LC-MSMS Analysis Research Articles

Rapid identification of antibiotic residues in bovine kidney using coated blade spray-mass spectrometry.

The use of certain antibiotics in food-producing animals is allowed in Europe following Regulation (EU) 2017/625. However, use could result in antibiotic residues in foodstuffs of animal origin. Maximum residue limits (MRLs) are in place to protect consumers. For monitoring purposes, animal matrices are tested to verify their compliance with these MRLs. Initially, matrices of (slaughtered) food animals are screened, often using a microbiological assay. Faster screening tests for antibiotics would be an advantage for control laboratories. Therefore, the present study describes, for the first time, the use of coated blade spray (CBS) followed by direct mass spectrometry (MS) analysis for the screening of tetracyclines, sulfonamides, quinolones, and macrolides residues from the renal area of intact bovine kidneys. An optimized workflow using two different desorption/ionization solutions per blade allowed screening of target compounds within 1min per sample. The proof-of-principle of the CBS-MS method is validated according to (EU) 2021/808, presenting CCβ screening values of 0.1 × MRL for 43 analytes, 0.5 × MRL for 4 analytes, and 2.5µgkg-1 for the prohibited substance dapsone, respectively. The developed method was successfully applied to seven official control samples of bovine kidneys. One of these samples was found to be positive using the CBS-MS method, which was confirmed as a true positive by LC-MSMS analysis. The developed method demonstrates that CBS devices can directly extract and analyze kidney samples for food safety testing.

Fast and Simple Extraction for LCMSMS Analysis of Oxytetracycline Residue in Pork

Pork is very popular as an animal-base protein source of peoples in the Lao PDR. The pig growth promotion, treatment and prevention of diseases frequently relies on the use of antibiotics. In terms of food safety, the analysis of antibiotic residue in pork would be therefore important. Oxytetracycline (OTC) is one of antibiotics used in the pig husbandry. In this study, a fast and simple extraction and analysis method for determining OTC residue in pork was developed. Briefly, 1.0 g of homogenized pork sample in a 50mL-centrifµge tube was extracted with 10 mL of CH3OH: H2O mixture (9:1 ratio) assisted with vortex for 10 min, treated with QuEChERS salt and subjected to centrifugation at 5000 rpm. The methanolic extract (1.5 mL) was then cleaned up with 2mL-dSPE tube (containing MgSO4, PSA, and C18), filtered through syringe filter 0.45 micron and analyzed with ESI-LCMS/MS optimized and operated in MRM positive mode. Calibration was prepared with OTC standard solutions (5-100 ng/g). The analytical method was validated with triplicated recovery tests at a spiked level of 50 ng/g. The results unveiled that the method gave very good recovery of 88.7±3.24%. The limits of detection (LOD) and quantification (LOQ) were achieved with 0.36 ± 0.02 and 1.19 ±0.04 ng/g. In consequence, the developed method would be suitable for OTC determination in pork samples.

Insightful analysis of cyanobacterial-assisted degradation of different polymers and their kinetics

The usage of plastics has increased reliably over the years. Worldwide plastic pollution has grown to 460 million tons annually, increasing global greenhouse gas emissions by 3.4 %. In recent years, several studies have been performed utilizing microorganisms for bioremediation. The aggregation of microplastics affects marine aquatic life, posing a severe threat to the aquatic environment and hurting the global food chain. Bioremediation of various polymers is cost-effective and environmentally feasible compared to chemical, physical, and biophysical methods. Cyanobacteria, also known as blue-green algae, are photosynthetic organisms and can generate value-added products from waste, i.e., waste valorization and minimization. The present study uses Cyanobacterial sp. (Anabaena & Nostoc) to degrade the synthetic polymers prepared in lab-scale conditions. Weight loss of the degraded polymers was examined, and polymeric characteristics were analyzed through SEM, FTIR, XRD, and AFM. The experimental study observed that the polymers degraded by about 70 % and 82 % by Nostoc and Anabaena sp. in about eight weeks (approx. Two months). Polymers are degraded into monomers such as vinyl alcohol, ethylene, terephthalic acid, polycaprolactone from polyethylene terephthalate, and polyvinyl alcohol. They further break down into carbon dioxide, hydrogen, and water. These are identified from the LCMS-MS analysis. The experimental data for the degradation of polymers in Cyanobacteria sp. and control media fitted the Sinclair and Peppas kinetic model well. The results inferred that those cyanobacteria could degrade the polymers very effectively since it is a potent bioremediatory agent that can remove different organic and inorganic contaminants from the environment.

Augmenting the antioxidant, anti-bacterial and anti-carcinogenic potential of Terminalia chebula and Terminalia bellirica after tannin acyl hydrolase mediated biotransformation

Tannins, the fourth most abundant plant compounds, comprise condensed and hydrolysable types. Proanthocyanidins are flavan-3-ol polymers, while hydrolysable tannins, categorized as gallotannins and ellagitannins, form esters of gallic/ellagic acid and poly-ol. Tannin acyl hydrolase catalyzes hydrolysable tannin breakdown, yielding gallic acid derivatives with antibacterial, anti-hyperglycemic, anti-inflammatory, antioxidant, anti-carcinogenic properties. In this study, we explored hydrolysable tannins in extracts from Terminalia chebula and Terminalia bellirica, focusing on their phenolic and tannin content, as well as enzymatic and biological activities. Quantitative assessments for Total Phenol Content (TPC), Tannic Acid Content (TAC), and Hydrolysable Tannin Content (HTC) were carried out with Terminalia chebula displaying maximum amount of all three i.e. 291.9mg GAE/g DW TPC, 266.7 mgTAE/g DW TAC and 246.6 mg TAE/g DW HTC. LCMS-MS analysis revealed 8 and 13 gallic acid derivatives from T. bellirica and T. chebula, respectively after enzymatic hydrolysis. Following enzymatic treatment, the IC50 values for T. bellirica were slightly higher than T. chebula. The tannase hydrolysed T. chebula demonstrated the highest zone of inhibition against E. coli whereas the un-hydrolysed T. chebula showed minimal inhibition across all four bacterial cultures under study. The hydrolysed extract of T. chebula exhibited significant cytotoxicity after a 20-min hydrolysis, demonstrating a substantial effect with a minimal IC50 value of 83.56 μg/ml against HCT-116 cell line. Our study quantitatively highlights the enhanced bioactive properties conferred by enzymatic hydrolysis of hydrolysable tannins and considerable health benefits associated with these compounds.

A comprehensive LCMS/MS characterization for the green extracted cucurbitane-triterpenoid glycosides from bitter melon (Momordica charantia) fruit

A first-time green extraction and LCMSMS analysis for karavilosides (KVs) VIII, X, and XI in different parts (skin, pith, and seed) of the fresh and dried fruit of bitter melon (BM) is reported herein. Ultrasonication for green extraction whereas, LCMS/MS for KVs quantification were used. More extract yield (675.80 ± 163.57 mg/g) was observed for the dried fruit parts compared to the fresh BM-fruit parts (513.20 ± 75.42 mg/g). The fresh skin (343.40 ± 54.07 mg/4g) and dried seeds (311.80 and 77.95 ± 38.98) exhibited more yield whereas, the solvent yield (mg/4mg) observed was; H2O (651.70) > EtOH (227.20) > EtAC (163.30) > ACT (146.80). The LCMS/MS yield for the KVs revealed a descending order; KVXI (2376.44 ppb) > KVX (639.17 ppb) > KVVIII (599.83 ppb). More correlation was seen for the solvent Vs extract yield whereas, the KVs revealed more correlation for the BM-fruit part (P = 0.05). The study comprehensively characterized the parts of fresh and dried BM-fruits in terms of extract yield and KVs amount.

Mechanistic Study of Reaction between 5-Sulfosalicylic Acid and Colloidal MnO2

The reaction between 5-sulfosalicylic acid (5-SSA) and colloidal MnO2 has been studied kinetically in acidic medium. During the reaction the concentration of 5-SSA was kept highly excess than the concentration of colloidal MnO2 to maintain the pseudo-first-order condition. The reaction was studied by following decrease in absorbance of colloidal MnO2 at 390 nm and the first order rate constants were determined from linear log (Abs.) against time plots. The linearity of the first-order plots did not involve any autocatalytic part. The oxidation of 5-sulfosalicylic acid (5-SSA) by colloidal MnO2 involves intervention of free radicals and Mn(III). The product of reaction in colloidal MnO2 was p-hydroxy benzene sulfonate ion as confirmed by LCMS-MS analysis. The oxidation by colloidal MnO2 consists of acid dependent and independent mechanisms. The results of added Mn(II), Na4P2O7 and acrylonitrile indicate involvement of Mn(III) and free radical during oxidation by colloidal MnO2. The probable mechanisms for acid dependent and independent paths were predicted and the rate expressions were also obtained. The activation parameters also support the proposed mechanisms.

The Wound-Healing Potential of the Endemic Plant Helianthemum canum (L.) Baumg: Preclinical Studies Supported with Phytochemical Profiling.

The study's objective is to clarify the probable mechanisms underlying the wound-healing properties of Helianthemum canum L. (Cistaceae), a traditional anti-inflammatory and wound-healing medicine. LC/MS-MS was used to perform phytochemical analyses on a 70 % methanol extract of the plant's aerial parts. In vivo, linear incision and circular excision models were used to evaluate the wound healing activity. For anti-inflammatory effect, in vivo acetic acid capillary permeability assay and in vitro Interleukin 1, Interleukin 6, and Interferon ɣ levels in LPS-induced FR skin fibroblast cell line were also evaluated. The extract significantly improved wound healing in experimental models, with tensile strength values of 27.8 % and a contraction value of 35.09 %. Histopathological examinations, hydroxyproline estimation, hyaluronidase, collagenase, and elastase enzyme inhibitory assays confirmed wound healing potential. Inflammatory cytokines were significantly inhibited in the LPS-induced FR cell line, with the highest effect seen on IL-6 (34.5±2.12 pg/mL). This study offered the first concrete proof that H. canum can be used to treat wounds by suggesting that the myricetin and quinic acid content identified by LCMS-MS analysis may be accountable for the effect of H. canum on wound contraction and hydroxyproline production.

Synthesis and Characterization of 1-(1-naphthyl)-5-phenyl-2,4-pentadien-1-one from trans-cinnamaldehyde and 1-acetonaphthone

Chalcone analogues (1-(1-naphthyl)-5-phenyl-2,4-pentadiene-1-one) have been synthesized through an aldol condensation reaction between cinnamaldehyde and 1-acetylnaphthalene. The method used in this study is the stirrer method, with the reaction conducted at room temperature for 24 hours using a NaOH catalyst in H2O/ethanol (50%, v/v). The purity of the product was analyzed using thin-layer chromatography on silica gel plates with n-hexane: ethyl acetate (9:1) employed as the solvent. The resulting product obtained was 0.88 g with a yield of 61.97%. Structure Analysis and elucidation of the synthesized compounds were performed by measuring the vibrations of the functional groups using FTIR, LCMSMS analysis, and determining the 1H and 13C atoms by HNMR and CNMR analysis. The FTIR, LCMSMS, 1H, and 13C NMR analysis identified that the synthesized product as chalcone analogues, specifically 1-(1-naphthyl)-5-phenyl-2,4-pentadiene-1-one.

Layered double hydroxide driven 1O2 non-radical or •OH radical process for the degradation, transformation and even mineralization of sulfamethoxazole via efficient peroxymonosulfate activation

As reported, PMS can be activated and decomposed into different reactive oxygen species such as 1O2, •OH and SO4•-, besides, coupling of non-radical (PMS direct oxidation) and radical (SO4•-) process could enhance the utilization efficiency of PMS. In this study, we proposed another strategy of 1O2-based non-radical and •OH-based radical process using layered double hydrotalcite (MgAl-LDH and MgAlCu-LDH) to regulate the degradation, transformation and even mineralization of sulfamethoxazole. Compared with PMS direct oxidation, SMX removal efficiency was increased from 41.8% to 71.7% by 1O2. However, there was almost no real degradation or mineralization since the ionization intermediates including C10H12N3O4S and C7H9N3O5S accumulated with reaction time according to DFT and LC-MSMS analysis. In comparison, 100% of SMX degradation was obtained via coupling 1O2 and •OH-radical process, in which the mineralization efficiency reached to 24.8%. Importantly, the above-mentioned intermediates of SMX were not detected and their accumulation were also significantly inhibited. Moreover, SMX degradation behavior and kinetics could be manipulated via direct oxidation, 1O2 and then •OH step by step, which was beneficial to enhance the PMS utilization efficiency. Finally, based on the results of zeta potential of MgAlCu-LDH, effect of solution pH on SMX degradation, the interfacial PMS activation process was responsible to regulate non-radical and radical process.

PMON54 Exosomes Isolated from Conditioned Media of Immortalized Kisspeptin Neurons Exert Diverse Effects on Central and Peripheral in Vitro Cell Models.

Abstract Previous work in our laboratory explored the proteomic contents of exosome-like extracellular vesicles (EVs) released into the media in vitro from immortalized Kisspeptin (KP) neuronal cell lines KTaV-3 (derived from female mouse AVPV) and KTaR-1 (derived from KNDy neurons in the ARC). EVs were isolated from conditioned media via ultracentrifugation or filtration kit and validated using a NanoCyte, and LCMS-MS analysis revealed that relative abundance of exosomal cargo varied dependent upon estrogen (E2) exposure in vitro, with ∼150-170 proteins up-regulated and ∼200-220 proteins downregulated by E2 in EVs of KTaR-1 and KTaV-3 KP neurons. Since E2-regulated KP exosomal proteins included candidates implicated in the regulation of synaptic plasticity and signaling (i.e. annexins, semaphorins, connexins), we investigated the effects of exposure to purified EVs on gene expression in immortalized GnRH neurons (GT1-7 cells). Notably, EVs from 24h E2-treated KTaV-3 neurons induced increased expression of kiss1r in GT1-7 cells, in contrast to KTaV-3 neurons not exposed to E2, suggesting that AVPV KP neurons may signal an increase in KP receptivity in GnRH neurons in vivo via non-neuronal communication. Additionally, increases in expression of the synaptic scaffolding protein PSD-95 (dlg4) were seen in GT1-7 cells treated with E2-treated KTaV-3 EVs, suggesting AVPV KP neurons may use extracellular vesicles to modulate GnRH neuronal synaptic plasticity over the estrous cycle. While EVs isolated from KTaR-1 conditioned media did not alter GT1-7 kiss1r or dlg4 levels, treatment (24h) resulted in induction of selective gap junction hemichannel expression in GT1-7 cells. KTaR-1 (but not KTaV-3) EVs increased expression of Cx26 (gjb2) irrespective of E2 exposure, while induction of Cx43 (gja1) expression in GT1-7 cells was only observed following treatment with E2-deprived KTaR-1 EVs. Further, we found that KTaR-1 media and EVs can affect osteoblast function in vitro, including increases in sp7 and runx2 expression, E2-dependent modulation of wnt10b, and formation of bone matrix (evaluated by Alizarin Red assay) in cultured osteoblast lines, supporting recent studies implicating ARC KP neurons in bone remodeling. Lastly, we found significant levels of immunomodulatory pentraxins (PTX3) in KP EVs, with abundance dependent upon prior E2 exposure in KTaR-1 KP neurons, as revealed by ELISA. Together, results from these studies suggest that EVs may represent additional intercellular communication pathways utilized by Kiss-1 neurons to elicit changes in nearby neuronal populations and potentially even in the periphery, affecting inflammation and bone remodeling. Future studies will address mechanisms involved in the E2 regulation of exosomal cargo in these critical neuronal populations. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

What is the added value of liquid chromatography-tandem mass spectrometry for routine urine drug screening in clinical situations?

Replacing the common two-step process of urine drug screening by a single-step methodology based on selective and quantitative liquid chromatography-tandem mass spectrometry (LC-MSMS) seems quite attractive. With this approach, it may be more appropriate to use the limit of analytical quantification instead of a cutoff concentration to report positive results. In this study, we want to evaluate the impact of this modification on the results of urine samples received in clinical situations. Results of all urine drug screens and associated confirmations performed between July 2021 and March 2022 for amphetamine, benzodiazepine and opiate groups, cannabis and cocaine metabolites and methadone were retrieved from the laboratory information system of the Medical Biology Department of Laboratoire National de Santé in Luxembourg. The samples came from hospitals, addiction centers, prisons and occupational health clinics. They were analyzed by an automated immunoassays, Cobas (Roche Diagnostics) and by a targeted quantitative screening assay, MassTox Drugs of Abuse testing, (Chromsystems) on a 6500QTRAP LC-MSMS (Sciex). We calculated the number of samples where results were discordant. Cases where no drugs were identified by LC-MSMS but results of the immunoassay were above the cut-off concentration (based on the screen test cut-off concentration of European guidelines) were considered as false positive (FP) results. Whereas cases where drugs were quantified by LC-MSMS but results of the immunoassay were below the cut-off concentration were considered as false negative (FN) results. The FP and FN results were 1 and 5% for cannabis metabolite ( n = 477), 0 and 14% for cocaine metabolite ( n = 189), 2 and 20% for methadone ( n = 211), 1 and 10% for benzodiazepines ( n = 489), 1 and 20% for opiates ( n = 149) and 48 and 2% for Amphetamines ( n = 92). The median concentrations of FN were 18 ng/ml (range from 15 to 31) for THC-COOH, 62 ng/ml (range from 24 to 121) for benzoylecgonin, 221 ng/ml (from 63 to 785) for methadone and 367 ng/ml (from 66 to 3174) for EDDP. In the benzodiazepine FN results (median concentration 69 ng/ml, range from 26-318), oxazepam, lorazepam and OH-alprazolam were the most frequently detected drugs. Among the opiate FN results, morphine associated or not with codeine was the most frequently quantified drug (median 81 ng/ml, range from 27 to 519). Four out of the 29 FN samples contained 6-monoacetylmorphine (from 17 to 136 ng/ml) in the absence of morphine, 2 samples were positive for pholcodine and one sample contained high concentration of meperidine. The two FN samples for amphetamines contained amphetamine (500 and 800 ng/ml). Nine out of the 42 amphetamines FP samples contained ritalinic acid (from 10,000 to 130,000 ng/ml). This targeted method improves the sensitivity but is restricted to a pre-defined list of drugs, although it covers a wider panel than commercial immunoassays, some relevant molecules may be missing. The hydrolysis protocol used before LC-MSMS analysis also explains the increase in sensitivity for benzodiazepines and opiates (allowing the detection of conjugated metabolites). Decreasing the threshold of detection for urine drug screening allowed for the extension of the drug detection window, improving the detection of drugs administered at low dose or those reacting poorly in immunoassay. This single step approach bypasses the main pitfalls of the immunoassays, namely the lack of analytical specificity (mainly for amphetamines) and a screening limited to well-established (parent) drugs, with consequences on patient management still only partially objectified. This targeted quantitative screening LC-MSMS method with its high sensitivity, accurate quantification, and associated internal quality control, achieves the required analytical performances expected in a clinical setting. The implementation of this approach will largely be conditioned by the optimization of the sample throughput to ensure a turn-around-times compatible with various clinical situations and the availability of the equipment and highly trained staff.

Reduction and Hydroxylation Metabolites of Mequindox in Holothurian Analysis by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

This research proposed and validated an LC-MSMS method for five reduction and hydroxylation metabolites of Mequindox (MEQ) as well as the precursor in holothurian samples. Specially, three hydroxylation metabolites (2-isoethanol-mequindox, M3, 2-isoethanol-1-desoxymequindox, M4 and 2-isoethanol-4-desoxymequindox, M5) are novel for analysis. Target compounds were extracted with methanol and ethyl acetate in turn without any complicated acidolysis, alkaline hydrolysis or enzymolysis steps. Samples were further purified with C18 solid-phase extraction cartridges for LC-MSMS analysis. Mean recoveries in spiked samples ranged from 81 to 107% with intra-day relative standard deviation (RSD) and inter-day RSD <11.2 and 9.9%, respectively. Limit of detection was determined based on signal-to-noise ratio ≥3 ranged from 0.16 to 2.11μgkg-1 for each target. The validated protocol was successfully applied for commercial holothurian samples with a positive rate at 13.3%. And concentrations of hydroxylation metabolites were higher than reduction metabolites and precursor MEQ in positive samples.

Development and validation of a sensitive LCMS/MS method for the simultaneous quantification of Azelnidipine and Chlorthalidone in human plasma

Highly resolved, selective and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was optimised for the simultaneous quantification and recently approved combined medication for the treatment of hypertension viz. azelnidipine and chlorthalidone in spiked human plasma. The study utilizes nifedipine as internal standard. In the extraction of analytes from spiked plasma, acetonitrile was used to induce protein precipitation and then liquid – liquid extraction was carried using ethyl acetate solvent followed by the LCMS-MS analysis. The analytes were separated on Inertsil ODS C18 (4.6 mm×100 mm, 5 μm) column at room temperature with 65:35 (v/v) ratio of acetonitrile and 0.1% formic acid as the mobile phase at 0.5 mL/min flow rate. Detection was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring with the transitions of m/z 583 → 331, 339 → 321 and 347 → 328 respectively for azelnidipine, chlorthalidone and nifedipine. The analytes were detected at a retention time of 1.72 min, 2.62 min and 3.12 min respectively for azelnidipine, chlorthalidone and nifedipine and the analysis was completed within run time of 5 min. The analysis was carried in the calibration range of 5-500 ng/mL with a very sensitive detection limit of 1.5 ng/mL. The method was validated as per FDA guidelines for bioanalytical method validation and the results in all the validation parameters were in the acceptable limit. Results proved that the method was selective, sensitive, accurate, precise and stable and hence can be applicable for the routine analysis of azelnidipine and chlorthalidone in biological samples.

AKTIVITAS EKSTRAK ETANOL DAUN SALAM [Syzygium polyanthum (Wight) Walp.] SEBAGAI PENGHAMBAT ENZIM α-GLUKOSIDASE DAN STUDI SECARA IN SILICO

Diabetes Mellitus (DM) is a degenerative disease that can cause various complications such as cardiovascular, stroke, and hypertension. Antidiabetic drugs that are commonly circulated in the market, have side effects that can harm other organs such as liver, kidneys and other organs. Bay leaves have properties as inhibitors of the α -glucosidase enzyme in vitro and in vivo. The aims of this study was determine the activity of ethanolic extract of bay leaves by ultrasonic extraction method in inhibiting the α -glucosidase enzyme in vitro and in silico. Bay leaves were extracted using 70% and 96% ethanol by ultrasonic method for 15 minutes, and filtered and concentrated using a rotary vacuum evaporator 4 times. The extract obtained was tested for phytochemical content and the activity of α -glucosidase inhibitory assay. The extract with the best IC50 value was then separated using column chromatography (SiO2, dichloromethane, methanol) and produced 7 fractions and tested for α -glucosidase enzyme inhibition. The best fraction (fraction 6) was analyzed by Liquid Chromatography Mas Spectra (LCMS-MS). The results showed that bay leaf extract containing alkaloids, flavonoids, tannins, steroids, phenols, and fraction-6 had the best IC50 of 30.82 ppm. The results of LCMS-MS analysis of chemical compounds as inhibitors of the α -glucosidase enzyme were delphinidin (m/z 303) and had a rerank score of -75.59. In the in silico test using PLANTS docking software.

LC-MS-MS Analysis of Δ9-THC, CBN and CBD in Hair: Investigation of Artifacts.

In forensic toxicology, high-performance liquid chromatography tandem mass spectrometry (LC-MS-MS) is increasingly used for the fast and sensitive measurement of a wide range of drugs. For our routine casework, a LC atmospheric pressure chemical ionization MS-MS method for the quantification of Δ9-tetrahydrocannabinol (Δ9-THC), cannabinol (CBN) and cannabidiol (CBD) in hair was established and fully validated. Separation was achieved using a Kinetex® C18 column (100 mm×2.1 mm, 100 Å, 1.7 μm, Phenomenex) at a flow rate of 0.5 mL/min. Measurements were performed on a QTRAP 5500 mass spectrometer (Sciex, Darmstadt, Germany). Unexpected signals were observed in authentic THC-positive hair samples. First, a signal with a slightly shifted retention time of THC whose origin could be assigned to the isomer Δ8-THC was detected. Second, additional peaks exhibiting the same fragments as CBN and Δ9-THC but eluting at different retention times were detected. Spiking experiments and enhanced product ion scans pointed to the origin of these additional signals as result of in-source decarboxylation of Δ9-tetrahydrocannabinolic acid A (Δ9-THCA-A) into Δ9-THC and further partial oxidation of Δ9-THC into CBN, respectively. Positive findings of Δ9-THCA-A in hair have been shown to derive from external contamination; therefore, the herein described artifacts may be used as indirect markers for external contamination.

Comprehensive metabolomic, lipidomic and pathological profiles of baobab (Adansonia digitata) fruit pulp extracts in diabetic rats

Baobab fruit pulp Adansonia digitata (AD) has received attention due to its numerous nutritional and medicinal values. In the current study, tentative identification was performed due to limited information available on its phytochemical composition. Phytochemicals from AD fruit pulp were obtained using successive organic solvent fractionation. The LC-MSMS analysis led to identification of 91 metabolites from methanol, butanol and ethyl acetate extracts. Moreover, 20 compounds were identified in the petroleum ether extract based on high resolution ion masses. In vitro antidiabetic and antioxidant properties of selected extracts were investigated using enzyme activity and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, respectively. Biological screening of the antidiabetic effects of target extracts was performed against streptozotocin-induced diabetes in experimental animals, following daily oral treatment for 3 successive weeks. Serum glucose, insulin, adiponectin, superoxide dismutase (SOD), lipid peroxide, cholesterol and HDL levels were measured. Finally, histopathological and immunohistochemical examinations of pancreas were carried out. Results revealed that animal groups treated daily with butanol (BuOH) and petroleum ether extracts of AD (oil) exhibited a significant improvement in carbohydrate and lipid metabolism as well as antioxidant effect. Both extracts revealed superior effects with respect to the total (TT) and ethyl acetate (EtOAc) extracts. Histopathological and immunohistochemical findings supported these results, showing marked protection of the pancreas. Thus, baobab oil and butanolic extract of the fruit pulp protected animals against STZ-induced diabetic changes, in addition to attenuation of lipid peroxidation, hypercholesterolemia and oxidation.

Multiclass analysis of emerging organic contaminants in tropical marine biota using improved QuEChERS extraction followed by LC MS/MS

Marine biota is one of the important components of an ecosystem, and it has been widely used as a pollution indicator for emerging organic contaminants (EOCs) in the coastal zone. Previous studies divulged the concentration of EOCs in various types of biota matrices at trace level detection. EOCs have been associated with several adverse human health effects. Bioaccumulation of EOCs in the food web has been the main concern, and thus a sensitive and selective method for trace quantification of EOCs in biota matrices need to be developed and optimized. Considering various matrix interferences for EOC analysis in marine biota, a combination technique of QuEChERS and solid phase extraction cleanup with detection based on liquid chromatography with tandem mass spectrometry (LC MS/MS) is presented in this study. A method for 16 compounds grouped into four different classes, namely, pharmaceutically active chemicals, phenolic endocrine disrupter compounds, estrogenic hormones, and pesticides, was developed and validated for biota extraction. Satisfactory extraction was obtained for the optimized method with percentage of recovery from 64% to 114% and excellent sensitivity with detection limit in the range of 0.02–3.50 ng/g. Linearity of the standards (in the solvent) in the LC MS-MS analysis ranged from 0.991 to 0.999. The relative standard deviation for intra-day and inter-day repeatability was less than 20%, indicating good-precision analysis. Assessment on the matrix effects showed ionization suppression for all the developed compounds. The developed method was verified by analyzing biota matrices collected from the Klang River estuary. Trace concentrations of EOCs, ranging from 0.05 to 10.76 ng/g, were found in those matrices. Of the 16 targeted compounds, 10 were detected, namely, diclofenac, bisphenol A, sulfamethoxazole, amoxicillin, E2, E1, progesterone, testosterone, primidone, and 4-octylphenol. The other compounds were below the method detection limit.

Quantitation and Validation of 34 Fentanyl Analogs from Liver Tissue Using a QuEChERS Extraction and LC-MS-MS Analysis.

Since 2013, drug overdose deaths involving synthetic opioids (including fentanyl and fentanyl analogs) have increased from 3,105 to 31,335 in 2018. Postmortem toxicological analysis in fentanyl-related overdose deaths is complicated by the high potency of the drug, often resulting in low analyte concentrations and associations with toxicity, multidrug use, novelty of emerging fentanyl analogs and postmortem redistribution. Objectives for this study include the development of a quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction and subsequent liquid chromatography-mass spectrometry--mass spectrometry analysis, validation of the method following the American Academy of Forensic Sciences Standards Board (ASB) standard 036 requirements and application to authentic liver specimens for 34 analytes including fentanyl, metabolites and fentanyl analogs. The bias for all 34 fentanyl analogs did not exceed ±10% for any of the low, medium or high concentrations and the %CV did not exceed 20%. No interferences were identified. All 34 analytes were within the criteria for acceptable percent ionization suppression or enhancement with the low concentration ranging from -10.2% to 23.7% and the high concentration ranging from -7.1% to 11.0%. Liver specimens from 22 authentic postmortem cases were extracted and analyzed with all samples being positive for at least one target analyte from the 34 compounds. Of the 22 samples, 17 contained fentanyl and metabolites plus at least one fentanyl analog. The highest concentration for a fentanyl analog was 541.4 μg/kg of para-fluoroisobutyryl fentanyl (FIBF). The concentrations for fentanyl (n = 20) ranged between 3.6 and 164.9 μg/kg with a mean of 54.7 μg/kg. The fentanyl analog that was most encountered was methoxyacetyl fentanyl (n = 11) with a range of 0.2-4.6 μg/kg and a mean of 1.3 μg/kg. The QuEChERS extraction was fully validated using the ASB Standard 036 requirements for fentanyl, metabolites and fentanyl analogs in liver tissue.

Quantitation of Fentanyl and Metabolites from Liver Tissue Using a Validated QuEChERS Extraction and LC-MS-MS Analysis.

According to the National Institute on Drug Abuse (NIDA), more than one hundred people die every day from opioid overdose. Overdose fatalities have risen as the availability of potent synthetic opioids, such as fentanyl, has increased. A forensic postmortem toxicological specimen is often in various stages of decomposition, experiencing autolysis and putrefaction, which complicates the extraction, creating a difficult challenge for toxicologists. Isolating the target drug, while creating an efficient and simplified analytical scheme, is a goal for most toxicology laboratories. The validation of a quick, easy, cheap, effective, rugged and safe extraction protocol is presented in this study as an alternative analytical method for efficient extraction and detection of fentanyl and its major metabolites: norfentanyl and despropionyl fentanyl (4-ANPP). The liquid Chromatography with tandem mass spectrometry analysis was validated following the American Academy of Forensic Sciences Standards Board (ASB) standard 036 proposed requirements. Evaluated parameters include selectivity, matrix effects (MEs), linearity, processed sample stability, bias, precision and proof of applicability using liver samples from authentic postmortem cases. MEs (represented as percent ionization suppression or enhancement) at low and high concentrations were -10.0% and 1.4% for fentanyl, -2.1% and -0.3% for 4-ANPP and 3.1% and 2.8% for norfentanyl, respectively. Bias for the three analytes ranged from -8.5% to -19.9% for the low concentrations, -3.6% to -14.7% for the medium concentrations and 1.5% to -16.1% for the high concentrations with all being within the ±20% guideline. Precision for the three analytes ranged from 2.2% to 15.1%. The linear range for the fentanyl and norfentanyl was 0.5-100 and 4-ANPP had a linear range of 0.4-80μg/kg. The authentic postmortem liver samples ranged in fentanyl concentrations from 56.6 to 462.3μg/kg with a mean of 149.2μg/kg (n=10). The range of norfentanyl concentrations were 1.9 to 50.0μg/kg with a mean of 14.1μg/kg (n=10). The range of 4-ANPP concentrations were 3.2 to 23.7μg/kg with a mean of 7.5μg/kg (n=7).

Method Validation of Seven Synthetic Cathinones by LC-MS/MS Analysis and the Prevalence of N-Ethylpentylone in Postmortem Casework.

N-ethylpentylone (NEP, ephylone, bk-EBDP) was the most prevalent synthetic cathinone detected by the Miami-Dade Medical Examiner Toxicology Laboratory from 2016-2018. There is limited information regarding the toxicity of NEP, however the few published reports suggest that NEP can cause serious toxic effects and sudden death. The purpose of this publication is to describe a validated LC-MS/MS method for seven synthetic cathinones (methylone, ethylone, butylone, dibutylone, α-PVP, pentylone, and NEP) and to present a detailed summary regarding the presence of NEP in postmortem casework at the Miami-Dade Medical Examiner Department. Post-mortem iliac blood, serum, liver, and brain specimens were prepared by solid-phase extraction with analysis by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS). Analyte linearity was established from 0.01 to 0.5 mg/L on a six-point calibration curve. A total of 101 NEP quantitations were performed using this method. Concentrations in postmortem case samples ranged from 0.01 mg/L to 2.7 mg/L. Iliac blood concentrations averaged 0.312 mg/L with a median of 0.137 mg/L (n=72) across all causes and manners of death. Approximately half of the cases were homicides in which the decedent was the victim of gunshot wounds or stabbing. Two of the three highest concentration cases of NEP (2.7 mg/L and 1.7 mg/L) involved 38-year old white males who were tasered by police prior to death. The psychostimulant effect of NEP may result in an excited delirium and/or hallucinogenic state. The concentration of NEP detected in accidental intoxication and polydrug cases overlapped with those attributed to other causes, including homicides and police involved deaths.