Direct sample preparation and simultaneous perfluoroacylation - Trimethylsilylation of biogenic monoamines along with their acidic metabolites for a single step analysis by GC-MS.

The GC-MS quantification of biogenic monoamines (BMAs), together with their acidic metabolites (ACMEs), in a single step, is presented here for the first time. This novel principle is based on the exceptional reactivity of the hexamethyldisilazane (HMDS) and perfluorocarboxylic acid (PFCA) couples [1,2], resulting in the simultaneous trimethylsilylation and acylation of BMAs and ACMEs. For this basic study, tyramine (TYR), 3-methoxytyramine (3-MeTYR), dopamine (DA), epinephrine (EP), normetanephrine (NORMNE), norepinephrine (NOREP), tryptamine (T), 3,4-dihydroxyphenylalanine (L-DOPA), 5-methoxytryptamine (5-MeT), serotonin (ST), and their ACMEs, such as homovanillic acid (HVA), vanillylmandelic acid (VMA), and 5-hydroxyindoleacetic acid (5-HIAA) were selected. These three ACMEs were derived from 3-MeTYR, NORMNE and ST, respectively. The mass fragmentation properties of the fully derivatized products proved to be of stoichiometric distribution. Informative high masses were obtained: such as the molecular ions [M]+= and/or their [M-CH3]+ alternatives. The exceptions were EP and NOREP which decomposed to the same specific, abundant mass of m/z 355 representing the C7H3-tri-OTMS ions formed by the loss of their nitrogen-containing moieties. The general rule of this new principle was confirmed by using trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA), or heptafluorobutyric acid (HFBA) with HMDS in parallel tests. In all three cases, derivatives of close retention properties in a stoichiometric manner were obtained. On the basis of the optimum separation characteristics between the BMA-ACME pairs, the HMDS & PFPA couple was preferred as the reagent of choice. Method validation was carried out, both with model solutions and in the presence of the urine matrices (without any preliminary extraction). Analytical performance characteristics for the model solutions like repeatability (RSD% 3.88-6.4), linearity (R2 0.991-0.999) and limit of quantitation (LOQ 8.8-103 ng/mL) were determined. Analytical performance characteristics for urine matrices were calculated by using the standard addition method applying the urine of a healthy volunteer and also analyzing urines of patients diagnosed with neurological diseases.

Alkylsilyl speciation and direct sample preparation of plant cannabinoids prior to their analysis by GC-MS.

A literature criticism is given on methods currently using gas chromatography mass spectrometry (GC-MS) to determine plant cannabinoids (p-CBDs). In this study, trialkylsilylation of seven p-CBDs (including their transformation products formed in the drug user's body) was compared applying various alkylsilyl reagents1 and the mass fragmentation properties of the corresponding derivatives were characterized. Derivatization, mass fragmentation and quantitation related model investigations were optimized as a function of the reaction times and conditions. Special emphasis was put (i) on the maximum responses of species, (ii) on the proportions of formed stable products, suitable for selective quantitation of all seven p-CBDs simultaneously. Results, as novel to the field confirmed that HMDS + TFA, for p-CBDs never applied reagent before, serves as their derivatization reagent of choice. These species were characterized by their retention, mass fragmentation and analytical performance characteristics. In model solutions with injected amounts in the range of 20 pg-2000 pg, repeatability (average 4.98% RSD, varying between 2.98 and 6.2% RSD), linearity (R2, 0.9956-0.9995), LOQ (20-80 pg/µL injected species) and recovery (95.2-104%) values were defined. The practical utility of this proposal, along with method development validation, was shown in a particularly unique manner and supported by the novel, extraction free, direct sample preparation working strategy. For this purpose, two Cannabis-type ruderalis (C-trd) plant tissues (C-trd1, C-trd2) were directly derivatized in the presence of the matrix. This process, which approaches green chemistry, performed without the use of organic solvents, was associated with the quantitation of self p-CBD contents of C-trd plant tissues. Applying 0.5-2.0 mg dried tissues, adding standards, the following self p-CBDs contents were confirmed: in C-trd1 6.6 µg/mg CBD, 4.4 µg/mg CBN and 1.3 µg/mg CBC, while in C-trd2 0.46 µg/mg CBD, 0.27 µg/mg CBC and 0.19 µg/mg CBG were found. The latter results were characterized by repeatability (2.52-4.99% RSD), linearity (R2, 0.9640-0.9997) and recovery (87.9-109%) data.

Structure-related related new approach in the gas chromatography/mass spectrometry analysis of cathinone type synthetic drugs.

A novel, structure-related derivatization principle has been developed in order to quantify cathinone-type synthetic drugs (CTSDs), focusing on the most common pentedrone (PENT), including also 4-fluoromethcathinone (4-FMC), methcathinone (MCTN), 4-methylethcathinone (4-MEC), 3,4-dimethylmethcathinone (3,4-DMMC), and 4-ethylmethcathinone (4-EMC). Firstly, oximated and, secondly, trimethylsilylated CTSD derivatives were characterized by mass fragmentation patterns using GC/MS that led to the development of a harmonized, quantitative, two-steps derivatization methodology. The two-step process involved i) oximation with hydroxylamine hydrochloride; and ii) trimethylsilylation with N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA). Next, the oximated-trimethylsilylated species were characterized by retention and mass fragmentation properties. Due to α-cleavage decomposition at their C1C2 bonds without exception, CTSDs uniformely exhibited a structure-related fragmentation pattern. The practical utility of this newly recognized mechanism was validated in urine samples employing an extraction-free and time-, work-, cost- and solvent-effective protocol in accordance with Green Chemistry. The centrifuged urines (10-40µL) were evaporated to dryness, followed by derivatization. The analytical performance of the methodology was characterized by repeatability (RSD%, varying between 1.43% and 5.44%), limit of quantitation (LOQ, 15-24µg/mL), linearity (R2, 0.9976-0.9998) and recovery (97-99%) values. The new principle was tested on drug users' urine: one specimen provided 56.8µg/mL PENT (3.8 RSD%). Simple trimethylsilylation of CTSDs confirmed their special fragmentation patterns, not yet described. The instantenous combination of the primarily formed, characteristic fragments with the mass m/z 44, led to the special equilibrium of products: confirming that direct trimethylsilylation of CTSDs is not suitable for their quantitation.

Trimethylsilyl speciations of cathine, cathinone and norephedrine followed by gas chromatography mass spectrometry: Direct sample preparation and analysis of khatamines.

A literature criticism is given on methods using currently gas chromatography mass spectrometry (GC/MS) to determine cathine (CAT), cathinone (CTN) and norephedrine (NE), jointly khatamines. In this study, khatamines' oximation, trimethylsilylation and mass fragmentation properties-applying N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), its trimethyliodosilane (TMIS) catalyst containing version (MSTFA(TMIS)), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and hexamethyldisilazane (HMDS)-was highlighted, at first. Derivatization, mass fragmentation and quantitation related, optimized model investigations have been carried out as a function of the reaction times and conditions. Special emphasis was put (i) on the stability of the primarily formed (CAT-2TMS, NE-2TMS, CTN-TMS(TMS-oximes)1,2), then transformed, fully derived (CAT-3TMS, NE-3MTS, CTN-2TMS(TMS-oximes)1,2) species, and, (ii) on the proportionally formed stable products, suitable to selective quantitation of all three natural amines, simultaneously. Results, as novelty to the field confirmed that (i) TMIS catalyzed trimethylsilyation triggers to form fully derivatized species unfortunately, in part only; while, (ii) khatamines' simultaneous quantitation needs to be carried out in a two steps derivatization process consisting of oximation (1st step, hydroxylamine in pyridine) and trimethylsilylation (2nd step, MSTFA), to the CAT-2TMS, NE-2TMS, CTN-TMS(TMS-oximes)1,2. These species were characterized with their retention, mass fragmentation and analytical performance properties, in model solutions and in the presence of plant tissues, as well: R(2), limit of quantitation (LOQ) data, expressed in pg/1µL injection basis, proved to be 62.5pg (CAT), 20pg (NE) and 62.5pg (CTN), respectively. The practical utility of proposal was enormously enhanced by the novel, direct sample preparation method. In this process, the freshly harvested, freeze-dried, then pulverized leaves of Catha edulis FORKS were directly derivatized, in the presence of the matrix. Reproducibility (in average 2.07 RSD% varying between 0.15 and 5.5 RSD%), linearity (0.9990-0.9994) and recovery (95.7-99.1%) values of the new sample preparation protocol was confirmed by the standard addition method for CAT, NE and CTN equally. From plant leaf, 0.061w/w% CAT and 0.014w/w% NE contents were obtained. In this tissue CTN was not found. Very likely attributable to the unfavorable climate for the plant: grown in Hungary of temperate zone and naturalized in the tropical Africa.

Quantitative Silylation Speciations of Primary Phenylalkyl Amines, Including Amphetamine and 3,4-Methylenedioxyamphetamine Prior to Their Analysis by GC/MS.

A novel, quantitative trimethylsilylation approach derivatizing 11 primary phenylalkyl amines (PPAAs), including amphetamine (A) and 3,4-methylenedioxyamphetamine (MDA), was noted. Triggering the fully derivatized ditrimethylsilyl (diTMS) species with the N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) reagent, a new principle was recognized followed by GC/MS. In the course of method optimization, the complementary impact of solvents (acetonitrile, ACN; ethyl acetate, ETAC; pyridine, PYR) and catalysts (trimethylchlorosilane, TMCS; trimethyliodosilane, TMIS) was studied: the role of solvent and catalyst proved to be equally crucial. Optimum, proportional, huge responses were obtained with the MSTFA/PYR = 2/1-9/1 (v/v) reagent applying catalysts; A and MDA needed the TMIS, while the rest of PPAAs provided the diTMS products also with TMCS. Similar to derivatives generated with hexamethyldisilazane and perfluorocarboxylic acid (HMDS and PFCA) ( Molnár et al. Anal. Chem. 2015 , 87 , 848 - 852 ), the fully silylated PPAAs offer several advantages. Both of our methods save time and cost by allowing for direct injection of analytes into the column; this is in stark contrast with the requirement to evaporate acid anhydrides by nitrogen prior to their injection. Efficiences of the novel catalyzed trimethylsilylation (MSTFA) and our recently introduced (now, for A and MDA extended) acylation principle were contrasted. Catalyzed trimethylsilylation led to diTMS derivatives resulting in on average a 1.7 times larger response compared to the corresponding acylated species. Catalyzed trimethylsilylation of PPAAs, A, and MDA were characterized with retention, mass fragmentation, and analytical performance properties (R(2), LOQ values). The practical utility of ditrimethylsilyation was shown by analyzing A in urine and mescaline (MSC) in cactus samples.