicology, gamma-hydroxybutyrate (GHB) still represents one of the most challenging drugs of abuse in terms of analytical detection and interpretation. Given its rapid elimination, the detection window of GHB in common matrices is short (maximum 12 h in urine). Additionally, the differentiation from naturally occurring endogenous GHB, is challenging. Thus, novel biomarkers to extend the detection window of GHB are urgently needed. The present study aimed at searching new potential biomarkers of GHB use by means of mass spectrometry (MS) metabolomic profiling in serum (up to 16.5 h) and urine samples (up to 8 h after intake) collected during a placebo-controlled crossover study in healthy men. MS data acquired by different analytical methods (reversed phase and hydrophilic interaction liquid chromatography; positive and negative electrospray ionization each) were filtered for significantly changed features applying univariate and mixed-effect model statistics. Complementary to a former study, conjugates of GHB with glycine, glutamate, taurine, carnitine and pentose (ribose) were identified in urine, with particularly GHB-pentose being promising for longer detection. None of the conjugates were detectable in serum. Therein, mainly energy metabolic substrates were identified, which may be useful for more detailed interpretation of underlying pathways but are too unspecific as biomarkers

Identification of new urinary gamma‐hydroxybutyric acid (GHB) markers applying untargeted metabolomics analysis following placebo‐controlled administration to humans

Gamma-hydroxybutyrate (GHB) is a short-chain fatty acid that occurs naturally in the mammalian brain and that is prescribed as a medication against narcolepsy or used as a drug of abuse. Particularly, its use as a knock-out drug in cases of drug facilitated crimes is of major importance in forensic toxicology. Because of its rapid metabolism and resulting narrow detection windows (<12 h in urine), detection of GHB remains challenging. Thus, there is an urgent call for new markers to improve the reliable detection of GHB use. In the framework of a randomized, placebo-controlled, crossover study in 20 healthy male volunteers, urine samples obtained 4.5 hours post-administration were submitted to untargeted mass spectrometry (MS, QTOF) analysis to identify possible new markers of GHB intake. MS data from four different analytical methods (reversed phase and hydrophilic interaction liquid chromatography; positive and negative electrospray ionization) were filtered for significantly changed features applying uni- and multivariate statistics. From the resulting 42 compounds of interest, eight were finally identified including conjugates of GHB with carnitine, glutamate, and glycine as well as the endogenous compounds glycolate and succinylcarnitine. While GHB conjugates were only detectable in the GHB, but not in the placebo group, glycolate and succinylcarnitine were present in both groups albeit significantly increased through GHB intake. Untargeted metabolomics proved as a suitable tool for the non-hypothesis driven identification of new GHB markers. However, more studies on actual concentrations, detection windows, and stability will be necessary to assess the suitability of these markers for routine application