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Acetic acid bacteria (AAB) and other members of the complex microbiotas, whose activity is essential for vinegar production, display biodiversity and richness that is difficult to study in depth due to their highly selective culture conditions. In recent years, liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) has emerged as a powerful tool for rapidly identifying thousands of proteins present in microbial communities, offering broader precision and coverage. In this work, a novel method based on LC–MS/MS was established and developed from previous studies. This methodology was tested in three studies, enabling the characterization of three submerged acetification profiles using innovative raw materials (synthetic alcohol medium, fine wine, and craft beer) while working in a semicontinuous mode. The biodiversity of existing microorganisms was clarified, and both the predominant taxa (Komagataeibacter, Acetobacter, Gluconacetobacter, and Gluconobacter) and others never detected in these media (Asaia and Bombella, among others) were identified. The key functions and adaptive metabolic strategies were determined using comparative studies, mainly those related to cellular material biosynthesis, energy-associated pathways, and cellular detoxification processes. This study provides the groundwork for a highly reliable and reproducible method for the characterization of microbial profiles in the vinegar industry. [ABSTRACT FROM AUTHOR]

Sulfite, a widely used food additive, is subject to regulated labeling. The extraction of sulfite as the stable hydroxymethylsulfonate (HMS) form and its quantitative analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been recognized for their good sensitivity, selectivity, and versatility across various food materials. This study aimed to develop a cost-effective and simpler method for sulfite quantitation, while maintaining the superior sensitivity and selectivity of mass spectrometry (MS). To achieve this, we introduced paper spray ionization (PSI), an ambient desorption ionization technique that could achieve the direct measurement of analytes without employing separation. We also employed a novel internal standard (IS) structurally similar to the analyte, replacing the more expensive isotopically labeled IS. Although the PSI-MS/MS method developed in this study exhibited slightly lower analytical performance compared to the conventional LC-MS/MS, it remained effective for sulfite analysis in dried fruits. [ABSTRACT FROM AUTHOR]

Flavonoids are important secondary metabolites found in Juglans mandshurica Maxim., which is a precious reservoir of bioactive substances in China. To explore the antitumor actions of flavonoids (JMFs) from the waste branches of J. mandshurica, the following optimized purification parameters of JMFs by macroporous resins were first obtained. The loading concentration, flow rate, and loading volume of raw flavonoid extracts were 1.4 mg/mL, 2.4 BV/h, and 5 BV, respectively, and for desorption, 60% ethanol (4 BV) was selected to elute JMFs-loaded AB-8 resin at a flow rate of 2.4 BV/h. This adsorption behavior can be explained by the pseudo-second-order kinetic model and Langmuir isotherm model. Subsequently, JMFs were identified using Fourier transform infrared combined with high-performance liquid chromatography and tandem mass spectrometry, and a total of 156 flavonoids were identified. Furthermore, the inhibitory potential of JMFs on the proliferation, migration, and invasion of HepG2 cells was demonstrated. The results also show that exposure to JMFs induced apoptotic cell death, which might be associated with extrinsic and intrinsic pathways. Additionally, flow cytometry detection found that JMFs exposure triggered S phase arrest and the generation of reactive oxygen species in HepG2 cells. These findings suggest that the JMFs purified in this study represent great potential for the treatment of liver cancer. [ABSTRACT FROM AUTHOR]

Bopyeo-tang (BPT) is composed of six medicinal herbs (Morus alba L., Rehmannia glutinosa (Gaertn.) DC., Panax ginseng C.A.Mey., Aster tataricus L.f., Astragalus propinquus Schischkin, and Schisandra chinensis (Turcz.) Baill.) and has been used for the treatment of lung diseases. This study focused on establishing an analytical method that can simultaneously quantify nine target compounds (i.e., hydroxymethylfurfural, mulberroside A, chlorogenic acid, calycosin-7-O-glucoside, 3,5-dicaffeoylquinic acid, quercetin, kaempferol, schizandrin, and gomisin A) from a BPT sample using high-performance liquid chromatography with a photodiode array detector (HPLC–PDA) and ultra-performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS). The separation of compounds in both analyses was performed on a C18 reversed-phase column using the gradient elution of water–acetonitrile as the mobile phase. In particular, the multiple reaction monitoring mode was applied for quick and accurate detection in UPLC–MS/MS analysis. As a result of analyzing the two methods, HPLC–PDA and UPLC–MS/MS, the coefficient of determination of the regression equation for each compound was ≥0.9952, and recovery was 85.99−106.40% (relative standard deviation (RSD) < 9.58%). Precision testing of the nine compounds was verified (RSD < 10.0%). The application of these analytical assays under optimized conditions for quantitative analysis of the BPT sample gave 0.01–4.70 mg/g. Therefore, these two assays could be used successfully to gather basic data for clinical research and the quality control of BPT. [ABSTRACT FROM AUTHOR]

The ultrasonic-assisted extraction (UAE) method was employed to separate Cinnamomum camphora proanthocyanidin-rich extracts (PCEs). This extraction process was optimized by the Box–Behnken design, and the optimal conditions, on a laboratory scale, were as follows: an ethanol concentration of 75%, a liquid-to-solid ratio of 24 mL/g, an ultrasonic time of 39 min, and an ultrasonic power of 540 W. Under the obtained conditions, the PCE yield extracted by UAE was higher than that from heat reflux extraction and soaking extraction. An ultra-performance liquid chromatography–tandem mass spectrometry analysis was employed to characterize the phloroglucinolysis products of the C. camphora PCEs, by which epigallocatechin, catechin, epicatechin, and (−)-epigallocatechin-3-O-gallate were identified as the terminal units; epigallocatechin, epicatechin, and (−)-epigallocatechin-3-O-gallate were recognized as extension units. The C. camphora PCEs possessed higher anti-ultraviolet activity in vitro compared with the commercially available sunscreen additive of benzophenone with respect to their ethanol solutions (sun protection factor of 27.01 ± 0.68 versus 1.96 ± 0.07 at a concentration of 0.09 mg/mL) and sunscreens (sun protection factor of 17.36 ± 0.62 versus 14.55 ± 0.47 at a concentration of 20%). These results demonstrate that C. camphora PCEs possess an excellent ultraviolet-protection ability and are promising green sunscreen additives that can replace commercial additives. [ABSTRACT FROM AUTHOR]

Despite the considerable steps taken in the last decade in the context of antineoplastic drug (AD) handling procedures, their mutagenic effect still poses a threat to healthcare personnel actively involved in compounding and administration units. Biological monitoring procedures usually require large volumes of sample and extraction solvents, or do not provide adequate sensitivity. It is here proposed a fast and automated method to evaluate the urinary levels of cyclophosphamide and iphosphamide, composed of a miniaturized solid phase extraction (µSPE) followed by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The extraction procedure, developed through design of experiments (DoE) on the ePrep One Workstation, required a total time of 9.5 min per sample, with recoveries of 77–79% and a solvent consumption lower than 1.5 mL per 1 mL of urine sample. Thanks to the UHPLC-MS/MS method, the limits of quantification (LOQ) obtained were lower than 10 pg/mL. The analytical procedure was successfully applied to 23 urine samples from compounding wards of four Italian hospitals, which resulted in contaminations between 27 and 182 pg/mL. [ABSTRACT FROM AUTHOR]

This report presents the development of a highly effective method employing high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) to investigate chlorantraniliprole's dissipation, risk assessment, and residue distribution in whole lychee fruit and its pulp. Mean recoveries of the samples ranged from 80 to 105%, exhibiting a relative standard deviation (RSD) of below 8%. The limits of quantification (LOQ) for lychee and pulp were determined as 0.001 mg/kg, and half-lives (t1/2) ranged from 8.0 to 12.2 days. Terminal residue concentrations in whole litchi and pulp were determined as 0.008–0.45 mg/kg and ≤0.001 mg/kg. These residues were treated twice and thrice at two different dosage levels with pre-harvest intervals (PHIs) of 7, 14, and 21 days. The potential chronic risk posed by chlorantraniliprole to humans was non-negligible, as indicated by the risk quotient (RQ) value not exceeding 1. Therefore, this study provides significant fresh data about the safe application of chlorantraniliprole in the production of lychee, which will help China develop maximum residual limits (MRLs). [ABSTRACT FROM AUTHOR]

β-casein, a protein in milk and dairy products, has two main variant forms termed as A1 and A2. A1 β-casein may have adverse effects on humans. The fact that there is only one amino acid variation at the 67th position between A1 and A2 β-casein makes it difficult to distinguish between them. In this study, a novel method using characteristic thermolytic peptides is developed for the determination of A1 and A2 β-casein in milk. Firstly, caseins extracted from milk samples are thermolytic digested at 60 °C without any denaturing reagents required for unfolding proteins, which simplifies the sample pretreatment procedure. The characteristic thermolytic peptides (i.e., fragments 66–76 and 59–76 for A1 and A2 β-casein, respectively) selected to specifically distinguish A1 and A2 β-casein only have eleven or eighteen amino acid moieties. Compared with tryptic characteristic peptides with a length of 49 amino acid moieties, these shorter thermolytic characteristic peptides are more suitable for LC-MS analysis. This novel method, with the advantages of high specificity, high sensitivity, and high efficiency, was successfully applied for the analysis of six milk samples collected from a local supermarket. After further investigation, it is found that this method would contribute to the development of A2 dairy products for a company and the quality inspection of A2 dairy products for a government. [ABSTRACT FROM AUTHOR]

The large-scale use of sulfonamide antimicrobials in human and veterinary medicine has seriously endangered the ecological environment and human health. The objective of this study was to develop and validate a simple and robust method for the simultaneous determination of seventeen sulfonamides in water using ultra-high performance liquid chromatography–tandem mass spectrometry coupled with fully automated solid-phase extraction. Seventeen isotope-labeled internal standards for sulfonamides were used to correct matrix effects. Several parameters affecting extraction efficiency were systematically optimized, and the enrichment factors were up to 982−1033 and only requiring about 60 min per six samples. Under the optimized conditions, this method manifested good linearity (0.05–100 μg/L), high sensitivity (detection limits: 0.01–0.05 ng/L), and satisfactory recoveries (79–118%) with acceptable relative standard deviations (0.3–14.5%, n = 5). The developed method can be successfully utilized for the determination of 17 sulfonamides in pure water, tap water, river water, and seawater. In total, six and seven sulfonamides were detected in river water and seawater, respectively, with a total concentration of 8.157–29.676 ng/L and 1.683–36.955 ng/L, respectively, and sulfamethoxazole was the predominant congener. [ABSTRACT FROM AUTHOR]

Herein, we used isotopic formaldehyde and sodium cyanoborohydride via reductive amination to label two methyl groups on primary amine to arrange the standards (h2-formaldehyde-modified) and internal standards (ISs, d2-formaldehyde-modified) of tryptophan and its metabolites, such as serotonin (5-hydroxytryptamine) and 5-hydroxytryptophan. These derivatized reactions with a high yield are very satisfactory for manufacturing standards and ISs. This strategy will generate one or two methyl groups on amine to create different mass unit shifts with 14 vs. 16 or 28 vs. 32 in individual compounds for biomolecules with amine groups. In other words, multiples of two mass units shift are created using this derivatized method with isotopic formaldehyde. Serotonin, 5-hydroxytryptophan, and tryptophan were used as examples to demonstrate isotopic formaldehyde-generating standards and ISs. h2-formaldehyde-modified serotonin, 5-hydroxytryptophan, and tryptophan are standards to construct calibration curves, and d2-formaldehyde-modified analogs such as ISs spike into samples to normalize the signal of each detection. We utilized multiple reaction monitoring modes and triple quadrupole mass spectrometry to demonstrate the derivatized method suitable for these three nervous biomolecules. The derivatized method demonstrated a linearity range of the coefficient of determinations between 0.9938 to 0.9969. The limits of detection and quantification ranged from 1.39 to 15.36 ng/mL. [ABSTRACT FROM AUTHOR]

Pentacyclic triterpenoids (PCTs), which possess a number of bioactive properties, are considered one of the most important classes of secondary plant metabolites. Their chromatographic determination in plant biomass is complicated by the need to separate a large number of structurally similar compounds belonging to several classes that differ greatly in polarity (monools, diols, and triterpenic acids). This study proposes a rapid, sensitive, and low-cost method for the simultaneous quantification of ten PCTs (3β-taraxerol, lupeol, β-amyrin, α-amyrin, betulin, erythrodiol, uvaol, betulinic, oleanolic, and ursolic acids) by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) using porous graphitic carbon (Hypercarb) as a stationary phase capable of hydrophobic retention and specific interactions with analytes. Revealing the effects of the mobile phase composition, pH, ionic strength, and column temperature on retention and selection of chromatographic conditions on this basis allowed for the effective separation of all target analytes within 8 min in gradient elution mode and attaining limits of detection in the range of 4–104 µg L−1. The developed method was fully validated and successfully tested in the determination of PCTs in common haircap (Polytrichum commune) and prairie sphagnum (Sphagnum palustre) mosses, and fireweed (Chamaenerion angustifolium) stems and leaves. [ABSTRACT FROM AUTHOR]

Large molecule protein therapeutics have steadily grown and now represent a significant portion of the overall pharmaceutical market. These complex therapies are commonly manufactured using cell culture technology. Sequence variants (SVs) are undesired minor variants that may arise from the cell culture biomanufacturing process that can potentially affect the safety and efficacy of a protein therapeutic. SVs have unintended amino acid substitutions and can come from genetic mutations or translation errors. These SVs can either be detected using genetic screening methods or by mass spectrometry (MS). Recent advances in Next-generation Sequencing (NGS) technology have made genetic testing cheaper, faster, and more convenient compared to time-consuming low-resolution tandem MS and Mascot Error Tolerant Search (ETS)-based workflows which often require ~6 to 8 weeks data turnaround time. However, NGS still cannot detect non-genetic derived SVs while MS analysis can do both. Here, we report a highly efficient Sequence Variant Analysis (SVA) workflow using high-resolution MS and tandem mass spectrometry combined with improved software to greatly reduce the time and resource cost associated with MS SVA workflows. Method development was performed to optimize the high-resolution tandem MS and software score cutoff for both SV identification and quantitation. We discovered that a feature of the Fusion Lumos caused significant relative under-quantitation of low-level peptides and turned it off. A comparison of common Orbitrap platforms showed that similar quantitation values were obtained on a spiked-in sample. With this new workflow, the amount of false positive SVs was decreased by up to 93%, and SVA turnaround time by LC-MS/MS was shortened to 2 weeks, comparable to NGS analysis speed and making LC-MS/MS the top choice for SVA workflow. [ABSTRACT FROM AUTHOR]

The presence of pesticide residues in herbs and the herbal products derived from them raises serious health concerns. This study was conducted to investigate the residual pesticide concentrations and assess potential human health risks from herbal medicines used in traditional Korean medicine clinics. A total of 40 samples of herbal decoctions were collected from 10 external herbal dispensaries. The pesticide residues were analyzed by the multiresidue method for 320 different pesticides using liquid chromatography tandem mass spectrometry (LC-MS/MS) and gas chromatography tandem mass spectrometry (GC-MS/MS). As a result of the monitoring, carbendazim was detected at 0.01 and 0.03 μg/g in eight samples and no pesticide was detected in the other herbal decoctions. Carbendazim was set for each individual item as less than 0.05 μg/g in Paeoniae radix, less than 0.05 μg/g in Cassiae semen, less than 2.0 μg/g in Lycii fructus, and less than 10 μg/g in Schisandrae fructus (dried). Therefore, the results of this study suggested that the detected pesticide residues in herbal decoctions could not be considered as posing a serious health risk. [ABSTRACT FROM AUTHOR]

Alternaria fungi are widely distributed plant pathogens that invade crop products, causing significant economic damage. In addition, toxic secondary metabolites produced by the fungi can also endanger consumers. Many of these secondary metabolites are chemically characterized as mycotoxins. In this study, Q Exactive Orbitrap mass spectrometry was used for the non-targeted analysis of the metabolome of seven Alternaria isolates cultured on Potato Carrot Agar (PCA), Potato Dextrose Agar (PDA) and Potato Sucrose Agar (PSA) medium. Due to the difficulty of detecting modified toxins, an analytical strategy with multiple visual analysis tools was also used to determine the presence of sulfate conjugated toxins, as well as to visualize the molecular network of Alternaria toxins. The results show that PSA medium exhibits more advantageous properties for the culture of Alternaria, with more toxigenic species and quantities and more obvious metabolic pathways. Based on high-resolution tandem mass spectrometry (MS/MS) data, the mycotoxins and their metabolites were mainly clustered into four groups: alternariol (AOH)/alternariol monomethyl ether (AME)/altenusin (ALU)/altenuene (ALT)/dehydroaltenusin (DHA)/Desmethyldehydroaltenusin (DMDA) families, Altertoxin-I (ATX-I) family, tentoxin (TEN) family and tenuazonic acid (TeA) family. Moreover, the PSA medium is more suitable for the accumulation of AOH, AME, ALU, ALT, DHA and DMDA, while the PDA medium is more suitable for the accumulation of ATX-I, TEN and TeA. This research may provide theoretical support for the metabolomics study of Alternaria. [ABSTRACT FROM AUTHOR]

Eleusine indica (L.) Gaertn is a perennial herb belonging to the Poaceae family. As the only species of Eleusine found abundantly in Malaysia, it is locally known as "rumput sambau" and has been traditionally used to treat various ailments including pain relief from vaginal bleeding, hastening the placenta delivery after childbirth, asthma, hemorrhoids, urinary infection, fever, and as a tonic for flu-related symptoms. A diverse array of biological activities have been reported for the plant, such as antimicrobial, cytotoxic, anticonvulsant, anti-inflammatory, analgesic, antipyretic, and hepatoprotective action. Despite many reports on its traditional uses and biological activities, limited chemical databases are available for the plant. Thus, the aims of this study were to annotate and identify the phytochemical constituents in the methanolic extract of E. indica through tandem LCMS-based analysis techniques using MZmine, GNPS, Compound Discoverer, and SIRIUS platforms. This technique managed to identify a total of 65 phytochemicals in the extract, comprising primary and secondary metabolites, and was verified by the isolation of one of the identified phytochemicals. The structural elucidation mainly using 1D and 2D NMR as well as comparison with values in the literature confirms the isolated phytochemical to be a 3-OH anomer of loliolide, a benzofuran-type of compound, which consequently increases the level of confidence in the applied technique. The research describes a useful method for the fast and simultaneous identification of phytochemicals in E. indica, contributing to the study of the chemical properties of the genus and family. [ABSTRACT FROM AUTHOR]

This study aimed to establish a simple and sensitive analytical method to simultaneously quantify donepezil (DPZ) and tadalafil (TAD) in rat plasma using lansoprazole (LPZ) as an internal standard (IS) by using liquid chromatography tandem mass spectrometry. The fragmentation pattern of DPZ, TAD, and IS was elucidated using multiple reaction monitoring in electrospray ionization positive ion mode for the quantification of precursor to production at m/z 380.1 → 91.2 for DPZ, m/z 390.2 → 268.1 for TAD, and m/z 370.3 → 252.0 for LPZ. The extracted DPZ and TAD from plasma using acetonitrile-induced protein precipitation was separated using Kinetex C18 (100 × 2.1 mm, 2.6 µm) column with a gradient mobile phase system consisting of 2 mM ammonium acetate and 0.1% formic acid in acetonitrile at a flow rate of 0.25 mL/min for 4 min. The selectivity, lower limit of quantification, linearity, precision, accuracy, stability, recovery, and matrix effect of this developed method was validated according to the guidelines of the U.S. Food and Drug Administration and the Ministry of Food and Drug Safety of Korea. The established method achieved acceptance criteria in all validation parameters, ensuring reliability, reproducibility, and accuracy, and was successfully implemented in a pharmacokinetic study on the co-administration of DPZ and TAD orally in rats. [ABSTRACT FROM AUTHOR]

The new direct oral anticoagulants (DOACs) are increasingly used to treat and prevent thromboembolic disorders, and monitoring concentrations may be valuable in some special scenarios to prevent clinical adverse events. This study aimed to develop generic methods for the rapid and simultaneous analysis of four DOACs in human plasma and urine. Protein precipitation and one-step dilution were used to prepare the plasma and urine; the extracts were injected to ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for analysis. Chromatographic separation was performed on an Acquity™ UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) with gradient elution of 7 min. A triple quadrupole tandem mass spectrometer with an electrospray ionization source was employed to analyze DOACs in a positive ion mode. The methods showed great linearity in the plasma (1~500 ng/mL) and urine (10~10,000 ng/mL) for all analytes (R2 ≥ 0.99). The intra- and inter-day precision and accuracy were within acceptance criteria. The matrix effect and extraction recovery were 86.5~97.5% and 93.5~104.7% in the plasma, while 97.0~101.9% and 85.1~99.5% in the urine. The stability of samples during the routine preparation and storage were within the acceptance criteria of less than ±15%. The methods developed were accurate, reliable, and simple for the rapid and simultaneous measurement of four DOACs in human plasma and urine, and successfully applied to patients and subjects with DOACs therapy for anticoagulant activity assessment. [ABSTRACT FROM AUTHOR]

Cyclodextrins are cyclic oligosaccharides that have received special attention due to their cavity-based structural architecture that imbues them with outstanding properties, primarily related to their capacity to host various guest molecules, from low-molecular-mass compounds to polymers. Cyclodextrin derivatization has been always accompanied by the development of characterization methods, able to unfold complicated structures with increasing precision. One of the important leaps forward is represented by mass spectrometry techniques with soft ionization, mainly matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). In this context, esterified cyclodextrins (ECDs) benefited also from the formidable input of structural knowledge, thus allowing the understanding of the structural impact of reaction parameters on the obtained products, especially for the ring-opening oligomerization of cyclic esters. The current review envisages the common mass spectrometry approaches such as direct MALDI MS or ESI MS analysis, hyphenated liquid chromatography-mass spectrometry, and tandem mass spectrometry, employed for unraveling the structural features and particular processes associated with ECDs. Thus, the accurate description of complex architectures, advances in the gas phase fragmentation processes, assessment of secondary reactions, and reaction kinetics are discussed in addition to typical molecular mass measurements. [ABSTRACT FROM AUTHOR]

Lupin alkaloids (LAs) represent a class of toxic secondary metabolites in plants, in particular in Lupinus spp.; they are produced as a defense mechanism due to their strong bitter taste and are very dangerous for human and animals. In this work, a sensitive and reliable high performance liquid chromatography—tandem mass spectrometry (HPLC-MS/MS) analytical method for the identification and quantification of thirteen lupin alkaloids was developed and validated according to FDA guidelines. Efficient extraction and clean-up steps, carried out by solid-phase extraction, were finely tuned on the basis of the characteristics of the analytes and lupin samples, providing good selectivity with minimized matrix interference. The effectiveness of the method was proven by the satisfactory recovery values obtained for most of the analytes and a matrix effect ≤23% for all tested levels. In addition, a sensitive and reliable determination of the target compounds was obtained; LOQs were between 1 and 25 µg Kg−1, i.e., below the requested maximum levels (<200 mg Kg−1). The method was applied to evaluate the LAs profile in different batches of raw L. albus L. samples, varying in size and across farming treatments. [ABSTRACT FROM AUTHOR]

Novel furoxan/coumarin hybrids were synthesized, and pharmacologic studies showed that the compounds displayed potent antiproliferation activities via downregulating both the phosphatidylinositide 3-kinase (PI3K) pathway and the mitogen-activated protein kinase (MAPK) pathway. To investigate the preclinical pharmacokinetic (PK) properties of three candidate compounds (CY-14S-4A83, CY-16S-4A43, and CY-16S-4A93), liquid chromatography, in tandem with the mass spectrometry LC-MS/MS method, was developed and validated for the simultaneous determination of these compounds. The absorption, distribution, metabolism, and excretion (ADME) properties were investigated in in vitro studies and in rats. Meanwhile, physiologically based pharmacokinetic (PBPK) models were constructed using only in vitro data to obtain detailed PK information. Good linearity was observed over the concentration range of 0.01–1.0 μg/mL. The free drug fraction (fu) values of the compounds were less than 3%, and the clearance (CL) values were 414.5 ± 145.7 mL/h/kg, 2624.6 ± 648.4 mL/h/kg, and 500.6 ± 195.2 mL/h/kg, respectively. The predicted peak plasma concentration (Cmax) and the area under the concentration-time curve (AUC) were overestimated for the CY-16S-4A43 PBPK model compared with the experimental ones (fold error > 2), suggesting that tissue accumulation and additional elimination pathways may exist. In conclusion, the LC-MS/MS method was successively applied in the preclinical PK studies, and the detailed information from PBPK modeling may improve decision-making in subsequent new drug development. [ABSTRACT FROM AUTHOR]

Natural products (NPs) have historically played a primary role in the discovery of small-molecule drugs. However, due to the advent of other methodologies and the drawbacks of NPs, the pharmaceutical industry has largely declined in interest regarding the screening of new drugs from NPs since 2000. There are many technical bottlenecks to quickly obtaining new bioactive NPs on a large scale, which has made NP-based drug discovery very time-consuming, and the first thorny problem faced by researchers is how to dereplicate NPs from crude extracts. Remarkably, with the rapid development of omics, analytical instrumentation, and artificial intelligence technology, in 2012, an efficient approach, known as tandem mass spectrometry (MS/MS)-based molecular networking (MN) analysis, was developed to avoid the rediscovery of known compounds from the complex natural mixtures. Then, in the past decade, based on the classical MN (CLMN), feature-based MN (FBMN), ion identity MN (IIMN), building blocks-based molecular network (BBMN), substructure-based MN (MS2LDA), and bioactivity-based MN (BMN) methods have been presented. In this paper, we review the basic principles, general workflow, and application examples of the methods mentioned above, to further the research and applications of these methods. [ABSTRACT FROM AUTHOR]

The incidence of colon cancer is increasing year over year, seriously affecting human health and quality of life in recent years. However, traditional Chinese medicine (TCM) has been utilized for the treatment of colon cancer. S. officinalis Saponins (S-Saponins), the potential compound of TCM, displays multiple biological activities in colon cancer treatment. In our study, ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) combined with multivariate statistical analysis were performed to analyze and identify raw and processed saponins. Then, MTT and cell migration assays were used to preliminarily explore the effects of saponins in vitro on colon cancer cells. The results showed that 29 differential saponins compounds under Paozhi were identified by UHPLC-MS/MS. Moreover, in vitro validation showed that Sprocessed better inhibited the proliferation and migration of colon cancer cells than Sraw. This study provides a basis for the determination of the chemical fundamentals of the efficacy changes during Paozhi through inferring the changes in saponin components and its possible transformation mechanisms before and after processing S. officinalis. Meanwhile, it also provides new insights into potential bioactive ingredients for the treatment of colon cancer. [ABSTRACT FROM AUTHOR]

Betulinic acid (BA) has been extensively studied in recent years mainly for its antiproliferative and antitumor effect in various types of cancers. Limited data are available regarding the pharmacokinetic profile of BA, particularly its metabolic transformation in vivo. In this study, we present the screening and structural investigations by ESI Orbitrap MS in the negative ion mode and CID MS/MS of phase I and phase II metabolites detected in mouse plasma after the intraperitoneal administration of a nanoemulsion containing BA in SKH 1 female mice. Obtained results indicate that the main phase I metabolic reactions that BA undergoes are monohydroxylation, dihydroxylation, oxidation and hydrogenation, while phase II reactions involved sulfation, glucuronidation and methylation. The fragmentation pathway for BA and its plasma metabolites were elucidated by sequencing of the precursor ions by CID MS MS experiments. [ABSTRACT FROM AUTHOR]

Hydroxylated polychlorinated biphenyls (OH-PCBs) are a group of metabolites biotransformed from polychlorinated biphenyls by animals with higher toxicities than their parent compounds. The present work developed and validated an analytical method for determinating penta-, hexa-, and hepta-chlorine substituted OH-PCBs in animal-derived food based on ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) with isotope-dilution. The target analytes were extracted with a 50% n-hexane/dichloromethane (v/v), purified by sulfuric acid-silica gel, and separated by 5% hydrated silica gel, achieving a final concentration of 100 times before injection to LC–MS/MS. The limits of detection (LOD) and quantification (LOQ) for target OH-PCBs were within the ranges of 0.003–0.010 μg/kg and 0.009–0.030 μg/kg, respectively. Average recoveries ranged between 76.7% and 116.5%, with relative standard deviations of less than 18.4%. The proposed method is simple, time-saving, sensitive, and accurate, making it a powerful tool for risk monitoring of OH-PCBs in animal-derived food. [ABSTRACT FROM AUTHOR]

This paper reports an important investigation and quantification of adulteration of sexual enhancement supplements with prescription medicines available in United Arab Emirates (UAE): tadalafil, sildenafil and vardenafil. A total of 158 sexual enhancement supplements were collected and analyzed in the current study. The samples were screened using REVERSE-phase liquid chromatography tandem high-resolution mass spectrometry/mass spectrometry (RP-HPLC-MS/MS). Of all sexual enhancements, 12.7% (95% CI: 7.4–18) contained undeclared sildenafil, 3.8% (95% CI: 0.78–6.81) contained undeclared tadalafil and 1.9% (95% CI: 0.25–4.05) contained undeclared vardenafil. Of all sexual enhancement supplements, 13.9% (95% CI: 8.5–19.4) contained significant concentrations of sildenafil, tadalafil or vardenafil. While the study found relatively low levels of undeclared pharmaceutical ingredients in the sexual enhancement dietary supplements available on the UAE market, it is likely that patients with ED tend to consume multiple such supplements daily, thereby exposing themselves to highly elevated cumulative levels. [ABSTRACT FROM AUTHOR]

The decoction turns into a complex multiphase system following exposure to high temperature and a complex chemical environment. However, the differences in the concentration of key active ingredients in different phase states and the release of drugs in sedimentary phase have yet to be elucidated. A simple ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous quantitative determination of brucine, strychnine, liquiritin, isoliquiritin, isoliquiritigenin and glycyrrhizic acid concentrations and it was applied to compare the content of different phases and measure the release characteristics of the sedimentary phase in "Glycyrrhiza glabra-Nux vomica" decoction (NGD). The results show that the method's selectivity, precision (intraday and interday ≤ 2%), matrix effect (101–108%), recovery and stability results were acceptable according to the guidelines. The method is sensitive and reliable. The content determination results show that the most toxic strychnine in the sedimentary phase accounted for 75.70% of the total components. The different components exhibited differential release in different media, and its components were released in the artificial intestinal fluid up to 81.02% in 12 h. Several components conformed to the primary kinetic model and the Ritger–Peppas model, and the most toxic compound exhibited slow release, thus conforming to the Ritger–Peppas model. This study provides a standard of reference for studies investigating reduction in toxicity of the combination of Glycyrrhiza glabra (Glycyrrhiza glabra L.) and Nux vomica (Strychnos nux-vomica L.). [ABSTRACT FROM AUTHOR]

A quantitative and qualitative method using a high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) detection approach was developed and validated for the analysis of tigecycline, four tetracyclines and their three 4-epimer derivatives in chicken muscle. Samples were extracted repeatedly with 0.1 mol/L Na2EDTA–McIlvaine buffer solution. After vortexing, centrifugation, solid-phase extraction, evaporation and reconstitution, the aliquots were separated using a C8 reversed-phase column (50 mm × 2.1 mm, 5 µm) with a binary solvent system consisting of methanol and 0.01 mol/L trichloroacetic acid aqueous solution. The typical validation parameters were evaluated in accordance with the acceptance criteria detailed in the guidelines of the EU Commission Decision 2002/657/EC and the U.S. Food and Drug Administration Bioanalytical Method Validation 05/24/18. The matrix-matched calibration curve was linear over the concentration range from the limit of quantitation (LOQ) to 400 μg/kg for doxycycline, and the calibration graphs for tetracycline, chlortetracycline, oxytetracycline, their 4-epimer derivatives and tigecycline showed a good linear relationship within the concentration range from the LOQ to 200 μg/kg. The limits of detection (LODs) for the eight targets were in the range of 0.06 to 0.09 μg/kg, and the recoveries from the fortified blank samples were in the range of 89% to 98%. The within-run precision and between-run precision, which were expressed as the relative standard deviations, were less than 5.0% and 6.9%, respectively. The applicability was successfully demonstrated through the determination of residues in 72 commercial chicken samples purchased from different sources. This approach provides a novel option for the detection of residues in animal-derived food safety monitoring. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop a method for the determination of glyphosate, its metabolite aminomethylphosphonic acid (AMPA), and glufosinate ammonium residues in beebread samples, which could then be used to assess bees' exposure to their residues. The complexity of beebread's matrix, combined with the specific properties of glyphosate itself, required careful selection and optimization of each analysis step. The use of molecularly imprinted solid-phase extraction (MIP-SPE) by AFFINIMIP glyphosate as an initial clean-up step significantly eliminated matrix components and ensured an efficient derivatization step. Colorless beebread extracts were derivatized by the addition of 9-fluorenylmethyl chloroformate (FMOC-Cl). After derivatization, in order to remove FMOC-OH and residual borate buffer, a solid-phase extraction (SPE) clean-up step using Oasis HLB was carried out. Instrumental analysis was performed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The method was validated according to the SANTE/11312/2021 guideline at concentrations of 5, 10, and 100 µg/kg, and satisfactory recovery (trueness) values (76–111%) and precision (RSDr) ≤ 18% were obtained. The limit of quantification (LOQ) was 5 µg/kg for AMPA and glufosinate ammonium and 10 µg/kg for glyphosate. The method was positively verified by the international proficiency test. Analysis of beebread samples showed the method's usefulness in practice. The developed method could be a reliable tool for the assessment of beebread's contamination with residues of glyphosate, its metabolite AMPA, and glufosinate ammonium. [ABSTRACT FROM AUTHOR]

The TDM of voriconazole which exhibits wide inter-individual variability is indispensable for treatment in clinic. In this study, a method that high-performance liquid chromatography tandem mass spectrometry cubed (HPLC-MS3) is first built and validated to quantify voriconazole in human plasma. The system is composed of Shimadzu Exion LCTM UPLC coupled with a Qtrap 5500 mass spectrometer. The separation of voriconazole is performed on a Poroshell 120 SB-C18 column at a flow rate of 0.8 mL/min remaining 7 min for each sample. The calibration curves are linear in the concentration range of 0.25–20 μg/mL. Intra-day and inter-day accuracies and precisions are within 8.0% at three concentrations, and the recoveries and matrix effect are all within accepted limits. In terms of stability, there is no significant degradation of voriconazole under various conditions. The HPLC-MS3 and HPLC-MRM (multiple reaction monitoring) methods are compared in 42 patients with Passing–Bablok regression and Bland–Altman plots, and the results show no significant difference between the two methods. However, HPLC-MS3 has a higher S/N (signal-to-noise ratio) and response than the MRM. Finally, the HPLC-MS3 assay is successfully applied to monitor the TDM (therapeutic drug monitoring) of voriconazole in human plasma, and this verifies that the dosing guidelines for voriconazole have been well implemented in the clinic and patients have received excellent treatment. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) and type 2 diabetes mellitus (T2DM) are common clinical conditions, and T2DM is an independent risk factor for HCC. Sorafenib and lenvatinib, two multi-targeted tyrosine kinase inhibitors, are first-line therapies for advanced HCC, while canagliflozin, a sodium-glucose co-transporter 2 inhibitor, is widely used in the treatment of T2DM. Here, we developed an ultra-performance liquid chromatography-tandem mass spectrometry method for the simultaneous determination of canagliflozin, sorafenib, and lenvatinib, and investigated the pharmacokinetic drug interactions between canagliflozin and sorafenib or lenvatinib in rats. The animals were randomly divided into five groups. Groups I–III were gavage administrated with sorafenib, lenvatinib, and canagliflozin, respectively. Group IV received sorafenib and canagliflozin; while Group V received lenvatinib and canagliflozin. The area under the plasma concentration-time curves (AUC) and maximum plasma concentrations (Cmax) of canagliflozin increased by 37.6% and 32.8%, respectively, while the apparent volume of distribution (Vz/F) and apparent clearance (CLz/F) of canagliflozin significantly decreased (30.6% and 28.6%, respectively) in the presence of sorafenib. Canagliflozin caused a significant increase in AUC and Cmax of lenvatinib by 28.9% and 36.2%, respectively, and a significant decrease in Vz/F and CLz/F of lenvatinib by 52.9% and 22.7%, respectively. In conclusion, drug interactions exist between canagliflozin and sorafenib or lenvatinib, and these findings provide a reference for the use of these drugs in patients with HCC and T2DM. [ABSTRACT FROM AUTHOR]

A novel method of ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed for the identification and quantification of four potential genotoxic impurities (PGIs) in the active pharmaceutical ingredients of TSD-1, a novel P2Y12 receptor antagonist. Four PGIs were named, 4-nitrobenzenesulfonic acid, methyl 4-nitrobenzenesulfonate, ethyl 4-nitrobenzenesulfonate, and isopropyl 4-nitrobenzenesulfonate. Following the International Conference of Harmonization (ICH) guidelines, this methodology is capable of quantifying four PGIs at 15.0 ppm in samples of 0.5 mg/mL concentration. This validated approach presented very low limits (0.1512–0.3897 ng/mL), excellent linearity (coefficients > 0.9900), and a satisfactory recovery range (94.9–115.5%). The method was sufficient in terms of sensitivity, linearity, precision, accuracy, selectivity, and robustness and, thus, has high practicality in the pharmaceutical quality control of TSD-1. [ABSTRACT FROM AUTHOR]

Aconiti Lateralis Radix Praeparata (Fu Zi) is the processed lateral root of Aconitum carmichaelii Debx, which is widely used in emergency clinics. Poisoning incidents and adverse reactions occur with the improper intake of Fu Zi. Metabolic characteristics of aconitum alkaloids of Fu Zi may vary, and the effects of Fu Zi in healthy and Long QT syndrome (LQTS) patients is unknown. In this experiment, 24 Sprague Dawley rats were randomly divided into three groups: 2.0, 1.0, and 0.5 g/kg dose groups, and blood samples were collected after the oral administration of Fu Zi extract. We used an ultra-high performance liquid chromatography-tandem mass spectrometry system to detect the concentrations of six aconitum alkaloids. Cell toxicity, calcium imaging, and patch-clamp recordings of human induced pluripotent stem cells-cardiomyocytes (hiPSC-CMs) of aconitine in healthy and LQTS were observed. We found that the AUC(0–48h), Cmax, and t1/2 of the six compounds increased with the multiplicative dosages; those in the high group were significantly higher than those in the low group. Aconitine concentration-dependently decreased the amplitude, which has no significant effect on the cell index of normal hiPSC-CMs. Aconitine at 5.0 μM decreased the cell index between 5–30 min for LQTS hiPSC-CMs. Meanwhile, aconitine significantly increased the frequency of calcium transients in LQTS at 5 μM. Aconitine significantly shortened the action potential duration of human cardiomyocytes in both normal and LQTS groups. These results show metabolic behaviors of aconitum alkaloids in different concentrations of Fu Zi and effects of aconitine in healthy and LQTS patients. [ABSTRACT FROM AUTHOR]

Geumgwesingihwan (GSH) is a traditional herbal prescription composed of eight medicinal herbs: Rehmannia glutinosa (Gaertn.) DC., Dioscorea japonica Thunb., Cornus officinalis Siebold and Zucc., Poria cocos Wolf, Paeonia suffruticosa Andrews, Alisma plantago-aquatica subsp. orientale (Sam.) Sam., Achyranthes bidentate Blume, and Plantago asiatica L. This study developed and validated an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method in the multiple reaction monitoring (MRM) mode for simultaneous determination of 14 compounds (allantoin, gallic acid, 5-(hydroxymethyl)furfural, geniposidic acid, oxypaeoniflorin, loganin, geniposide, paeoniflorin, ecdysterone, verbascoside, cornuside, benzoylpaeoniflorin, paeonol, and alisol B acetate) in GSH. The chromatographic separation of all marker analytes was carried out on an Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm) using gradient elution of a mobile phase of distilled water–acetonitrile containing 0.1% acetic acid. The newly established UPLC–MS/MS MRM method was validated by evaluating the linearity, the limits of detection and quantification, recovery, and precision. All markers were detected at concentrations of 6.94–4126.28 mg/kg. In addition, the recovery was 76.65–119.49% and the relative standard deviation value of the precision was 0.19–9.91%. The newly developed and validated UPLC–MS/MS assay will provide useful information for quality assessment of GSH. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive muscle loss, leading to difficulties in movement. Mutations in the DMD gene that code for the protein dystrophin are responsible for the development of DMD disorder, where the synthesis of this protein is completely halted. Therefore, circulating dystrophin protein could be a promising biomarker of DMD disease. Current methods for diagnosing DMD have sensitivity, specificity, and reproducibility limitations. Herein, a quantitative liquid chromatography–tandem spectrometry (LC–MS/MS) technique in multiple reaction monitoring (MRM) mode was designed and validated for accurate dystrophin protein measurement in a dried blood spot (DBS). The method was successfully validated on the basis of international guidelines regarding calibration curves, precision, and accuracy. In addition, patients and healthy controls were used to test the amount of dystrophin protein circulating in DBS samples as a potential biomarker for DMD disorders. DMD patients were found to have considerably lower levels than controls. To the best of our knowledge, this is the first study to report dystrophin levels in DBS through LC–MS/MS as a diagnostic marker for DMD to the proposed MRM method, providing a highly specific and sensitive approach to dystrophin quantification in a DBS that can be applied in DMD screening. [ABSTRACT FROM AUTHOR]

Coronary heart disease (CHD), which has developed into one of the major diseases, was reported to be treated by the target of peroxisome proliferators-activate receptor γ (PPAR-γ). As a natural medicine long used in the treatment of CHD, there are few studies on how to screen the target active compounds with high specific activity from Choerospondias axillaris. To advance the pace of research on target-specific active compounds in natural medicines, we have combined magnetic ligand fishing and functionalized nano-microspheres to investigate the active ingredients of PPAR-γ targets in Choerospondias axillaris. The PPAR-γ functionalized magnetic nano-microspheres have been successfully synthesized and characterized by vibrating sample magnetometer (VSM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The specificity, reusability, and reproducibility of the nano-microspheres were investigated with the help of the specific binding of rosiglitazone to PPAR-γ. In addition, the incubation temperature and the pH of the buffer solution in the magnetic ligand fishing were optimized to improve the specific adsorption efficiency of the analytes. Finally, with the aid of ultraperformance liquid chromatography plus Q-Exactive Orbitrap tandem mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS/MS), the 16 active ligands including 9 organic acids, 5 flavonoids, and 2 phenols were found in the ethanolic extracts of Choerospondias axillaris. Therefore, the study can provide a successful precedent for realizing the designated extraction and rapid isolation of target-specific active ingredient groups in the complex mixtures. [ABSTRACT FROM AUTHOR]

The aquaculture industry has become a sustainable source of food for humans. Remaining challenges include disease issues and ethical concerns for the discomfort and stress of farmed fish. There is a need for reliable biomarkers to monitor welfare in fish, and the stress hormone cortisol has been suggested as a good candidate. This study presents a novel method for measurement of cortisol in fish feces based on enzymatic hydrolysis, liquid–liquid extraction, derivatization, and finally instrumental analysis by liquid chromatography coupled with tandem mass spectrometry. Hydrolysis and extraction conditions were optimized. Cortisol appeared to be mostly conjugated to sulfate and less conjugated to glucuronic acid in the studied samples of feces from farmed Atlantic salmon. The method was suitable for quantification of cortisol after enzymatic deconjugation by either combined glucuronidase and sulfatase activity, or by glucuronidase activity alone. The limit of detection was 0.15 ng/g, the limit of quantification was 0.34 ng/g, and the method was linear (R2 > 0.997) up to 380 ng/g, for measurement of cortisol in wet feces. Method repeatability and intermediate precision were acceptable, both with a coefficient of variation (CV) of 11%. Stress level was high in fish released into seawater, and significantly reduced after eight days. [ABSTRACT FROM AUTHOR]

Blood levels of the vitamin D3 (D3) metabolites 25-hydroxyvitamin D3 (25(OH)D3), 24R,25-dihydroxyvitamin D3, and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) are recognized indicators for the diagnosis of bone metabolism-related diseases, D3 deficiency-related diseases, and hypercalcemia, and are generally measured by liquid-chromatography tandem mass spectrometry (LC-MS/MS) using an isotope dilution method. However, other D3 metabolites, such as 20-hydroxyvitamin D3 and lactone D3, also show interesting biological activities and stable isotope-labeled derivatives are required for LC-MS/MS analysis of their concentrations in serum. Here, we describe a versatile synthesis of deuterium-labeled D3 metabolites using A-ring synthons containing three deuterium atoms. Deuterium-labeled 25(OH)D3 (2), 25(OH)D3-23,26-lactone (6), and 1,25(OH)2D3-23,26-lactone (7) were synthesized, and successfully applied as internal standards for the measurement of these compounds in pooled human serum. This is the first quantification of 1,25(OH)2D3-23,26-lactone (7) in human serum. [ABSTRACT FROM AUTHOR]

Withania somnifera is a traditional Indian herb described under the 'Rasayana' class in Ayurveda, which gained immense popularity as a dietary supplement in the USA, Europe, Asia, and the Indian domestic market. Despite enormous research on the pharmacological effect of withanosides and withanolides, bioanalytical method development and pharmacokinetics remained challenging and unexplored for these constituents due to isomeric and isobaric characteristics. In current research work, molecular descriptors, pharmacokinetic, and toxicity prediction (ADMET) of these constituents were performed using Molinspiration and admetSAR tools. A rapid, selective, and reproducible bioanalytical method was developed and validated for seven withanosides and withanolides as per USFDA/EMA guidelines, further applied to determine pharmacokinetic parameters of Withania somnifera root extract (WSE) constituents in male Sprague Dawley rats at a dose of 500 mg/kg. Additionally, an ex vivo permeability study was carried out to explore the absorption pattern of withanosides and withanolides from the intestinal lumen. In silico, ADMET revealed oral bioavailability of withanosides and withanolides following Lipinski's rules of five with significant absorption from the gastrointestinal tract and the ability to cross the blood-brain barrier. Upon oral administration of WSE, Cmax was found to be 13.833 ± 3.727, 124.415 ± 64.932, 57.536 ± 7.523, and 7.283 ± 3.341 ng/mL for withanoside IV, withaferin A, 12-Deoxy-withastramonolide, and withanolide A, respectively, with Tmax of 0.750 ± 0.000, 0.250 ± 0.000, 0.291 ± 0.102, and 0.333 ± 0.129 h. Moreover, at a given dose, withanoside V, withanolide B, and withanone were detected in plasma; however, the concentration of these constituents was found below LLOQ. Thus, these four major withanoside and withanolides were quantified in plasma supported by ex vivo permeation data exhibiting a time-dependent absorption of withanosides and withanolides across the intestinal barrier. These composite findings provide insights to design a clinical trial of WSE as a potent nutraceutical. [ABSTRACT FROM AUTHOR]

β-Lactam antibiotics are most commonly used in the critically ill, but their effective dosing is challenging and may result in sub-therapeutic concentrations that can lead to therapy failure and even promote antimicrobial resistance. In this study, we present the analytical tool enabling specific and sensitive determination of the sole biologically active fraction of piperacillin and imipenem in biological material from the critically ill. Thin-film microextraction sampling technique, followed by rapid liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis, was optimized and validated for the quantitative determination of antibiotics in blood and bronchoalveolar lavage (BAL) specimens collected from intensive care unit (ICU) patients suffering from ventilation-associated pneumonia (n = 18 and n = 9, respectively). The method was optimized and proved to meet the criteria of US Food and Drug Administration (FDA) guidelines for bioanalytical method validation. Highly selective, sensitive, accurate and precise analysis by means of thin-film microextraction–LC-MS/MS, which is not affected by matrix-related factors, was successfully applied in clinical settings, revealing poor penetration of piperacillin and imipenem from blood into BAL fluid (reflecting the site of bacterial infection), nonlinearity in antibiotic binding to plasma-proteins and drug-specific dependence on creatinine clearance. This work demonstrates that only a small fraction of biologically active antibiotics reach the site of infection, providing clinicians with a high-throughput analytical tool for future studies on personalized therapeutic drug monitoring when tailoring the dosing strategy to an individual patient. [ABSTRACT FROM AUTHOR]

The high-throughput quantitation of cannabinoids is important for the cannabis industry. As medicinal products increase, and research into compounds that have pharmacological benefits increase, and the need to quantitate more than just the main cannabinoids becomes more important. This study aims to provide a rapid, high-throughput method for cannabinoid quantitation using a liquid chromatography triple-quadrupole mass spectrometer (LC-QQQ-MS) with an ultraviolet diode array detector (UV-DAD) for 16 cannabinoids: CBDVA, CBDV, CBDA, CBGA, CBG, CBD, THCV, THCVA, CBN, CBNA, THC, Δ8-THC, CBL, CBC, THCA-A and CBCA. Linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, recovery and matrix effect were all evaluated. The validated method was used to determine the cannabinoid concentration of four different Cannabis sativa strains and a low THC strain, all of which have different cannabinoid profiles. All cannabinoids eluted within five minutes with a total analysis time of eight minutes, including column re-equilibration. This was twice as fast as published LC-QQQ-MS methods mentioned in the literature, whilst also covering a wide range of cannabinoid compounds. [ABSTRACT FROM AUTHOR]

This study aimed to define a consortium of lactic acid bacteria (LAB) that will bring added value to dried fresh cheese through specific probiotic properties and the synthesis of bioactive peptides (biopeptides). The designed LAB consortium consisted of three Lactobacillus strains: S-layer carrying Levilactobacillus brevis D6, exopolysaccharides producing Limosilactobacillus fermentum D12 and plantaricin expressing Lactiplantibacillus plantarum D13, and one Enterococcus strain, Enterococcus faecium ZGZA7-10. Chosen autochthonous LAB strains exhibited efficient adherence to the Caco-2 cell line and impacted faecal microbiota biodiversity. The cheese produced by the LAB consortium showed better physicochemical, textural and sensory properties than the cheese produced by a commercial starter culture. Liquid chromatography coupled with matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (LC-MALDI-TOF/TOF) showed the presence of 18 specific biopeptides in dried fresh cheeses. Their identification and relative quantification was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring (MRM). The results also showed that their synthesis resulted mainly from β-casein and also α-S1 casein degradation by proteolytic activities of the LAB consortium. The designed LAB consortium enhanced the functional value of the final product through impact on biopeptide concentrations and specific probiotic properties. [ABSTRACT FROM AUTHOR]

Bu-yang-huan-wu-tang (BYHWT) is a popular Traditional Chinese Medicine formula consisting of seven herbal medicines (Astragalus membranaceus, Angelica sinensis, Paeonia lactiflora, Ligusticum chuanxiong, Carthamus tinctorius, Amygdalus persica and Pheretima aspergillum), that has been used in China for centuries to overcome stroke-induced disability. To ensure the consistency of quality, a reliable analytical method is required, therefore, we developed a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for quantitative analysis of the major constituents in BYHWT. The herbal ingredients consisting of the cycloartane-type triterpene glycosides of astragaloside I, astragaloside II and astragaloside IV; isoflavones of formononetin, ononin calycosin, calycosin-7-O-β-D-glucoside; ligustilide and paeoniflorin were separated on a C18 column with gradient elution of methanol/10 mM ammonium acetate buffer-formic acid (100:0.1, v/v). This study was performed by a mass spectrometer using electrospray ionization (ESI) with positive ionization ions monitored in the multiple reaction-monitoring (MRM) mode. The linearity, accuracy, precision, limit of detection (LOD) and lower limit of quantification (LLOQ) were validated for this quantification method, and the sensitivity, reliability and reproducibility were all confirmed. The experiments provided a good method for analyzing BYHWT extracts. This study also quantitated the active components in various brands of commercially available products. The results indicated that the pharmaceutical industrial products of BYHWT exhibited considerable variation in their contents of the herbal compounds. [ABSTRACT FROM AUTHOR]

Quantitative and qualitative analyses of cell protein composition using liquid chromatography/tandem mass spectrometry are now standard techniques in biological and clinical research. However, the quantitative analysis of protein–protein interactions (PPIs) in cells is also important since these interactions are the bases of many processes, such as the cell cycle and signaling pathways. This paper describes the application of Skyline software for the identification and quantification of the biotinylated form of the biotin acceptor peptide (BAP) tag, which is a marker of in vivo PPIs. The tag was used in the Proximity Utilizing Biotinylation (PUB) method, which is based on the co-expression of BAP-X and BirA-Y in mammalian cells, where X or Y are interacting proteins of interest. A high level of biotinylation was detected in the model experiments where X and Y were pluripotency transcription factors Sox2 and Oct4, or heterochromatin protein HP1γ. MRM data processed by Skyline were normalized and recalculated. Ratios of biotinylation levels in experiment versus controls were 86 ± 6 (3 h biotinylation time) and 71 ± 5 (9 h biotinylation time) for BAP-Sox2 + BirA-Oct4 and 32 ± 3 (4 h biotinylation time) for BAP-HP1γ + BirA-HP1γ experiments. Skyline can also be applied for the analysis and identification of PPIs from shotgun proteomics data downloaded from publicly available datasets and repositories. [ABSTRACT FROM AUTHOR]

Ammodaucus leucotrichus is a spontaneous plant endemic of the North African region. An efficient selective pressurized liquid extraction (PLE) method was optimized to concentrate neuroprotective extracts from A. leucotrichus fruits. Green solvents were tested, namely ethanol and water, within a range of temperatures between 40 to 180 °C. Total carbohydrates and total phenolics were measured in extracts, as well as in vitro antioxidant capacity (DPPH radical scavenging), anticholinesterase (AChE) and anti-inflammatory (LOX) activities. Metabolite profiling was carried out by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-q-TOF-MS/MS), identifying 94 compounds. Multivariate analysis was performed to correlate composition with bioactivity. A remarkable effect of the temperature using water was observed: the higher temperature, the higher extraction yield, the higher total phenolic content, as well as the higher total carbohydrates content. The water extract obtained at 180 °C, 10.34 MPa and 10 min showed meaningful anti-inflammatory (IC50LOX = 39.4 µg/mL) and neuroprotective activities (IC50AChE = 55.6 µg/mL). The Principal Components Analysis (PCA) and the cluster analysis correlated these activities with the presence of carbohydrates and phenolic compounds. [ABSTRACT FROM AUTHOR]

Acyclovir (ACV) is an effective and selective antiviral drug, and the study of its toxicology and the use of appropriate detection techniques to control its toxicity at safe levels are extremely important for medicine efforts and human health. This review discusses the mechanism driving ACV's ability to inhibit viral coding, starting from its development and pharmacology. A comprehensive summary of the existing preparation methods and synthetic materials, such as 5-aminoimidazole-4-carboxamide, guanine and its derivatives, and other purine derivatives, is presented to elucidate the preparation of ACV in detail. In addition, it presents valuable analytical procedures for the toxicological studies of ACV, which are essential for human use and dosing. Analytical methods, including spectrophotometry, high performance liquid chromatography (HPLC), liquid chromatography/tandem mass spectrometry (LC-MS/MS), electrochemical sensors, molecularly imprinted polymers (MIPs), and flow injection–chemiluminescence (FI-CL) are also highlighted. A brief description of the characteristics of each of these methods is also presented. Finally, insight is provided for the development of ACV to drive further innovation of ACV in pharmaceutical applications. This review provides a comprehensive summary of the past life and future challenges of ACV. [ABSTRACT FROM AUTHOR]

Methylphenidate is a powerful central nervous system stimulant with a high potential for abuse in horse racing. The detection of methylphenidate use is of interest to horse racing authorities for both prior to and during competition. The use of hair as an alternative sampling matrix for equine anti-doping has increased as the number of detectable compounds has expanded. Our laboratory developed a liquid chromatography–high-resolution mass spectrometry method to detect the presence of methylphenidate in submitted samples. Briefly, hair was decontaminated, cut, and pulverized prior to liquid–liquid extraction in basic conditions before introduction to the LC-MS system. Instrumental analysis was conducted using a Thermo Q Exactive mass spectrometer using parallel reaction monitoring using a stepped collision energy to obtain sufficient product ions for qualitative identification. The method was validated and limits of quantitation, linearity, matrix effects, recovery, accuracy, and precision were determined. The method has been applied to confirm the presence of methylphenidate in official samples submitted by racing authorities. [ABSTRACT FROM AUTHOR]

The colored grain of wheat (Triticum aestivum L.) contains a large number of polyphenolic compounds that are biologically active ingredients. The purpose of this work was a comparative metabolomic study of extracts from anthocyaninless (control), blue, and deep purple (referred to here as black) grains of seven genetically related wheat lines developed for the grain anthocyanin pigmentation trait. To identify target analytes in ethanol extracts, high-performance liquid chromatography was used in combination with Bruker Daltonics ion trap mass spectrometry. The results showed the presence of 125 biologically active compounds of a phenolic (85) and nonphenolic (40) nature in the grains of T. aestivum (seven lines). Among them, a number of phenolic compounds affiliated with anthocyanins, coumarins, dihydrochalcones, flavan-3-ols, flavanone, flavones, flavonols, hydroxybenzoic acids, hydroxycinnamic acids, isoflavone, lignans, other phenolic acids, stilbenes, and nonphenolic compounds affiliated with alkaloids, carboxylic acids, carotenoids, diterpenoids, essential amino acids, triterpenoids, sterols, nonessential amino acids, phytohormones, purines, and thromboxane receptor antagonists were found in T. aestivum grains for the first time. A comparative analysis of the diversity of the compounds revealed that the lines do not differ from each other in the proportion of phenolic (53.3% to 70.3% of the total number of identified compounds) and nonphenolic compounds (46.7% to 29.7%), but diversity of the compounds was significantly lower in grains of the control line. Even though the lines are genetically closely related and possess similar chemical profiles, some line-specific individual compounds were identified that constitute unique chemical fingerprints and allow to distinguish each line from the six others. Finally, the influence of the genotype on the chemical profiles of the wheat grains is discussed. [ABSTRACT FROM AUTHOR]

The rapid source identification and environmental risk assessment (ERA) of hundreds of chemicals of emerging concern (CECs) in river water represent a significant analytical challenge. Herein, a potential solution involving a rapid direct-injection liquid chromatography–tandem mass spectrometry method for the quantitative determination of 102 CECs (151 qualitatively) in river water is presented and applied across six rivers in Germany and Switzerland at high spatial resolution. The method required an injection volume of only 10 µL of filtered sample, with a runtime of 5.5 min including re-equilibration with >10 datapoints per peak per transition (mostly 2 per compound), and 36 stable isotope-labelled standards. Performance was excellent from the low ng/L to µg/L concentration level, with 260 injections possible in any 24 h period. The method was applied in three separate campaigns focusing on the ERA of rivers impacted by wastewater effluent discharges (1 urban area in the Basel city region with 4 rivers, as well as 1 semi-rural and 1 rural area, each focusing on 1 river). Between 25 and 40 compounds were quantified directly in each campaign, and in all cases small tributary rivers showed higher CEC concentrations (e.g., up to ~4000 ng/L in total in the R. Schwarzach, Bavaria, Germany). The source of selected CECs could also be identified and differentiated from other sources at pre- and post- wastewater treatment plant effluent discharge points, as well as the effect of dilution downstream, which occurred over very short distances in all cases. Lastly, ERA for 41 CECs was performed at specific impacted sites, with risk quotients (RQs) at 1 or more sites estimated as high risk (RQ > 10) for 1 pharmaceutical (diclofenac), medium risk (RQ of 1–10) for 3 CECs (carbamazepine, venlafaxine, and sulfamethoxazole), and low risk (RQ = 0.1–1.0) for 7 CECs (i.e., RQ > 0.1 for 11 CECs in total). The application of high-throughput methods like this could enable a better understanding of the risks of CECs, especially in low flow/volume tributary rivers at scale and with high resolution. [ABSTRACT FROM AUTHOR]

The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts. [ABSTRACT FROM AUTHOR]

An ultra-high performance liquid chromatography tandem mass spectrometry method was developed and validated for the sensitive determination and unambiguous confirmation of residues of per and polyfluorinated alkyl substances (PFAS) in breastmilk, retail milk and infant formulas following two sample preparation methods. Sample pre-treatment was carried out by a simplified QuEChERS method without requiring dSPE or any further clean-up. The method was validated in accordance with the requirements of Commission Decision 657/2002/EC with slight modifications. The method displayed good linearity with R2 ranging from 0.9843–0.9998 for all target PFAS. The recovery and within-laboratory reproducibility of the method (n = 63) were in the range 60–121% and 5–28%, respectively. The decision limit, detection capability and limit of quantitation ranged from 30–60 ng kg−1 to 40–100 ng kg−1 and 5–50 ng kg−1, respectively. Acceptable matrix effect values in the range −45–29% were obtained with uncertainty of measurement lower than 25% for all target PFAS. The method displays its suitability for the sensitive and high-throughput confirmatory analysis of C4–C14 PFAS in breastmilk, dairy milk and infant formulas. [ABSTRACT FROM AUTHOR]