LC MS-MS Research Articles

Characterization and quantification of peptaibol produced by novel Trichoderma spp: Harnessing their potential to mitigate moisture stress through enhanced biochemical and physiological responses in black pepper (Piper nigrum L.).

Trichoderma spp. is primarily applied to manage biotic stresses in plants. Still, they also can mitigate abiotic stresses by the stimulation of antioxidative protective mechanisms and enhanced synthesis of secondary metabolites. The study optimized the conditions to enhance peptaibol production by novel Trichoderma spp, characterized and quantified peptaibol- alamethicin using HPLC and LC MS-MS. The present study investigated these isolates efficacy in enhancing growth and the associated physio-biochemical changes in black pepper plants under moisture stress. Under in vitro conditions, out of 51 isolates studied, six isolatesviz., T. asperellum (IISR NAIMCC 0049), T. erinaceum (IISR APT1), T. harzianum (IISR APT2), T. harzianum (IISR KL3), T. lixii (IISR KA15) and T. asperellum (IISR TN3) showed tolerance to low moisture levels (5, 10 and 20%) and higher temperatures (35 and 40°C). In vivo evaluation on black pepper plants maintained under four different moisture levels (Field capacity [FC]; 75%, 50%, and 25%) showed that the plants inoculated with Trichoderma accumulated greater quantities of secondary metabolites viz., proline, phenols, MDA and soluble proteins at low moisture levels (50% and 25% FC). In the present study, plants inoculated with T. asperellum and T.harzianum showed significantly increased growth compared to uninoculated plants. The shortlisted Trichoderma isolates exhibited differences in peptaibol production and indicated that the peptide might be the key factor for their efficiency as biocontrol agents. The present study also demonstrated that Trichoderma isolates T. harzianum and T. asperellum (IISR APT2 & NAIMCC 0049) enhanced the drought-tolerant capabilities of black pepper by improving plant growth and secondary metabolite production.

Imatinib pharmacokinetics and creatine kinase levels in chronic myeloid leukemia patients: implications for therapeutic response and monitoring

BackgroundImatinib treatment for certain cancers can lead to elevated creatine kinase (CK) levels, potentially indicating muscle injury, and ongoing research aims to understand the correlation between imatinib levels and creatine kinase to assess its impact on treatment response.MethodsThis single-center observational study involved 76 chronic myeloid leukemia (CML) patients receiving imatinib treatment, focusing on evaluating drug and metabolite levels using liquid chromatography–mass spectrometry (LC–MS-MS) instrumentation. Serum CK and creatine kinase-MB (CK-MB) levels were assessed using Colorimetric kits.ResultsCK and CK-MB levels were measured, CK showed a median value of 211.5 IU/l and CK-MB showed a median value of 4.4 IU/l. Comparing low and high CK groups, significant differences were found in peak and trough plasma concentrations of imatinib and its metabolites. Correlations between CK levels and pharmacokinetic parameters were explored, with notable associations identified. Binary logistic regression revealed predictors influencing the therapeutic response to imatinib and categorized expected CK levels into high or low, with peak levels of imatinib emerging as a significant predictor for CK level categorization.ConclusionThe study highlights the link between imatinib’s pharmacokinetics and elevated CK levels, indicating a possible correlation between specific metabolites and improved treatment response. Individualized monitoring of CK levels and imatinib pharmacokinetics could enhance care for CML patients.

A validated dilute-and-shoot LC–MS-MS urine screening for the analysis of 95 illicit drugs and medicines: Insights from clinical and forensic Brazilian cases

Abstract Urine toxicological analysis is a relevant tool in both clinical and forensic scenarios, enabling the diagnosis of acute poisonings, elucidation of deaths, verification of substance use in the workplace and identification of drug-facilitated crimes. For these analyses, the dilute-and-shoot technique associated with liquid chromatography coupled with tandem mass spectrometry (LC–MS-MS) is a promising alternative since it has demonstrated satisfactory results and broad applicability. This study developed and validated a comprehensive LC–MS-MS screening method to analyze 95 illicit drugs and medicines in urine samples and application to clinical and forensic Brazilian cases. The dilute-and-shoot protocol was defined through multivariate optimization studies and was set using 100 µL of sample and 300 µL of solvent. The total chromatographic run time was 7.5 min. The method was validated following the recommendations of the ANSI/ASB Standard 036 Guideline. The lower limits of quantification varied from 20 to 100 ng/mL. Within-run and between-run precision coefficient of variations% were <20%, and bias was within ± 20%. Only 4 of the 95 analytes presented significant ionization suppression or enhancement (>25%). As proof of applicability, 839 urine samples from in vivo and postmortem cases were analyzed. In total, 90.9% of the analyzed samples were positive for at least one substance, and 78 of the 95 analytes were detected. The most prevalent substances were lidocaine (40.2%), acetaminophen (38.0%) and benzoylecgonine (31.5%). The developed method proved to be an efficient and simplified alternative for analyzing 95 therapeutic and illicit drugs in urine samples. Additionally, the results obtained from sample analysis are essential for understanding the profile of Brazilian substance use, serving as a valuable database for the promotion of health and safety public policies.

Analyses of the brown stain on the Parthenon Centaur head in Denmark

In 1688 two sculptural fragments, a head of bearded man and a head of an unbearded youth, arrived in Copenhagen, sent from Athens as a gift to King Christian 5. They were placed in the Royal Kunstkammer, their provenance given as the Temple of Artemis in Ephesos, one of the Seven Wonders of the World. Almost a hundred and fifty years later, in the early 1820’s they were noticed and studied by two scholars independently visiting the Kunstkammer. However, both concluded that the two heads belonged to one of the metopes decorating the south side of the Parthenon temple on the Acropolis in Athens, showing fighting between Greeks and the mythical Centaurs, part man and part horse. In the 1830’s another sculptural fragment, a horse’s hoof, obtained through the German archaeologist and state antiquary of Greece, Ludwig Ross, reached Copenhagen. It was forwarded by the Danish consul to Athens, C.T. Falbe, as a gift to King Christian 8. The inventory reads: ‘… was found on the Acropolis near the Parthenon temple and is supposed to belong to one the Centaurs on the metopes.’ The present paper focuses solely on the head of the Centaur.A brown stain was noticed on the Parthenon marbles as early as 1830 by the British Museum and has ever since eluded a deeper understanding of its genesis despite many investigations and attempts of analyses. A quite similar brown stain can be observed on the Centaur’s head in Copenhagen as well.The present study reports analyses by LA-ICP-MS, SEM–EDX, µXRD, GC–MS, and LC–MS-MS, as well as optical microscopy of five small samples sequestered in 1999 from the Centaur head curated by the National Museum of Denmark. Our analyses show that the brown stain consists of two consecutively added surficial layers of the calcium oxalate minerals whewellite and weddellite. Despite a thorough search using proteomics, we have found no viable organic precursor material for the oxalates. Our results do not solve the mystery of the formation of the brown stain, but they do further qualify the structure and characterization of the brown stain.

Label-free quantitative proteomic profiling reveals differential plasma protein expression in patients with obesity after treatment with liraglutide.

Treatment and management of obesity is clinically challenging. The inclusion of GLP-1 receptor agonists (GLP1RA) in the medical management of obesity has proven to be efficacious. However, mechanisms underlying the molecular changes arising from GLP1RA treatment in patients with obesity remain to be elucidated. A single-center, prospective study was undertaken to evaluate the changes in the plasma proteins after liraglutide 3 mg therapy in twenty patients (M/F: 7/13) with obesity (mean BMI 40.65 ± 3.7 kg/m2). Anthropometric and laboratory parameters were measured, and blood samples were collected at two time points: baseline, before initiating treatment (pretreatment group, PT), and after three months of receiving the full dose liraglutide 3 mg (posttreatment group, PoT). An untargeted label-free LC MSMS mass spectrometric approach combined with bioinformatics and network pathway analysis was used to determine changes in the proteomic profiles. The mean age of the study participants was 36.0 ± 11.1 years. A statistically significant change was observed in weight, BMI and HbA1c levels between the PT and PoT groups (paired t-test, P < 0.001). A significant dysregulation was noted in the abundances of 151 proteins (31 up and 120 downregulated) between the two groups. The potential biomarkers were evaluated using receiver operating characteristic (ROC) curves. The top ten proteins (area under the curve (AUC) of 0.999 (95% CI)) were identified as potential biomarkers between PT and PoT groups and included Cystatin-B, major vault protein, and plastin-3, which were upregulated, whereas multimerin-2, large ribosomal P2, and proline-rich acidic protein 1 were downregulated in the PoT group compared with the PT group. The top network pathway identified using ingenuity pathway analysis (IPA), centered around dysregulation of MAPK, AKT, and PKc signaling pathways and related to cell-to-cell signaling and interaction, cellular assembly and organization, cellular compromise and a score of 46 with 25 focus proteins. Through label-free quantitative proteomic analysis, our study revealed significant dysregulation of plasma proteins after liraglutide 3 mg treatment in patients with obesity. The alterations in the proteomic profile between the PT and PoT groups demonstrated a decrease in levels of proteins involved in inflammation and oxidative stress pathways. On the other hand proteins involved in the glycolytic and lipolytic metabolic pathways as well as those participating in cytoskeletal and endothelial reorganization were observed to be increased. Understanding actions of liraglutide at a molecular and proteomic levels provides a holistic look into how liraglutide impacts metabolism, induces weight loss and improves overall metabolic health.

Neuroprotective and nephroprotective effects of Ircinia sponge in polycyclic aromatic hydrocarbons (PAHs) induced toxicity in animal model: a pharmacological and computational approach

The present study investigated the neuroprotective and nephroprotective effects of the sponge Ircinia sp. ethyl acetate extract (ISPE) against persistent aromatic pollutants in vitro and in vivo. Different exponential experimental assays were applied to this study. An in vitro study to investigate the potential therapeutic effect of ISPE using antioxidants (for example, ABTS and DPPH) and anti-Alzheimer assays (inhibition of acetylcholinesterase); the in-vivo study was designed to evaluate the protective effect of ISPE as neuroprotective and nephroprotective against the destructive effect of PAH. Several assays included oxidative assays (LPO), antioxidant biomarkers (GSH, GST), and inflammatory and neurodegenerative biomarkers (PTK,SAA). Additionally, the results were confirmed using histopathological examination. The in silico screening study improved the in vitro and in vivo findings through interaction between the aryl hydrocarbon receptor (AHR) and the polyphenolic content of ISPE extract, which was determined using LCMSM. The results and discussion showed that ISPE exhibited a promising antioxidant and anti-acetylcholinesterase activity as evidenced by IC50 values of 49.74, 28.25, and 0.18 µg/mL in DPPH, ABTS, and acetylcholinesterase inhibition assays, respectively. In vivo, the study showed that animals receiving ISPE before poly aromatic hydrocarbons administration PAHs (Prot, ISPE) showed significant amelioration in kidney functions manifested by the reduction of serum urea, uric acid, and creatinine by 40.6%, 66.4%, and 134.8%, respectively, concerning PAH-injected mice (HAA). Prot, ISPE revealed a decline in malondialdehyde (MDA) and total proteins (TP) in kidney and brain tissues by 73.63% and 50.21%, respectively, for MDA and 59.82% and 80.41%, respectively, for TP with respect to HAA. Prot, ISPE showed significant elevation in reduced glutathione (GSH) and glutathione transferase (GST) in kidney and brain tissues and reduction in the inflammatory and pre-cancerous biomarkers, namely, serum protein tyrosine kinases (PTKs) and serum amyloid A (SAA). These findings were further supported by histopathological examination of kidney and brain tissues, which revealed normal structure approaching normal control. Metabolic profiling of ISPE using LC–MS-MS showed the presence of fourteen polyphenolic compounds belonging mainly to phenolic acids and flavonoids. In silico study revealed that all the tested compounds exerted certain binding with the aryl hydrocarbon receptor, where rutin showed the best fitting (ΔG = − 7.6 kcal/mol−1) with considerable pharmacokinetic and pharmacodynamic properties revealed from in silico ADME (Absorption, Distribution, Metabolism, and Excretion) study. Hence, it can be concluded that the Ircinia sponge showed a promising protective effect versus kidney and brain toxicity triggered by PAHs.Graphical

Anthocyanin metabolism in Nelumbo: translational and post-translational regulation control transcription

BackgroundLotus (Nelumbo Adans.) is used as an herbal medicine and the flowers are a source of natural flavonoids. ‘Da Sajin’, which was firstly found in the plateau area, is a natural mutant in flower color with red streamers dyeing around white petals.ResultsThe LC–MS-MS results showed that eight anthocyanin compounds, including cyanidin 3-O-glucoside, cyanidin 3-O-galactoside, malvidin 3-O-galactoside, and malvidin 3-O-glucoside, were differentially enriched in red-pigmented tissues of the petals, whereas most of these metabolites were undetected in white tissues of the petals. Transcriptome profiling indicated that the relative high expression levels of structural genes, such as NnPAL, NnF3H, and NnANS, was inconsistent with the low anthocyanin concentration in white tissues. Members of the NnMYB and NnbHLH transcription factor families were presumed to play a role in the metabolic flux in the anthocyanin and proanthocyanidin biosynthetic pathway. The expression model of translational initiation factor, ribosomal proteins and SKP1–CUL1–F-box protein complex related genes suggested an important role for translational and post-translational network in anthocyanin biosynthesis. In addition, pathway analysis indicated that light reaction or photo destruction might be an important external cause for floral color determination in lotus.ConclusionsIn this study, it is supposed that the natural lotus mutant ‘Da Sajin’ may have originated from a red-flowered ancestor. Partial loss of anthocyanin pigments in petals may result from metabolic disorder caused by light destruction. This disorder is mainly regulated at post translation and translation level, resulting in a non-inherited phenotype. These results contribute to an improved understanding of anthocyanin metabolism in lotus, and indicate that the translational and post-translational regulatory network determines the metabolic flux of anthocyanins and proanthocyanidins under specific environmental conditions.

A simultaneous, high-throughput and sensitive method for analysing 13 neonicotinoids and metabolites in urine using a liquid chromatography–tandem mass spectrometry

A simultaneous, high-throughput and sensitive method for analysing nine neonicotinoid pesticides (NEOs) and four metabolites (NEOms) in urine using a liquid chromatography-tandem mass spectrometry (LC–MSMS) was developed. The method detection limit (MDL) and lowest concentration minimum reporting limit (LCMRL) of the nine NEOs were 0.0013–0.048 ng/ml and 0.0050–0.17 ng/ml, respectively. The MDL and LCMRL of the four NEOms were 0.0052–0.52 ng/ml and 0.011–1.6 ng/ml, respectively. Intermediate precision for the nine NEOs and four NEOms was 7.5–12.5% and 7.4–10.9%, respectively. Accuracy for the nine NEOs and four NEOms was 3.83–5.60% and 3.01–29.2%, respectively. The developed method was applied to analyse urine samples collected from participants of a large-scale birth cohort study, namely, the Japan Environment and Children's Study (JECS).•The NEO and NEOm concentrations in 100 µl urine samples were analysed using a highly sensitive LC–MSMS.•Automated solid phase extraction in a 96-well plate was utilised to achieve high-throughput analysis.•Intermediate precision and accuracy were less than 12.5% and 94.8–99.1%, respectively.

New Synthetic Cannabinoid ADB-BUTINACA-Related Death of a Police Dog.

The popularity of synthetic cannabinoids puts police sniffer dogs at risk of accidental introduction of such substances into the body. The extreme efficacy and potency of many new synthetic cannabinoids are associated with a high risk of serious poisonings and even deaths. The paper presents the toxicological findings in an intoxication of a police dog, in which a new synthetic cannabinoid ADB-BUTINACA was detected and quantified in postmortem materials. The screening analyses were performed by liquid chromatography with tandem mass spectrometry (LC-MS-MS) and liquid chromatography--quadrupole/time-of-flight mass spectrometry (LC-QTOF-MS). LC-MS-MS was also used for quantitative analyses, while LC-QTOF-MS for metabolite identification. Due to unusual matrices, the standard addition method was used for the quantitative determination of ADB-BUTINACA. The determined concentrations of ADB-BUTINACA in blood, lung, stomach, liver and kidney were 8.1 ng/mL, 6.4 ng/g, 1.5 ng/g, 1.8 ng/g and 0.4 ng/g, respectively. Apart from ADB-BUTINACA, the monohydroxylated metabolites and the dihydrodiol metabolite were detected and identified in all analyzed materials, and moreover the product of N-debutylation was found in blood and liver. The described case presents the identification and quantitation of a new synthetic cannabinoid ADB-BUTINACA in postmortem dog specimens. Although the cause of death was acute gastric dilatation, it cannot be ruled out that this process was the result of synthetic cannabinoid inhalation. Due to dogs' sensitivity to cannabinoids, ADB-BUTINACA poisoning cannot be excluded either. The described case suggests that ADB-BUTINACA elicits serious adverse effects in dogs. The article also indicates the dangers to which police dogs coming into contact with extremely potent drugs may be exposed.

Fingerprints: A New Specimen for Innovative Applications for the Detection of Xenobiotics.

Fingerprints are invisible traces that result from a deposition of sweat and sebum present on the papillary ridges. As sweat and sebum contain drugs, fingerprints are promising since collection is rapid, non-invasive and difficult to falsify. Very limited data are available in the literature, and therefore, it seems opportune to study the transfer of xenobiotics onto the items taken in hand via the fingerprints. Two studies were implemented using the ballpoint pen as a model. The objective of the first study was to compare the nicotine concentrations found on the pens of three smokers and three non-smokers. Five pens, belonging to each subject and used regularly, were rubbed with a cotton swab dipped in methanol and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). The second study was to analyze the transfer via fingerprints of four volunteers, after administration of 30 mg of codeine. The objective was to determine the feasibility of this study and the time corresponding to the highest concentration of codeine. Over a 24-h period, new pens were handled for 5 min by the four volunteers, rubbed with a cotton swab dipped in methanol, and then analyzed by LC-MS-MS. The nicotine study showed a major difference between the nicotine concentrations obtained from smokers (between 6 and 276 ng/pen) and non-smokers (between 2 and 4 ng/pen). After administration of 30 mg of codeine, the analysis of the pens of the four volunteers allowed to demonstrate the presence of codeine up to 24 h between 9 and 544 pg/pen. Normal hygiene practices did not influence the final result. The highest concentration was observed after 2 h. Morphine was also detected (between 19 and 33 pg/pen). These preliminary results should be considered a demonstration of the interest of fingerprints testing to document drug exposure.

Ommochrome Wing Pigments in the Monarch Butterfly Danaus plexippus (Lepidoptera: Nymphalidae).

Monarch butterflies (Danaus plexippus) use bright orange coloration to warn off predators as well as for sexual selection. Surprisingly the underlying pigment compounds have not been previously characterized. We used LCMS and fragmentation MS (including MSMS and MSn) of extracted pigments from nonmigratory summer-generation female monarch forewings to identify and provide relative quantitation of various orange pigments from D. plexippus. We observed seven ommochrome pigments, with xanthommatin and decarboxylated xanthommatin being the most abundant followed by xanthommatin methyl ester. Among the seven pigments, we also observed molecules that correspond to deaminated forms of these three amine-containing pigments. To the best of our knowledge, these deaminated compounds have not been previously discovered. A seventh pigment that we observed was α-hydroxyxanthommatin methyl ester, previously described in other nymphalid butterflies. We also show that chemical reduction of pigment extracts results in a change of their color from yellow to red, concomitant with the appearance of dihydro-xanthommatin and similarly reduced forms of the other pigment compounds. These findings indicate that monarchs may employ differences in the redox states of these pigments in order to achieve different hues of orange.

Multidimensional Chromatography and Its Applications in Food Products, Biological Samples and Toxin Products: A Comprehensive Review

Food, drugs, dyes, extracts, and minerals are all made up of complex elements, and utilizing unidimensional chromatography to separate them is inefficient and insensitive. This has sparked the invention of several linked chromatography methods, each of them with distinct separation principles and affinity for the analyte of interest. Multidimensional chromatography consists of the combination of multiple chromatography techniques, with great benefits at the level of efficiency, peak capacity, precision, and accuracy of the analysis, while reducing the time required for the analysis. Various coupled chromatography techniques have recently emerged, including liquid chromatography–gas chromatography (LC–GC), gas chromatography–gas chromatography (GC–GC), liquid chromatography–liquid chromatography (LC–LC), GCMS–MS, LCMS–MS, supercritical fluid techniques with chromatography techniques, and electro-driven multidimensional separation techniques. In this paper, the different coupled chromatography techniques will be discussed, along with their wide spectrum of applications for food, flavor, and environmental analysis, as well as their usefulness for the pharmaceutical, color, and dyes industries.

QBD APPROACH IN HPLC METHOD DEVELOPMENT AND VALIDATION OF TAMOXIFEN

The number of new drugs introduced into the market is increasing day by day. These drugs may be either new entities or partial structural modification of the existing one. Very often there is a time lag from the date of introduction of a drug into the market to the date of its inclusion in pharmacopoeias. This happens because of the possible uncertainties in the continuous and wider usage of these drugs, reports of new toxicities (resulting in their withdrawal from the market), development of patient resistance and introduction of better drugs by competitors. Under these conditions, standards and analytical procedures for these drugs may not be available in the pharmacopoeias. It becomes necessary, therefore to develop newer analytical methods for such drugs. Using QbD, pharmaceutical quality is assured by understanding and controlling formulation and manufacturing variables, degradation products and related impurities in bulk drugs, pharmaceutical formulations and biological samples such as whole blood, plasma, serum and urine. Instances of these include estimation of tamoxifen through the HPLC method with UV detector (48 -50), florescence detector (51), diode array detector (52.53), photodiode array detector (54), UPLC-ESIQ-MS (55), UPLC–MS-MS (56), LC–MS-MS method (57-60), etc.

Analysis of Mitragynine and Speciociliatine in Umbilical Cord by LC-MS-MS for Detecting Prenatal Exposure to Kratom.

Kratom is an herbal drug that is legal in the USA. While it is marketed as a safer alternative to opioids, it can cause opioid-like withdrawal symptoms when discontinued after regular use. Several case studies have shown that kratom exposure in utero can lead to symptoms in newborns consistent with neonatal abstinence syndrome. Here, we present a validated method for the detection of kratom in umbilical cord by liquid chromatography--tandem mass spectrometry. The umbilical cord is homogenized in solvent and kratom analytes are purified by solid phase extraction (strong cation exchange). Diastereomeric kratom alkaloids mitragynine (MG), speciociliatine (SC), speciogynine and mitraciliatine are separated by reverse phase chromatography on a phenyl-hexyl column. Applying this method to residual umbilical cords submitted to our laboratory for drug testing, 29 positive specimens exhibiting varied kratom analyte distributions were observed. MG and SC were the most abundant kratom analytes and were selected as biomarkers of kratom exposure. A cutoff concentration of 0.08 ng/g was established for both MG and SC.

Evaluation of Alcohol Markers in Urine and Oral Fluid after Regular and Hard Kombucha Consumption.

Although kombucha is a popular fermented beverage, the presence of alcohol markers has not been well studied despite being potential indicators of unintentional impairment. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) were measured in oral fluid and urine collected after consumption of regular or hard kombucha. Participants drank within 20 min and provided all urine voids for 12 h, the first urine voids on days 2 and 3 and oral fluid specimens at fixed time points for 48 h. Screening employed liquid chromatography-tandem mass spectrometry (LC-MS-MS; EtS, 25 ng/mL cutoff [oral]; 100 ng/mL cutoff [urine]; EtG, 500 ng/mL cutoff [urine] and immunoassay (IA; EtG, 500 ng/mL cutoff [urine]). After consuming regular kombucha (n = 12 participants), EtS was not detected in oral fluid but both markers were detected by LC-MS-MS in urine specimens within the first five voids from 83% of participants with median (range) concentrations of 240 (100-3,700) ng/mL for EtS and 830 (530-2,200) ng/mL for EtG. Neither marker was positive by IA nor LC-MS-MS after day 1. After consuming hard kombucha (n = 7 participants), 2 (2.8%) of the 70 collected oral fluid specimens tested positive for EtS 3 h after consumption; however, 21 (30%) had EtS levels above the limit of detection (LOD, 10 ng/mL) after 0.5-8 h. Both markers were detected in urine specimens from all participants with median (range) concentrations of 3,381 (559-70,250) ng/mL for EtS and 763 (104-12,864) ng/mL For EtG. Urine specimens were negative for EtG and EtS by the end of the 48-hour study.

Toxicological Investigation of a Case Series Involving the Synthetic Cathinone α-Pyrrolidinohexiophenone (α-PHP) and Identification of Phase I and II Metabolites in Human Urine.

α-Pyrrolidinohexiophenone (α-PHP) is a derivative of the class of α-pyrrolidinophenones, a subgroup of synthetic cathinones. These substances are the second most abused drugs of new psychoactive substances. Here, we report the toxicological investigation of a series of 29 authentic forensic and clinical cases with analytically confirmed intake of α-PHP including two cases of drug testing in newborns using meconium. The age range of subjects where serum samples were available was 23-51 years (median 39.5), and 90% were male. Serum α-PHP concentrations, determined by a validated LC-MS-MS method, showed a high variability ranging from 1 to 83 ng/mL (mean, 40 ng/mL; median, 36 ng/mL). Comprehensive toxicological analysis revealed co-consumption of other psychotropic drugs in almost all cases with frequent occurrence of opiates (60%), benzodiazepines (35%), cannabinoids (30%), and cocaine (20%). Hence, forensic and clinical symptoms like aggressive behavior, sweating, delayed physical response, and impaired balance could not be explained by the abuse of α-PHP alone but rather by poly-intoxications. Liquid chromatography-quadrupole time-of-flight mass spectrometry and gas chromatography-mass spectrometry were used to investigate the metabolism of α-PHP in vivo using authentic human urine samples. Altogether, 11 phase I metabolites and 5 phase II glucuronides could be identified by this approach. Apart from the parent drug, most abundant findings in urine were the metabolites dihydroxy-pyrrolidinyl-α-PHP and dihydro-α-PHP and, to a lesser extent, 2'-oxo-dihydro-α-PHP and 2'-oxo-α-PHP. Monitoring of these metabolites along with the parent drug in forensic and clinical toxicology could unambiguously prove the abuse of the novel designer drug α-PHP.

The Origin and Implications of Artifact Ions in Bioanalytical LC–MS

Liquid chromatography–mass spectrometry (LC–MS) with electrospray ionization (ESI) is a widely used bioanalytical technique with both qualitative and quantitative applications. Ions are created by electrically charging a stream of droplets from the LC system, which evaporate and leave ions that are transferred to the mass spectrometer. Ideally, these are only from the analyte, but background ions, such as metals, impurities and coeluted species, can react with analytes producing adducts, such as [M + Na]+, [M + K]+, and multimers (2M + H+, 3M + H+, and so forth). Although well known, the extent of adduct ion formation and the implications for quantitative analysis and analyte characterization by tandem MS (MS/MS) are not fully appreciated. We summarize the problem and identify areas that should be considered when developing or using electrospray LC–MS.

Urinary Excretion Profile of Cannabinoid Analytes Following Acute Administration of Oral and Vaporized Cannabis in Infrequent Cannabis Users.

Traditionally, smoking has been the predominant method for administering cannabis, but alternative routes of administration have become more prevalent. Additionally, research examining urinary cannabinoid excretion profiles has primarily focused on 11-nor-9-carboxy-∆9-tetrahydrocannabinol (∆9-THC-COOH), a metabolite of ∆9-tetrahydrocannabinol (∆9-THC), as the primary analyte. The aim of the current study was to characterize the urinary excretion profile of ∆9-THC-COOH, ∆9-THC, ∆8-tetrahydrocannabinol (∆8-THC), 11-hydroxy-∆9-tetrahydrocannabinol (11-OH-∆9-THC), ∆9-tetrahydrocannabivarin (THCV), 11-nor-∆9-tetrahydrocannabivarin-9-carboxlic acid (THCV-COOH), cannabidiol (CBD), cannabinol (CBN) and 8,11-dihydroxytetrahydrocannabinol (8,11-diOH-∆9-THC) following controlled administration of both oral and vaporized cannabis. Participants (n = 21, 11 men/10 women) who were infrequent cannabis users ingested cannabis-containing brownies (0, 10 and 25 mg ∆9-THC) and inhaled vaporized cannabis (0, 5 and 20 mg ∆9-THC) across six double-blind outpatient sessions. Urinary concentrations of ∆9-THC analytes were measured at baseline and for 8 h after cannabis administration. Sensitivity, specificity and agreement between the three immunoassays (IAs) for ∆9-THC-COOH (cutoffs of 20, 50 and 100 ng/mL) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analyses (confirmatory cutoff concentrations of 15 ng/mL) were assessed. Urinary concentrations for ∆9-THC-COOH, ∆9-THC, 11-OH-∆9-THC, THCV, CBN and 8,11-diOH-∆9-THC all peaked at 5-6 h and 4 h following oral and vaporized cannabis administration, respectively. At each active dose, median maximum concentrations (Cmax) for detected analytes were quantitatively higher after oral cannabis administration compared to vaporized. Using current recommended federal workplace drug-testing criteria (screening via IA with a cutoff of ≥50 ng/mL and confirmation via LC-MS-MS at a cutoff of ≥15 ng/mL), urine specimens tested positive for ∆9-THC-COOH in 97.6% of oral sessions and 59.5% of vaporized sessions with active ∆9-THC doses. These data indicate that while ∆9-THC-COOH may serve as the most consistent confirmatory analyte under the current drug-testing guidelines, future work examining 11-OH-∆9-THC under similar parameters could yield an alternative analyte that may be helpful in distinguishing between licit and illicit cannabis products.

Identification of Proteases and Protease Inhibitors in Seeds of the Recalcitrant Forest Tree Species Quercus ilex.

Proteases and protease inhibitors have been identified in the recalcitrant species Quercus ilex using in silico and wet methods, with focus on those present in seeds during germination. In silico analyses showed that the Q. ilex transcriptome database contained 2,240 and 97 transcripts annotated as proteases and protease inhibitors, respectively. They belonged to the different families according to MEROPS,1 being the serine and metallo ones the most represented. The data were compared with those previously reported for other Quercus species, including Q. suber, Q. lobata, and Q. robur. Changes in proteases and protease inhibitors alongside seed germination in cotyledon and embryo axis tissues were assessed using proteomics and in vitro and in gel activity assays. Shotgun (LC–MSMS) analysis of embryo axes and cotyledons in nonviable (NV), mature (T1) and germinated (T3) seeds allowed the identification of 177 proteases and 12 protease inhibitors, mostly represented by serine and metallo types. Total protease activity, as determined by in vitro assays using azocasein as substrate, was higher in cotyledons than in embryo axes. There were not differences in activity among cotyledon samples, while embryo axis peaked at germinated T4 stage. Gel assays revealed the presence of protease activities in at least 10 resolved bands, in the Mr range of 60–260 kDa, being some of them common to cotyledons and embryo axes in either nonviable, mature, and germinated seeds. Bands showing quantitative or qualitative changes upon germination were observed in embryo axes but not in cotyledons at Mr values of 60–140 kDa. Proteomics shotgun analysis of the 10 bands with protease activity supported the results obtained in the overall proteome analysis, with 227 proteases and 3 protease inhibitors identified mostly represented by the serine, cysteine, and metallo families. The combined use of shotgun proteomics and protease activity measurements allowed the identification of tissue-specific (e.g., cysteine protease inhibitors in embryo axes of mature acorns) and stage-specific proteins (e.g., those associated with mobilization of storage proteins accumulated in T3 stage). Those proteins showing differences between nonviable and viable seeds could be related to viability, and those variables between mature and germinated could be associated with the germination process. These differences are observed mostly in embryo axes but not in cotyledons. Among them, those implicated in mobilization of reserve proteins, such as the cathepsin H cysteine protease and Clp proteases, and also the large number of subunits of the CNS and 26S proteasome complex differentially identified in embryos of the several stages suggests that protein degradation via CNS/26S plays a major role early in germination. Conversely, aspartic proteases such as nepenthesins were exclusively identified in NV seeds, so their presence could be used as indicator of nonviability.

Fast and Sensitive Method for the Determination of 17 Designer Benzodiazepines in Hair by Liquid Chromatography-Tandem Mass Spectrometry.

In recent years, identification and analysis of designer benzodiazepines have become a challenge in forensic toxicology. These substances are analogs of the classic benzodiazepines, but their pharmacology is not well known, and many of them have been associated with overdoses and deaths. As a result, there has been a surge in efforts to develop analytical methods to determine these compounds in different biological samples. Our aim was to develop and validate a fast, sensitive and specific method for determining 17 designer benzodiazepines (adinazolam, clobazam, clonazolam, delorazepam, deschloroetizolam, diclazepam, etizolam, flualprazolam, flubromazepam, flubromazolam, flunitrazolam, N-desmethylclobazam, nifoxipam, nitrazolam, meclonazepam, pyrazolam and zolazepam) in hair by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Hair samples were decontaminated and pulverized, and a 20 mg aliquot was incubated in methanol in an ultrasound bath (1 h, 25°C). The supernatant was evaporated and reconstituted in 200 µL of mobile phase, and the extracts were filtered (nano-filter vials) before injection into LC-MS-MS. All analytes were eluted from the chromatographic column in 8 min, and two multiple-reaction monitoring (MRM) transitions were used to identify each compound. The limits of quantification were 5 or 25 pg/mg depending on the analyte, and the calibration functions were linear to 200 pg/mg. Imprecision was <19.2% (n = 15), and bias was from -13.7 to 18.3% (n = 15). All the analytes yielded high extraction efficiencies >70% and displayed ion suppression between -62.8% and -23.9% (n = 10). The method was applied to 19 authentic cases. Five samples were positive for flualprazolam (<LOQ-> 200 pg/mg) and/or etizolam (47.4-88.5 pg/mg). In conclusion, the present validated method has proven to be fast, sensitive, specific and capable of determining 17 designer benzodiazepines in hair using LC-MS-MS.