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This study developed and validated a robust UPLC-MS/MS method for quantifying cyclic glycine–proline (cGP) in mangrove-derived Penicillium and Aspergillus strains. The method demonstrated excellent linearity, precision, and recovery, with detection limits as low as 4.8 ng/mL. Penicillium pedernalense extract achieved a cGP content of 67.45 ± 1.11 ng/mL, with a corresponding fermentation yield of 29.31 ± 0.61 mg/L. This surpassed Penicillium steckii, which reached a content of 31.71 ± 0.31 ng/mL, with a yield of 8.51 ± 0.15 mg/L. This quantitative approach for metabolite analysis provides a viable method for screening these fungal strains, highlighting their potential for sustainable production of cyclic glycine–proline (cGP).

Pears are popular among consumers for their juicy and delicious taste. In this study, the sugar and organic acid compositions of pear fruits with different texture types during development were determined by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), and fruit texture traits were determined by a texture analyzer. The results showed that the dominant sugar in soft and crispy types of pear fruits was fructose. The main difference between pears was the second-highest sugar component; glucose content was higher in crispy-flesh pear fruits while sucrose content was higher in soft-flesh pear fruits. The composition of organic acid components in both texture types of pear fruits was similar. The turning points of changes in the content of sucrose, sorbitol, glucose and quinic acid were different between different-textured pear varieties. A Pearson correlation analysis showed that sugar and organic acid components were significantly correlated with single fruit weight and soluble solid contents (SSCs), respectively. There was a high correlation among texture traits, individual sugars and organic acids. A partial least squares discriminant analysis (PLS-DA) VIP score plot showed that the differential traits with scores greater than 1 were total soluble sugars/total organic acids (TSSs/TAs), fracture and malic acid/citric acid (MA/CA), which could distinguish pear fruits of different texture types better and reflect the uneven quality differences among pear fruits adapted to different origins. The comprehensive analysis results of the flesh texture parameters and sugar and organic acid components are in line with objective reality and will provide a reference for quantitative indicators of the sensory evaluation of pear varieties as well as for molecular mechanism research on trait differences.

Pterocephin A is a natural triterpenoid saponin isolated from Pterocephalus hookeri, a traditional Tibetan medicine with slight toxicity, which can induce liver injury in rats. This study aimed to establish a sensitive and reliable UPLC-MS/MS method for exploring the toxicokinetics and tissue distribution of pterocephin A following single intravenous and intragastric administration. Pterocephin A and prosapogenin 1C (internal standard, IS) were extracted using a simple protein precipitation technique with methanol as the precipitant for plasma samples and methanol/acetonitrile = 1:1 (v/v) for tissue samples. UPLC separation was achieved by gradient elution with 0.3 mL/min and a mobile phase consisting of 5 mM ammonium formate (A) and acetonitrile (B) (0–2 min 30% B; 2–4 min: 30–80% B; 4–5 min: 80–98% B; 5–6.5 min: 98% B; 6.5–7 min: 98–30% B; and 7–8 min: 30% B, v/v) with a column temperature of 35 °C. MS spectrometry adopted negative ion scanning mode, primary MS spectrometry adopted full scan monitoring mode, and secondary MS spectrometry adopted targeted MS2 scan monitoring mode. The assay exhibited a linear dynamic range of 0.02–15 μg/mL for pterocephin A in biological samples, with the low limit of quantification set at 0.02 μg/mL. Non-compartmental toxicokinetic parameters indicated that pterocephin A was well absorbed into the systemic circulation and had a long residual time after intravenous (10 mg/kg) and intragastric (60 mg/kg) administration, as it could still be detected after 72 h. Tissue distribution analysis revealed detectable levels of pterocephin A in various tissues, and a high concentration was maintained in the liver after intravenous (10 mg/kg) administration, with the highest concentration being 610.95 ± 25.73 ng/mL and a specific distribution pattern of liver > lung > kidney > intestine > spleen > testes > heart > stomach. The toxicokinetic process and tissue distribution characteristics of pterocephin A were expounded in this study, which can provide relevant data support for further research and clinical application of pterocephin A with its slight toxicity.

Metformin is the gold standard substrate for evaluating potential inhibitors of the organic cation transporters (OCTs). Here, we established a UPLC-MS/MS assay to quantify metformin in cell pellets with a range of 0.05–50 ng/mL using 6-deuterated metformin as an internal standard. We used an ion-pairing chromatographic approach with heptafluorobutyric acid, making use of a reverse-phase column, and overcame the associated ion-suppression via previously established post-column injection of aqueous ammonia. The assay was validated according to the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) recommendations for bioanalytical methods. The established extraction procedure was simple, very fast and ensured almost 100% recovery of the analyte. The exceptionally sharp peak form and retention of the ion-pairing chromatography are superior to other methods and allow us to measure as sensitively as 0.05 ng/mL. We used the herein established and validated method to develop a cellular OCT inhibition assay by using metformin as a substrate and human embryonic kidney cells (HEK) overexpressing the OCTs 1-3. The method presented may be useful for identifying new OCT inhibitors, but also for drug–drug interactions and other pharmacokinetic studies, where accurate quantification of low metformin amounts in relevant tissues is mandatory.

The aim of the present study was to establish a simple and reliable ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method and apply it for the determination of pharmacokinetics of moxidectin-loaded microspheres (MOX-MS) in rats. Plasma samples were processed using a simplified liquid–liquid extraction method and were separated using an Agilent Zorbax Eclipse Plus C18 column (50 mm × 2.1 mm, 1.8 μm) with a mobile phase consisting of a 10 mM ammonium formate solution with 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.4 mL/min for 5 min. Avermectin B1a was used as an internal standard (IS). The sample was injected at a volume of 10 μL with a column temperature of 35 °C and detected in a positive ion mode. A good linear response across the concentration range of 1.00–200 ng/mL (r2 > 0.99) and a lower limit of quantification (LLOQ) of 1.00 ng/mL were achieved. The extraction recovery of moxidectin exceeded 94.1%, the matrix effect was between 91.2% and 96.2%, the accuracy ranged from 100.1 to 103.6%, and the relative standard deviation (RSD) did not exceed 15% for the intra- and inter-day accuracy and precision. The pharmacokinetic results showed that MOX-MS significantly decreased Cmax, prolonged T1/2, and improved bioavailability. The developed method significantly reduced the assay volume, shortened detection time, simplified sample processing methods and saved assay costs, which may contribute to the development of the new antiparasitic drug.

Phellodendron amurense Rupr. is a precious medicinal tree species in northeast China. However, P. amurense resources have been severely destroyed due to uncontrolled overharvest and the limited innovation of new germplasms by traditional cross-breeding. In this study, polyploid breeding was introduced to the improvement program of P. amurense. Fifty-four tetraploid plants of P. amurense were first produced by colchicine-induced adventitious bud chromosome doubling in stem segment explants. The induction frequency reached 36.16% (1.0 g L−1 colchicine solution for 48 h treatment) and 50.00% (2.0 g L−1 colchicine solution for 24 h treatment), respectively, showing the high efficiency of the somatic chromosome doubling based on the organogenesis system. Tetraploidization resulted in significant phenotypic variation, such as larger and thicker leaves, thicker stems, and bigger stomata. Ultra-performance liquid chromatography–mass spectrometry (UPLC–MS/MS) analysis identified 59 differentially accumulated alkaloids (DAAs) between the leaf and stem samples of tetraploids, including 32 upregulated and 27 downregulated in stems. For both leaf and stem samples, 18 DAAs were identified between diploids and tetraploids, with 16 DAAs upregulated in tetraploid leaves and 8 upregulated in tetraploid stems, suggesting that polyploidization caused significant alterations in alkaloid contents in leaves and stems of P. amurense. The contents of the main medicinal compounds, such as berberine, jatrorrhizine, phellodendrine, and palmatine, increased significantly in the leaf and/or stem samples after polyploidization. This finding implied that polyploid breeding might be an effective approach for improving P. amurense, beneficial to preserving and exploiting natural resources.

Despite a higher safety profile compared to vitamin K antagonists, rivaroxaban therapy is still connected with multiple adverse effects, such as a high risk of bleeding. Thus, therapeutic drug monitoring (TDM) of rivaroxaban concentrations is suggested. An alternative to plasma samples can be dried blood spots (DBS), which minimize the cost of sample storage and transport. In this study, we developed a UPLC-MS/MS method for the analysis of rivaroxaban in DBS and plasma samples. Chromatographic separation was achieved on a Zorbax Eclipse Plus C18 column (2.1 × 100 mm; 3.5 µm, Agilent Technologies Inc., Santa Clara, CA, USA) with a mobile phase consisting of water and acetonitrile, both containing 0.1% formic acid. The analytes were detected using a positive ionization mode by multiple reaction monitoring. We validated the method according to ICH guidelines. The precision and accuracy were satisfactory. Extraction recovery was approximately 57% and 66% for DBS and plasma samples, respectively. A high correlation between rivaroxaban concentrations in plasma and DBS samples collected from patients was confirmed with Deming regression. The suitability of both sampling techniques for the rivaroxaban TDM was also verified by Bland–Altman plots based on DBS-predicted and observed plasma concentrations. In addition, we found a significant relationship between rivaroxaban concentrations and coagulation parameters, including prothrombin time (PT) and international normalized ratio (INR).

The aim of individuals consuming health supplements is to attain a robust state through nutritional regulation. However, some unscrupulous manufacturers, motivated by profit, fraudulently incorporate drugs or unauthorized components with therapeutic effects into the product for instant product performance enhancement. The long-term use of these products may inadvertently inflict harm on human health and fail to promote nutritive healthcare. The illegal inclusion of these substances is prevalent in kidney-tonifying and sexuality-enhancing products. Developing effective analytical methods to identify these products and screen for illegal added ingredients can effectively prevent such products from reaching and remaining on the market. A target screening method for the detection and quantification of 90 phosphodiesterase type 5 inhibitors (PDE-5is) in 5 kinds of health products was developed and validated. The type of dietary supplements varied from tablets, capsules, and protein powder to wine and beverages. Sample preparation was completed with a one-step liquid phase extraction. The screening process of 90 PDE-5is was done efficiently within 25 min by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) using the dynamic multiple reaction monitoring (dMRM) technique. The LODs of 90 PDE-5is were detected at levels ranging from 25 to 85 ng/g or ng/mL. This novel targeting methodology was effective and can be applied to routine market supervision. Among 286 batches of samples, 8 batches were found to be positive. Three kinds of PDE-5is were first detected in healthy products. The screening method demonstrated herein will be a promising and powerful tool for rapid screening of PDE-5is.

Millions of individuals throughout the world suffer from lymphatic filariasis (LF), which is a morbid disease caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori. These infections belong to tissue-invading nematodes and are one of the major neglected tropical diseases that often result in permanent and enduring disability among individuals in endemic regions. Due to combination therapy, LF eradication has drastically decreased infections globally. The development of blood micro-sampling techniques allowing precise quantitation of drugs in blood would facilitate pharmacokinetic (PK) studies in remote populations. Therefore, an LC-MS/MS bioanalytical method was utilized to analyze albendazole (ABZ), albendazole sulfone (ABZ-ON), albendazole sulfoxide (ABZ-OX), and probenecid (PR) in plasma and dried blood spots. Solid-phase extraction was utilized to extract the analyte from both plasma and blood-spiked DBS. Analytes of interest were eluted with a gradient mobile system using 0.05% formic acid in water (A) and 0.05% formic acid in methanol (B) and separated using a reversed-phase Acquity ®BEH C18 UPLC column (100 × 2.1 mm, 1.7 µm). Precision and accuracy at each QC level were within the acceptable limit, i.e., ±15% for all analytes in both the matrices. Tests for stability under laboratory and storage conditions indicated that no notable changes were observed for plasma and DBS. The LC-MS/MS method demonstrated its capability to consistently identify all target analytes (ABZ, ABZ-ON, ABZ-OX, and PR) at low concentrations, even at the small specimen volumes obtained from DBS cards. This confirms the efficacy and durability of DBS cards as a micro-sampling technique. Moreover, it enhances collection efforts for therapeutic drug monitoring in remote locations for patients infected with lymphatic filariasis.

A total of 769 wheat kernels collected from six provinces in China were analyzed for beauvericin (BEA) and four enniatins (ENNs), namely, ENA, ENA1, ENB and ENB1, using a solid phase extraction (SPE) technique with ultra-high performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). The results show that the predominant toxin was BEA, which had a maximum of 387.67 μg/kg and an average of 37.69 μg/kg. With regard to ENNs, the prevalence and average concentrations of ENB and ENB1 were higher than those of ENA and ENA1. The geographical distribution of BEA and ENNs varied. Hubei and Shandong exhibited the highest and lowest positive rates of BEA and ENNs (13.46% and 87.5%, respectively). However, no significant difference was observed among these six provinces. There was a co-occurrence of BEA and ENNs, and 42.26% of samples were simultaneously detected with two or more toxins. Moreover, a significant linear correlation in concentrations was observed between the four ENN analogs (r range: 0.75~0.96, p < 0.05). This survey reveals that the contamination and co-contamination of BEA and ENNs in Chinese wheat kernels were very common.

Mycotoxins, natural toxins produced by fungi, contaminate nearly 80% of global food crops. Alternaria mycotoxins, including alternariol (AOH), alternariol monomethylether (AME), and tenuazonic acid (TeA), present a health concern due to their prevalence in various plants and fruits. Exposure to these toxins exceeds the threshold of toxicological concern in some European populations, especially infants and toddlers. Despite this, regulatory standards for Alternaria toxins remain absent. The lack of toxicokinetic parameters, reference levels, and sensitive detection methods complicates risk assessment and highlights the necessity for advanced biomonitoring (HBM) techniques. This study addresses these challenges by developing and validating ultra-high performance liquid chromatography method coupled with tandem mass spectrometry to quantify AOH, AME, TeA, and their conjugates in multiple biological matrices. The validated method demonstrates robust linearity, precision, recovery (94–111%), and sensitivity across urine (LOD < 0.053 ng/mL), capillary blood (LOD < 0.029 ng/mL), and feces (LOD < 0.424 ng/g), with significantly lower LOD for TeA compared to existing methodologies. The application of minimally invasive microsampling techniques for the blood collection enhances the potential for large-scale HBM studies. These advancements represent a step toward comprehensive HBM and exposure risk assessments for Alternaria toxins, facilitating the generation of data for regulatory authorities.

Barley is rich in phenolic compounds, providing health benefits and making it a valuable addition to a balanced diet. However, most studies focus on these compounds at barley’s final maturity, neglecting their synthesis during grain development and its impact on barley quality for food applications. This study investigates phenolic profiles during grain development in four hull-less barley genotypes with different grain colors, specifically bred for food applications. The objectives were to determine the phenolic profile and identify the optimal maturity stage for maximum phenolic content and antioxidant capacity. Using UPLC-MS/MS and in vitro antioxidant capacity assays, results show that total phenolic compounds decrease as grain matures due to increased synthesis of reserve components. Flavan-3-ols, phenolic acids, and flavone glycosides peaked at immature stages, while anthocyanins peaked at physiological maturity. The harvest stage had the lowest phenolic content, with a gradient from black to yellow, purple, and blue genotypes. Antioxidant capacity fluctuated during maturation, correlating positively with phenolic compounds, specially bound phenolic acids and anthocyanins. These findings suggest that early harvesting of immature grain can help retain bioactive compounds, promoting the use of immature barley grains in foods. To support this market, incentives should offset costs associated with decreased grain weight.

The concept of a “circular bioeconomy” holds great promise for the health, cosmetic, and nutrition sectors by re-using Castanea sativa (Mill.) by-products. This sustainable resource is rich in bioactive secondary metabolites with antioxidant and anti-inflammatory properties. By transforming these by-products into high-value products for human health, we can promote sustainable economic growth and reduce the environmental impact of traditional waste disposal, adding value to previously underutilized resources. In the present study, we investigated the antioxidant capacity, phytochemical composition, and in vitro antioxidant and anti-inflammatory activity of C. sativa burr (CSB) aqueous extract. The spectrophotometric study revealed high total phenolic content (TPC) values with significant antioxidant and anti-radical properties. Using UPLC-MS/MS techniques, the phytochemical investigation identified 56 metabolites, confirming the presence of phenolic compounds in CSBs. In addition, CSBs significantly downregulated pro-inflammatory mediators in LPS-stimulated RAW 264.7 macrophage cells without significant cell toxicity. Lastly, in silico studies pinpointed three kinases from RAW 264.7 cells as binding partners with ellagic acid, the predominant compound found in our extract. These findings strongly advocate for the recycling and valorization of C. sativa by-products, challenging their conventional classification as mere “waste”.

Yeokwisan (YWS) is an herbal medicine prescription consisting of six oriental herbal medicines, developed to treat reflux esophagitis. We focused on developing an analytical method capable of simultaneously quantifying 13 compounds in YWS samples using high-performance liquid chromatography–photodiode array detection (HPLC–PDA) and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) and exploring their antioxidant effects. All compounds examined in both analytical systems were chromatographically separated on a SunFireTM C18 (4.6 × 250 mm, 5 μm) column and an Acquity UPLC BEH C18 (2.1 × 100 mm, 1.7 μm) column using gradient elution of a water–acetonitrile mobile phase. Antioxidant effects were evaluated based on radical scavenging activity (DPPH and ABTS tests) and ferrous ion chelating activity. In two analytical methods, the coefficient of determination of the regression equation was ≥0.9965, the recovery range was 81.11–108.21% (relative standard deviation (RSD) ≤ 9.33%), and the precision was RSD ≤ 11.10%. Application of the optimized analysis conditions gave quantitative analysis results for YWS samples of 0.02–100.36 mg/g. Evaluation of the antioxidant effects revealed that baicalein and baicalin exhibit significant antioxidant activity, suggesting that they play an important role in the antioxidant effects of YWS.

Perilla frutescens var. acuta (Lamiaceae) is widely used not only as an oil or a spice, but also as a traditional medicine to treat colds, coughs, fever, and indigestion. As an ongoing effort, luteolin-7-O-diglucuronide (1), apigenin-7-O-diglucuronide (2), and rosmarinic acid (3) isolated from P. frutescens var. acuta were investigated for their anti-adipogenic and thermogenic activities in 3T3-L1 cells. Compound 1 exhibited a strong inhibition against adipocyte differentiation by suppressing the expression of Pparg and Cebpa over 52.0% and 45.0%, respectively. Moreover, 2 inhibited the expression of those genes in a dose-dependent manner [Pparg: 41.7% (5 µM), 62.0% (10 µM), and 81.6% (50 µM); Cebpa: 13.8% (5 µM), 18.4% (10 µM), and 37.2% (50 µM)]. On the other hand, the P. frutescens var. acuta water extract showed moderate thermogenic activities. Compounds 1 and 3 also induced thermogenesis in a dose-dependent manner by stimulating the mRNA expressions of Ucp1, Pgc1a, and Prdm16. Moreover, an LC-MS/MS chromatogram of the extract was acquired using UHPLC-MS2 and it was analyzed by feature-based molecular networking (FBMN) and the Progenesis QI software (version 3.0). The chemical profiling of the extract demonstrated that flavonoids and their glycoside derivatives, including those isolated earlier as well as rosmarinic acid, are present in P. frutescens var. acuta.

A novel co-bonded octyl and pyridine silica (OPS) sorbent was prepared and applied for the solid phase extraction (SPE) of cyclopiazonic acid (CPA, a type of mycotoxin) in feed and agricultural products for the first time. A simple mixed-ligand one-pot reaction strategy was employed for OPS sorbent preparation. Nitrogen adsorption–desorption measurements, elemental analysis (EI), thermal gravimetric analysis (TGA), and Fourier transform infrared spectroscopy (FT-IR) analysis demonstrated the successful immobilization of octyl and quaternary ammonium groups onto the surface of silica gel. The large specific surface area, high-density functional groups, and mixed-mode anion-exchange characteristics of these silica particles made them the ideal material for the efficient extraction of CPA. Additionally, the OPS sorbents displayed excellent batch-to-batch reproducibility, satisfactory reusability, and low cost. The SPE parameters were optimized to explore the ionic and hydrophobic interactions between CPA and the functional groups, and the ultra-high performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UPLC-MS/MS) parameters were optimized to obtain a desirable extraction efficiency and high sensitivity to CPA. Meanwhile, the OPS sorbent presented a satisfactory extraction selectivity and low matrix effect. Under the optimized conditions, our developed CPA detection method was used to determine CPA level in rice, wheat flour, corn flour, peanut, and feed samples, exhibiting a lower detection limit, better linearity, higher sensitivity, and satisfactory extraction recovery rate than previously reported methods. Therefore, our method can be preferentially used as a method for the detection of CPA in agricultural products and feeds.

Utilizing plant-based resources, particularly their by-products, aligns with sustainability principles and circular bioeconomy, contributing to environmental preservation. The therapeutic potential of plant extracts is garnering increasing interest, and this study aimed to demonstrate promising outcomes from an extract obtained from an underutilized plant waste. Chaetomorpha linum, an invasive macroalga found in the Orbetello Lagoon, thrives in eutrophic conditions, forming persistent mats covering approximately 400 hectares since 2005. The biomass of C. linum undergoes mechanical harvesting and is treated as waste, requiring significant human efforts and economic resources—A critical concern for municipalities. Despite posing challenges to local ecosystems, the study identified C. linum as a natural source of bioactive metabolites. Phytochemical characterization revealed lipids, amino acids, and other compounds with potential anti-inflammatory activity in C. linum extract. In vitro assays with LPS-stimulated RAW 264.7 and TNF-α/IFN-γ-stimulated HaCaT cells showed the extract inhibited reactive oxygen species (ROS), nitric oxide (NO), and prostaglandin E2 (PGE2) productions, and reduced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions via NF-κB nuclear translocation, in RAW 264.7 cells. It also reduced chemokines (TARC/CCL17, RANTES/CCL5, MCP-1/CCL2, and IL-8) and the cytokine IL-1β production in HaCaT cells, suggesting potential as a therapeutic candidate for chronic diseases like atopic dermatitis. Finally, in silico studies indicated palmitic acid as a significant contributor to the observed effect. This research not only uncovered the untapped potential of C. linum but also laid the foundation for its integration into the circular bioeconomy, promoting sustainable practices, and innovative applications across various industries.

Hydroxytyrosol (HT) is a well-known compound for its bioactive properties. It is naturally present in olives, olive oil, and wine. Its presence in wines is partly due to its production during alcoholic fermentation by yeast through a hydroxylation of tyrosol formed through the Ehrlich pathway. This work aims to explore the influence of yeast assimilable nitrogen (YAN) and glucose content as precursors of HT formation during alcoholic fermentation. Commercial Saccharomyces cerevisiae QA23 and its metabolically engineered strain were used to ferment synthetic must. Each strain was tested at two different YAN concentrations (210 and 300 mg L−1) and two glucose concentrations (100 and 240 g L−1). This work confirms that the less YAN and the more glucose, the higher the HT content, with fermentations carried out with the metabolically engineered strain being the ones with the highest HT content (0.6 mg L−1).

The use of different rootstocks has a significant effect on the content of flavor components and overall fruit quality. However, little information is available about the metabolic basis of the nutritional value of citrus plants. In this study, UPLC-MS/MS (ultra-performance liquid chromatography-tandem mass spectrometry) was performed to analyze the metabolites of three late-maturing hybrid mandarin varieties (‘Gold Nugget’, ‘Tango’ and ‘Orah’) grafted on four rootstocks (‘Trifoliate orange’, ‘Carrizo citrange’, ‘Red tangerine’ and ‘Ziyang Xiangcheng’). A total of 1006 metabolites were identified through OPLS-DA (Orthogonal Partial Least Squares-Discriminant Analysis) analysis. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis revealed the most critical pathways among the different pathways associated with genes grafted on the four rootstocks that were differentially activated, including tryptophan metabolism and sphingolipid metabolism in ‘Gold Nugget’; tryptophan metabolism, phenylpropanoid biosynthesis and sphingolipid metabolism in ‘Tango’; and pantothenate and CoA biosynthesis- and photosynthesis-related biosynthesis in ‘Orah’. A considerable difference between the different rootstocks was also observed in the accumulation of lipids, phenolic acids and flavonoids; further analysis revealed that the rootstocks regulated specific metabolites, including deacetylnomylinic acid, sudachinoid A, amoenin evodol, rutaevin, cyclo (phenylalanine-glutamic acid), cyclo (proline-phenylalanine), 2-hydroxyisocaproic acid, and 2-hydroxy-3-phenylpropanoic acid. The results of this study provide a useful foundation for further investigation of rootstock selection for late-maturation hybrid mandarin varieties.

Honey is well-known as a food product that is rich in active ingredients and is very popular among consumers. Free amino acids (FAAs) are one of the important nutritional components of honey, which can be used not only as a nutritional indicator of honey but also as an indicator of plant source identification. In this study, the contents of 20 FAAs in seven types of honey from 11 provinces in China were examined for the first time. The 20 FAAs were analyzed by ultra-performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS). By analyzing 93 honey samples from seven types of honey, the FAAs were found to range from 394.4 mg/kg (linden honey) to 1771.7 mg/kg (chaste honey). Proline ranged from 274.55 to 572.48 mg/kg, and methionine was only present in some of the linden honey, chaste honey, acacia honey, and rape honey. Evaluated by amino acid principal component analysis, multifloral grassland honey had the highest overall evaluation score, acacia and jujube honey were the most similar, while chaste honey was the least similar to the other types of honey. In addition, DNA was extracted from 174 Xinjiang grassland honey samples and different plant leaves for PCR and sequencing to identify the species of nectar plants. As a result, 12 families and 25 species of honey plants were identified. The results confirmed the diversity of FAAs in dissimilar types and sources of honey. This study provides a reference for expanding honey quality standards and verifying the authenticity of honey.

Bopyeo-tang (BPT), comprising six medicinal plants, has been used for the treatment of respiratory diseases such as pulmonary fibrosis and chronic obstructive pulmonary disease. In this study, we developed and validated a quantitative method for the quality assessment of BPT using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS). Eighteen marker compounds were separated on an Acquity UPLC BEH C18 reversed-phase column (2.1 mm × 100 mm, 1.7 μm) via gradient elution with a 0.1% aqueous formic acid–acetonitrile mobile phase. The multiple-reaction monitoring mode was used to improve analysis speed and accuracy. The coefficients of determination, limits of detection, and limits of quantitation of the 18 marker compounds were 0.9991–0.9996, 0.36–24.45 μg/L, and 1.07–73.35 μg/L, respectively. The recovery was 85.19–110.25%, and the relative standard deviation of precision was ≤9.01%. When applied to a typical BPT sample, the method revealed a range of concentrations from below the quantitative limit (one compound only) to a maximum of 3.20 mg/freeze-dried g. This method will be used for quality control of BPT preparations.

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.

Background: Paeonia lactiflora Pall. (PLP) is a plant with excellent ornamental and therapeutic value that can be utilized in traditional Chinese medicine as Paeoniae Radix Alba (PRA) and Paeoniae Radix Rubra (PRR). PRA must undergo the “peeling” process, which involves removing the cork and a portion of the phloem. PLP’s biological function is strongly linked to its secondary metabolites, and the distribution of metabolites in different regions of the PLP rhizome causes changes in efficacy when PLP is processed into various therapeutic compounds. Methods: The metabolites of the cork (cor), phloem (phl), and xylem (xyl) were examined in the roots of PLP using a metabolomics approach based on UPLC-Q-Exactive-Orbitrap-MS/MS (UPLC-MS/MS), and the differential metabolites were evaluated using multivariate analysis. Results: Significant changes were observed among the cor, phl, and xyl samples. In both positive and negative ion modes, a total of 15,429 peaks were detected and 7366 metabolites were identified. A total of 525 cor-phl differential metabolites, 452 cor-xyl differential metabolites, and 328 phl-xyl differential metabolites were evaluated. Flavonoids, monoterpene glycosides, fatty acids, sugar derivatives, and carbohydrates were among the top 50 dissimilar chemicals. The key divergent metabolic pathways include linoleic acid metabolism, galactose metabolism, ABC transporters, arginine biosynthesis, and flavonoid biosynthesis. Conclusion: The cor, phl, and xyl of PLP roots exhibit significantly different metabolite types and metabolic pathways; therefore, “peeling” may impact the pharmaceutical effect of PLP. This study represents the first metabolomics analysis of the PLP rhizome, laying the groundwork for the isolation and identification of PLP pharmacological activity, as well as the quality evaluation and efficacy exploration of PLP.

Sea buckthorn pomace (SBP) is a by-product of sea buckthorn processing that is rich in bioactive compounds. In this study, different active ingredients were extracted by using different solvents (water, methanol, ethanol, glycerol, ethyl acetate, and petroleum ether) combined with an ultrasonic assisted method. The correlation between the active ingredients and antioxidant properties of the extract was studied, which provided a research basis for the comprehensive utilization of SBP. This study revealed that the 75% ethanol extract had the highest total phenolic content (TPC) of 42.86 ± 0.73 mg GAE/g, while the 75% glycerol extract had the highest total flavonoid content (TFC) of 25.52 ± 1.35 mg RTE/g. The ethanol extract exhibited the strongest antioxidant activity at the same concentration compared with other solvents. The antioxidant activity of the ethanol, methanol, and glycerol extracts increased in a concentration-dependent manner. Thirteen phenolic compounds were detected in the SBP extracts using UPLC-MS/MS analysis. Notably, the 75% glycerol extract contained the highest concentration of all identified phenolic compounds, with rutin (192.21 ± 8.19 μg/g), epigallocatechin (105.49 ± 0.69 μg/g), and protocatechuic acid (27.9 ± 2.38 μg/g) being the most abundant. Flavonols were found to be the main phenolic substances in SBP. A strong correlation was observed between TPC and the antioxidant activities of SBP extracts. In conclusion, the choice of solvent significantly influences the active compounds and antioxidant activities of SBP extracts. SBP extracts are a valuable source of natural phenolics and antioxidants.

This study aims to explore the effects of frying conditions on the formation of HAs and PAHs in crispy pork spareribs, a popular meat commodity sold on Taiwan’s market. Raw pork spareribs were marinated, coated with sweet potato powder, and fried in soybean oil and palm oil at 190 °C/6 min or 150 °C/12 min, followed by an analysis of HAs and PAHs via QuEChERS coupled with UPLC-MS/MS and GC-MS/MS, respectively. Both HAs and PAHs in pork spareribs during frying followed a temperature- and time-dependent rise. A total of 7 HAs (20.34–25.97 μg/kg) and 12 PAHs (67.69–85.10 μg/kg) were detected in pork spareribs fried in soybean oil and palm oil at 150 °C/12 min or 190 °C/6 min, with palm oil producing a higher level of total HAs and a lower level of total PAHs than soybean oil. The content changes of amino acid, reducing sugar, and creatinine played a vital role in affecting HA formation, while the degree of oil unsaturation and the contents of precursors including benzaldehyde, 2-cyclohexene-1-one, and trans,trans-2,4-decadienal showed a crucial role in affecting PAH formation. The principal component analysis revealed that HAs and PAHs were formed by different mechanisms, with the latter being more liable to formation in pork spareribs during frying, while the two-factorial analysis indicated that the interaction between oil type and frying condition was insignificant for HAs and PAHs generated in crispy pork spareribs. Both CcdP (22.67–32.78 μg/kg) and Pyr (16.70–22.36 μg/kg) dominated in PAH formation, while Harman (14.46–17.91 μg/kg) and Norharman (3.41–4.55 μg/kg) dominated in HA formation in crispy pork spareribs during frying. The outcome of this study forms a basis for learning both the variety and content of HAs and PAHs generated during the frying of pork spareribs and the optimum frying condition to minimize their formation.

Ginseng Radix et Rhizoma Rubra (Panax ginseng C.A. Mey, Hongshen, in Chinese) and Ophiopogonis Radix (Ophiopogon japonicus (L.f) Ker-Gawl., Maidong, in Chinese) are traditional Chinese herbal pairs, which were clinically employed to enhance the immune system of cancer patients. This study employed the pharmacokinetic and pharmacodynamic (PK–PD) spectrum-effect association model to investigate the antitumor active substances of P. ginseng and O. japonicus (PG–OJ). The metabolic processes of 20 major bioactive components were analyzed using Ultra-Performance Liquid Chromatography–Mass Spectrometry/Mass Spectrometry (UPLC–MS/MS) in the lung tissue of tumor-bearing mice treated with PG–OJ. The ELISA method was employed to detect the levels of TGF-β1, TNF-α, and IFN-γ in the lung tissue of mice at various time points, and to analyze their changes after drug administration. The results showed that all components presented a multiple peaks absorption pattern within 0.083 to 24 h post-drug administration. The tumor inhibition rate of tumor and repair rate of IFN-γ, TNF-α, and TGF-β1 all increased, indicating a positive therapeutic effect of PG–OJ on A549 tumor-bearing mice. Finally, a PK–PD model based on the GBDT algorithm was developed for the first time to speculate that Methylophiopogonanone A, Methylophiopogonanone B, Ginsenoside Rb1, and Notoginsenoside R1 are the main active components in PG–OJ for lung cancer treatment.

DAPB, a new molecule including danshensu, borneol, and a mother nucleus of ACEI (Angiotensin-converting enzyme inhibitors), is being developed as an antihypertensive candidate compound. A rapid, accurate, and sensitive ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was established and validated for the determination of DAPB in rat plasma. Chromatographic separation was performed on an Agilent SB-C18 column after protein precipitation by acetonitrile with a mobile phase consisting of acetonitrile and deionized water with 0.02% formic acid and 5 mM NH4F (v/v) at a flow rate of 0.2 mL/min. Quantification was performed using electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode. The method was linear over the range of 2–1000 ng/mL. The intra- and inter-day precision was within 12%, with accuracies less than 7%. Stability was within the acceptable limits under various storage and processing conditions. No apparent matrix effect was detected. The validated method was applied to the pre-clinical pharmacokinetic study of DAPB after oral administration of 30 mg/kg and intravenous administration of 6 mg/kg in rats.

The calcium-dependent serine endoprotease PACE4 is evaluated as a therapeutic target for prostate cancer. The peptide Ac-[d-Leu]LLLRVK-amba inhibits PACE4 with high affinity and has shown efficacy in preclinical mice xenograft models of prostate cancer. To support in vivo examinations of the potential therapeutic peptide Ac-[d-Leu]LLLRVK-amba, we established a highly sensitive assay for its quantification in mouse whole blood microsamples based on UPLC-MS/MS determination. Ac-[d-Leu]LLLRVK-amba was very labile during sample processing, which was particularly pronounced in plasma. High resolution mass spectrometric investigations of the metabolism/degradation in plasma revealed that no peptide bond hydrolysis generated products were formed, leaving the cause of the observed consumption of the peptide elusive. As a consequence, whole-blood quantification was developed relying on the immediate snap-freezing of blood samples after collection and immediate sample processing after serial thawing to ensure accurate and reliable quantification. The assay was validated according to the applicable recommendations of the FDA and EMA in a range of 10–10,000 ng/mL and applied to determine the pharmacokinetics of Ac-[d-Leu]LLLRVK-amba after intravenous and intraperitoneal administration to mice. Individual pharmacokinetic profiles were assessed using four microsamplings per animal. Intraperitoneal absorption was found to be efficient, demonstrating that this well-manageable route of administration is feasible for preclinical efficacy experiments with Ac-[d-Leu]LLLRVK-amba.

Despite potential health implications, data on the presence of Glyphosate (GLY) and other non-GLY herbicides in human matrices remain scarce. This study aimed to develop a simple and cost-effective methodology for detecting and quantifying GLY, its primary biodegradation product; aminomethylphosphonic acid (AMPA); and glufosinate (GLU) in plasma and urine of environmentally and occupationally exposed populations from the province of Córdoba (Argentina). Different alternatives of pre-treatment, derivatization with FMOC-Cl, solid phase extraction, and final sample conditioning steps were evaluated to improve the quantification of the herbicides by a high-performance liquid chromatography system coupled to a triple-quadrupole mass spectrometer. Recoveries ranged from 39 to 84% in both matrices, while limits of quantification were 3, 1, and 0.3 ng/mL and 3.6, 5.1, and 0.3 ng/mL for AMPA, GLY, and GLU in plasma and urine, respectively. In plasma samples, GLY was the most frequently detected analyte (32%), followed by GLU (10%). In urine samples, GLU was the most frequently detected herbicide (13%), followed by GLY (6%). No differences between group or matrix correlations were found. This study is the first report of GLU in human biological matrices and should be used to establish baseline values for future surveillance systems.

Coffee cherries contain a crucial flavor-precursor and chemical substances influencing roasted bean quality, yet limited knowledge exists on metabolite changes during cherry ripening. Our study identified 1078 metabolites, revealing 46 core differential metabolites using a KEGG pathway analysis. At the GF vs. ROF stage, amino acid synthesis dominated; ROF vs. BRF featured nucleotide catabolism; BRF vs. PRF exhibited glycoside and flavonoid synthesis; and PRF vs. PBF involved secondary metabolite synthesis and catabolism. The PRF stage emerged as the optimal cherry-harvesting period. A correlation analysis identified core differential metabolites strongly linked to taste indicators, suggesting their potential as taste markers. Notably, nucleotides and derivatives exhibited significant negative correlations with glycosides and flavonoids during ripening. This research systematically analyzed flavor and active substances in green coffee beans during cherry ripening, offering valuable insights into substance formation in Coffea arabica L.

Korla fragrant pear (Pyrus sinkiangensis Yü) fruits have a unique flavor and are rich in phenolic acids, flavonoids, amino acids, and other nutrients. At present, the molecular basis of the quality differences among Korla fragrant pear fruits with a convex calyx and rough skin (RS), calyx shedding (SD), and a convex calyx (CV) remains unknown. To analyze the main metabolic components of Korla fragrant pear fruits and compare the antioxidant activities of these three fruits with different qualities, we used nutrient composition analysis and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS)-based widely targeted metabolomics approaches to analyze the changes in the quality characteristics of the pulp of these three Korla fragrant pear fruits with different appearances. The nutrient composition analysis showed that the fructose and glucose contents were not significantly different, and sucrose and vitamin C contents were significantly higher in SD fruits compared with CV and RS fruits. However, the levels of flavor substances such as titratable acids, total phenols, and total flavonoids were high in the pulp of RS fruits. The metabolomics results identified 1976 metabolites that were clustered into 12 categories, and phenolic acid and flavonoid metabolites were the most abundant. The differentially accumulated metabolites (DAMs) in the fruits with different appearances were screened by multivariate statistical methods, and a total of 595 DAMs were detected. The analysis identified 300 DAMs between the CV and SD fruits, 246 DAMs between the RS and CV fruits, and 405 DAMs between the RS and SD groups. SD fruits contained the most metabolites with a high relative content, especially phenolic acids, lipids, amino acids and derivatives, alkaloids, and organic acids. Compared with CV fruits, flavonoid metabolism was more active in RS fruits, which also had a higher content of flavonoids, whereas the fewest metabolites were found in CV fruits, which also displayed less flavonoid accumulation. KEGG pathway enrichment analysis revealed that the DAMs were mainly enriched in the metabolic pathways of flavone and flavonol biosynthesis, confirming that CV fruits have decreased flavone and flavonol biosynthesis and accumulate fewer flavonoids than RS fruits, which may explain the less bitter and astringent flavor of CV fruits. However, the flavonoid content in RS fruits was very high, which may be one of the reasons why RS fruits have a harder pulp and are less juicy, more slaggy, and less flavorful. Moreover, the analysis of the antioxidant activity showed that during fruit development and maturation, RS fruits had stronger antioxidant activity than SD and CV fruits. These results provide a theoretical basis for improving the fruit quality of Korla fragrant pears and the processing of pear pulp.

Trichoderma, a well-known and extensively studied fungal genus, has gained significant attention for its remarkable antagonistic abilities against a wide range of plant pathogens. In this study, a total of 108 Trichoderma isolates were screened through in vitro dual antagonistic assays and culture filtrate inhibition against Fusarium graminearum. Of these, the YNQJ1002 displayed noteworthy inhibitory activities along with thermal stability. To validate the metabolic differences between YNQJ1002 and GZLX3001 (with strong and weak antagonism, respectively), UPLC-TOF-MS/MS mass spectrometry was employed to analyze and compare the metabolite profiles. We identified 12 significantly up-regulated metabolites in YNQJ1002, which include compounds like Trigoneoside, Torvoside, trans,trans-hepta-2,4,6-trienoic acid, and Chamazulene. These metabolites are known for their antimicrobial properties or signaling roles as components of cell membranes. Enriched KEGG analysis revealed a significant enrichment in sphingolipid metabolism and linoleic acid metabolism, as well as autophagy. The results demonstrated that YNQJ1002’s abundance of antimicrobial substances, resulting from specific metabolic pathways, enhanced its superior antagonistic activity against F. graminearum. Finally, YNQJ1002 was identified using the ITS, tef1-1α, and rpb2 regions, with MIST system sequence matching confirming its classification within the species. Overall, we have obtained a novel strain, T. asperellum YNQJ1002, which is rich in metabolites and shows potential antagonistic activity against F. graminearum. This study has opened promising prospects for the development of innovative Trichoderma-derived antifungal compounds, featuring a unique mechanism against pathogens.

Determining the influence of environmental factors on the stability of drugs is very helpful when choosing excipients, storage conditions or packaging materials. In addition, information about possible toxic degradation products enables detecting and avoiding the harmful side effects of the drug. We used the thin-layer chromatographic-densitometric procedure for the assay of five coxibs, conducted degradation studies in various environments and at different temperatures along with the determination of pharmacokinetic parameters. The results were subjected to chemometric analysis, to investigate and visualize the similarities and differences of the studied coxibs. Samples of the tested drug were also analyzed by UPLC-MS/MS in order to identify degradation products, and determine possible drug degradation pathways. Using the human liver cancer HepG2 cell line, the hepatotoxic effect of the degradation products was also determined. It was observed that all substances were relatively stable under the analyzed conditions and degraded more in acidic than alkaline environments. Robenacoxib is the drug that decomposes the fastest, and cimicoxib turned out to be the most stable. Robenacoxib also showed significant hepatotoxicity at the highest tested concentration, which correlates with the high degree of its degradation, and the probable formation of a more hepatoxic product. The obtained mass spectra of compounds formed as a result of hydrolysis of the protonated drug leading to the formation of several product ions, which enabled us to propose probable degradation pathways.

To systematically and comprehensively investigate the metabolic characteristics of coloring substances and floral aroma substances in Camellia oleifera petals with different colors, ultrahigh-performance liquid chromatography–mass spectrometry (UPLC–MS/MS) and headspace solid phase microextraction and gas chromatography–mass spectrometry (HS–SPME–GC–MS) metabolomics methods were applied to determine the metabolic profiles of white, candy-pink and dark-red petals. The results revealed that 270 volatile organic compounds were detected, mainly terpenoids, heterocyclic, esters, hydrocarbons, aldehydes, and alcohols, in which phenylethyl alcohol, lilac alcohol, and butanoic acid, 1-methylhexyl ester, hotrienol, alpha-terpineol and 7-Octen-4-ol, 2-methyl-6-methylene-, (S)-, butanoic acid, 2-methyl-, 2-methylbutyl ester, 2,4-Octadienal, (E,E)- could act as the floral scent compounds. A total of 372 flavonoid compounds were identified, and luteolin, kaempferol, cyanidin and peonidin derivatives were considered as the main coloring substances for candy-pink and dark-red petal coloration. In conclusion, this study intuitively and quantitatively exhibited the variations in flower color and floral scent of C. oleifera petal with different colors caused by changes in variations of flavonoids and volatile organic compound composition, and provided useful data for improving the sensory quality and breeding of C. oleifera petals.

Acalabrutinib, commercially known as Calquence®, is a pharmacological molecule that has robust inhibitory activity against Bruton tyrosine kinase. The medicine in question was carefully developed by the esteemed pharmaceutical company AstraZeneca. The FDA granted authorization on 21 November 2019 for the utilization of acalabrutinib (ACB) in the treatment of small lymphocytic lymphoma (SLL) or chronic lymphocytic leukemia (CLL) in adult patients. The aim of this study was to develop a UPLC–MS/MS method that is effective, accurate, environmentally sustainable, and has a high degree of sensitivity. The methodology was specifically developed with the intention of quantifying ACB in human liver microsomes (HLMs). The methodology described above was subsequently utilized to assess the metabolic stability of ACB in HLMs in an in vitro environment. The validation procedures for the UPLC–MS/MS method in the HLMs were conducted in accordance with the bioanalytical method validation criteria established by the U.S.- DA. The utilization of the StarDrop software (version 6.6), which integrates the P450 metabolic module and DEREK software (KB 2018 1.1), was employed for the purpose of evaluating the metabolic stability and identifying potential hazardous alarms associated with the chemical structure of ACB. The calibration curve, as established by the ACB, demonstrated a linear correlation across the concentration range of 1 to 3000 ng/mL in the matrix of HLMs. The present study conducted an assessment of the accuracy and precision of the UPLC–MS/MS method in quantifying inter-day and intra-day fluctuations. The inter-day accuracy demonstrated a spectrum of values ranging from −1.00% to 8.36%, whilst the intra-day accuracy presented a range of values spanning from −2.87% to 4.11%. The t1/2 and intrinsic clearance (Clint) of ACB were determined through in vitro testing to be 20.45 min and 39.65 mL/min/kg, respectively. The analysis concluded that the extraction ratio of ACB demonstrated a moderate level, thus supporting the recommended dosage of ACB (100 mg) to be administered twice daily for the therapeutic treatment of persons suffering from B-cell malignancies. Several computational tools have suggested that introducing minor structural alterations to the butynoyl group, particularly the alpha, beta-unsaturated amide moiety, or substituting this group during the drug design procedure, could potentially enhance the metabolic stability and safety properties of novel derivatives in comparison to ACB.

This study developed and validated a robust UPLC-MS/MS method for quantifying cyclic glycine-proline (cGP) in mangrove-derived Penicillium and Aspergillus strains. The method demonstrated excellent linearity, precision, and recovery, with detection limits as low as 4.8 ng/mL. Penicillium pedernalense extract achieved a cGP content of 67.45 ± 1.11 ng/mL, with a corresponding fermentation yield of 29.31 ± 0.61 mg/L. This surpassed Penicillium steckii , which reached a content of 31.71 ± 0.31 ng/mL, with a yield of 8.51 ± 0.15 mg/L. This quantitative approach for metabolite analysis provides a viable method for screening these fungal strains, highlighting their potential for sustainable production of cyclic glycine-proline (cGP).

Mycotoxins and pesticides are the most concerning chemical contaminants that can affect the quality of Pu-erh tea during its production and storage. This study presents a method that can simultaneously determine 31 pesticide residues and six mycotoxins in Pu-erh tea within 11 min using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) after QuEChERS extraction. The lower limit of quantification (LOQ) for all analytes ranged between 0.06 and 50 ppb. Recoveries for each pesticide and mycotoxin ranged between 62.0 and 130.3%, with intra- and inter-day precisions lower than 15%. Good linear relationships were obtained, with correlation coefficients of r2 > 0.991 for all analytes. The established method was applied to 31 Pu-erh tea samples, including raw and ripened Pu-erh tea with different storage times. As a result, pesticide residues were not detected in any of the collected samples, and the mycotoxins detected in the samples were well below the official maximum residue limits (MRLs). Notably, the levels of aflatoxin B1 (AFB1), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) were lower than 1 ppb in the samples stored for more than 30 years.

Toxic compounds such as heterocyclic amines (HAs) and polycyclic aromatic hydrocarbons (PAHs) can be produced during food processing, especially meat products. This study aims to monitor the formation of HAs and PAHs in fried pork fiber, a common meat product in Taiwan, at different processing conditions. A total of six experimental groups, including raw pork tenderloin, dried pork filaments, sesame oil-stir-fried pork at 160 °C for 15 min, sesame oil-stir-fried pork at 200 °C for 6 min, lard-stir-fried pork at 160 °C for 15 min, and lard-stir-fried pork at 200 °C for 6 min, were prepared and analyzed for formation of HAs via UPLC-MS/MS and PAHs via GC-MS/MS in triplicate. Frying in sesame oil or lard showed a greater content of total HAs in fried pork fiber processed at 160 °C for 15 min than at 200 °C for 6 min. However, in the same heating conditions, pork fiber fried in sesame oil produced a higher level of total HAs than that fried in lard. Of the various HAs in fried pork fiber, both Harman and Norharman were generated in the highest amount. The precursors, including reducing sugar, amino acid, and creatine/creatinine, played a vital role in HAs formation in fried pork fiber. For total PAHs, the highest level was shown for pork fiber fried in lard at 200 °C/6 min, followed by frying in sesame oil at 200 °C/6 min and 160 °C/15 min, and in lard at 160 °C/15 min. Like HAs, at the same heating condition, a greater content of total PAHs was produced in pork fiber fried in sesame oil than in lard. Notably, the highly toxic benzo[a]pyrene was undetected in fried pork fiber. The PAH precursor benzaldehyde was shown to generate at a much higher level than 2-cyclohexene-1-one and trans,trans-2,4-decadienal in fried pork fiber, and it should play a more important role in PAH formation. Principal component analysis (PCA) also revealed that the formation mechanism of HAs and PAHs in fried pork fiber was different.

Lonicerae japonicae Flos (LJF) possesses a good anti-respiratory syncytial virus (RSV) effect. However, the material basis of LJF in treating RSV is still unclear. In this study, a sensitive and accurate quantitative method based on UHPLC-QQQ MS was established and validated for the simultaneous determination of the 15 ingredients from LJF in RSV-infected mice plasma. Multiple reaction monitoring was performed for quantification of the standards and of the internal standard in plasma. All the calibration curves show good linear regression within the linear range (r2 > 0.9918). The method validation results, including specificity, linearity, accuracy, precision, extraction recovery, matrix effect, and stability of 15 ingredients, are all within the current acceptance criteria. This established method was successfully applied to the pharmacokinetic study of 15 compounds from LJF. Furthermore, the repair rate of lung index and the improvement rate of IFN-γ and IL-6 improved after administration of the LJF, indicating that LJF possessed a positive effect on the treatment of RSV infection. Finally, by combining Spearman and Grey relation analysis, isochlorogenic acid B, isochlorogenic acid C, secoxyloganin, chlorogenic acid, and loganic acid are speculated to be the main effective ingredients of LJF in treating RSV. This study lays the foundation for attempts to reveal the mechanisms of the anti-RSV effect of LJF.

Before use as medicines, most traditional Chinese medicine (TCM) plants are processed and decocted. During processing, there may be some changes in pesticide residues in TCM. In recent years, reports have studied the changes of pesticides during the processes of boiling, drying and peeling of TCM materials but have rarely involved special processing methods for TCM, such as ethanol extraction and volatile oil extraction. The changes of carbendazim, carbofuran, pyridaben and tebuconazole residues in common processing methods for P. cablin products were systemically assessed in this study. After each processing step, the pesticides were quantitated by UPLC-MS/MS. The results showed amount decreases in various pesticides to different extents after each processing procedure. Processing factor (PF) values for the four pesticides after decoction, 75% ethanol extraction and volatile oil extraction were 0.02~0.75, 0.40~0.98 and 0~0.02, respectively, which indicated that residual pesticide concentrations may depend on the processing technique. A risk assessment according to the hazard quotient with PF values showed that residual pesticide amounts in P. cablin were substantially lower than levels potentially posing a health risk. Overall, these findings provide insights into the safety assessment of P. cablin.

Dietary isoflavones, a type of phytoestrogens, have gained importance owing to their health-promoting benefits. However, the beneficial effects of isoflavones are mediated by smaller metabolites produced with the help of gut bacteria that are known to metabolize these phytoestrogenic compounds into Daidzein and Genistein and biologically active molecules such as S-Equol. Identifying and measuring these phytoestrogens and their metabolites is an important step towards understanding the significance of diet and gut microbiota in human health and diseases. We have overcome the reported difficulties in quantitation of these isoflavones and developed a simplified, sensitive, non-enzymatic, and sulfatases-free extraction methodology. We have subsequently used this method to quantify these metabolites in the urine of mice using UPLC-MS/MS. The extraction and quantitation method was validated for precision, linearity, accuracy, recoveries, limit of detection (LOD), and limit of quantification (LOQ). Linear calibration curves for Daidzein, Genistein, and S-Equol were set up by performing linear regression analysis and checked using the correlation coefficient (r2 > 0.995). LOQs for Daidzein, Genistein, and S-Equol were 2, 4, and 2 ng/mL, respectively. This UPLC-MS/MS swift method is suitable for quantifying isoflavones and the microbial-derived metabolite S-Equol in mice urine and is particularly useful for large numbers of samples.

Entrectinib is an oral selective inhibitor of the neurotrophic T receptor kinase (NTRK). It is used in the treatment of solid tumors in NTRK gene fusion lung cancer. The study aimed to develop and validate an analytical method for quantifying entrectinib plasma by UPLC-MS/MS using quizartinib as an internal standard. The method involves liquid–liquid extraction of entrectinib from plasma using tert butyl methyl ether. The mass-to-charge transitions were 561.23 → 435.1 for entrectinib and 561.19 → 114.1 for quizartinib. The method was successfully validated according to ICH and FDA guidelines. The method has a low quantification limit of 0.5 ng/mL, and the calibration curves constructed over a wide range of 0.5–1000 ng/mL showed good linearity (≥0.997). This method exhibits a tenfold increase in sensitivity compared with the previous method. The method is also accurate, precise, and reproducible, as evidenced by the inter-day and intra-day accuracy and precision values of 82.24–93.33% and 3.64–14.78%, respectively. Principles of green analytical chemistry were considered during all analytical steps to ensure safety. The greenness of the methods was evaluated using two assessment tools. These tools are the Analytical Eco-Scale and the analytical greenness metric approach (AGREE). The results were satisfactory and compatible with the criteria of these tools for green assessment. This method is green, accurate, precise, and reproducible. The method can be used to quantitate entrectinib in plasma and its pharmacokinetics in preclinical, and therapeutic drug monitoring.

Safinamide is an orally active, selective monoamine oxidase-B inhibitor with dopaminergic and non-dopaminergic properties approved by the European Medicine Agency and US Food and Drug Administration for the treatment of mid- to late-stage fluctuating Parkinson’s disease (PD) used in combination with other PD medications such as levodopa. In this study, an analytical quality by design (AQbD) approach was applied to optimize an LC-MS/MS bioanalytical method to determine safinamide in human plasma. A full 33 factorial design was used to optimize safinamide separation conditions, with a method first screened and optimized using chromatographic responses, including peak area and retention time. The results showed that temperature had a significant indirect effect on retention time and peak area (p < 0.05), while ammonium acetate concentration had an insignificant indirect impact on peak area or retention time. However, the temperature was significantly agonistic to the effect of buffer concentration (p < 0.05). The resultant optimized chromatography conditions utilized 9.0 mM ammonium acetate buffer and acetonitrile (22.0:78.0) as mobile phases at a column temperature of 23.2 °C. The assay was linear from 0.1–1000 ng/mL, met acceptance criteria for inter- and intra-assay precision and accuracies across three quality controls, and was successfully applied to in vitro microsomal metabolic stability. The UPLC/MS/MS method was found to be adequately sensitive and suitable for routine safinamide pharmacokinetic studies.

Peaches are the most significant temperate fruit crop worldwide. However, peach fruits are susceptible to fungal and mycotoxin contamination. Consequently, monitoring the residual levels of multiple mycotoxins in peaches and related products is essential. In this study, a novel method based on QuEChERS extraction, followed by ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) detection, was developed for analyzing 14 mycotoxins in peaches and peach products from China. Matrix-matched calibrations were employed to accurately quantify the mycotoxins and compensate for matrix effects. Recoveries for the target analytes ranged from 84.6% to 117.6%, with intra-day and inter-day precision below 20%. The limits of quantification were 2 or 5 μg/L for the 14 mycotoxins. This method was utilized to detect the presence of target mycotoxins in 109 fresh peaches, 100 diseased peaches, and 89 peach products from China. Six mycotoxins were identified in the rotten parts of the diseased peaches, with concentrations ranging from 5.2 to 1664.3 µg/kg. In the remaining parts of the diseased peach samples, only two toxins, alternariol (AOH) and alternariol monomethyl ether (AME), were quantified at levels of 15.3 µg/kg and 15.5 µg/kg, respectively. No mycotoxins were detected in fresh peaches. For peach products, all contamination levels were below the quantitative limits and significantly lower than the maximum legal limits established for the products.

3,4-dichloroaniline (3,4-DCA) and 3,5-dichloroaniline (3,5-DCA) are, respectively, the primary metabolites deriving from the breakdown of phenylurea herbicides and dicarboximide fungicides in both soils and plants, whose residues in vegetable products have a heightened concern considering their higher health risks to humans and greater toxicity than the parent compounds in the environment. In this study, a sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the simultaneous determination of 3,4-DCA and 3,5-DCA residues in chive products based on the optimization of HPLC-MS/MS chromatographic and mass-spectrometric conditions using the standard substances and the modified QuEChERS preparation technique. The preparation efficiency of 3,4-DCA and 3,5-DCA from chive samples showed that acetonitrile was the best extractant. The combination of the purification agent graphite carbon black + primary secondary amine and the eluting agent acetonitrile + toluene (4:1, v/v) had a satisfactory purification effect. The linear correlation coefficients (R2) were more than 0.996 with the six concentration range of 0.001–1.000 mg/L for 3,4-DCA and 3,5-DCA. The limit of detection and limit of quantitation of this method was 0.6 and 2.0 µg/kg for 3,4-DCA, as well as 1.0 and 3.0 µg/kg for 3,5-DCA, respectively. The matrix effect range of 3,4-DCA and 3,5-DCA in chive tissues was from −9.0% to −2.6% and from −4.4% to 2.3%, respectively. The fortified recovery of 3,4-DCA and 3,5-DCA in chive samples at four spiked levels of 0.001–1.000 mg/kg was 75.3–86.0% and 78.2–98.1%, with the relative standard deviation of 2.1–8.5% and 1.4–11.9%, respectively. The limit of detection (LOD) and limit of quantification (LOQ) of the method were 0.6, 2.0, and 1.0, 3.03 for 4-DCA and 3,5-DCA, respectively. This study highlights that the analytical method established here can efficiently and sensitively detect residues of 3,4-DCA and 3,5-DCA residues for monitoring chive products. The method was successfully applied to 60 batches of actual vegetable samples from different regions.

Sanjoin-tang (SJIT) is an ancient oriental medicine prescription listed in the Jinguiyaolue that is mainly used for the treatment of primary insomnia. This study was conducted to develop and validate an ultra-performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS) simultaneous analysis method for the quality control of SJIT using 18 target compounds. The 18 analytes were separated on an Acquity UPLC BEH C18 column maintained at 45 °C using a mobile phase composed of distilled water and acetonitrile. The MS system was used to simultaneously detect all analytes using the multiple reaction monitoring (MRM) method of Xevo TQ-XS coupled with an electrospray ionization source. The concentrations of the 18 analytes investigated in the SJIT samples ranged from below the limit of detection to 9.553 mg/g. In conclusion, the validated UPLC–MS/MS MRM analysis method can be used to obtain basic data to establish chemical-nonclinical linkage efficacy and for the clinical research and quality evaluation of SJIT.

Five volatile thiol compounds (methanethiol, ethanethiol, 2-mercapto-1-ethanol, 2-furfurylthiol, and 2-methyl-3-furanethiol) in fermented grains of sauce-aroma baijiu were determined using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). The samples were pre-treated using a modified QuEChERS method. 4,4′-Dithiodipyridine (DTDP) derivatization reaction improved the detectability and stability of volatile thiol compounds. From the end of the first round to the end of the seventh round of fermentation and different fermentation states from the fifth round of fermented grains of the sauce-aroma baijiu production process were analyzed. The results showed that the concentrations of methanethiol (67.64–205.37 μg/kg), ethanethiol (1.22–1.76 μg/kg), 2-furfurylthiol (0.51–3.03 μg/kg), and 2-methyl-3-furanthiol (1.70–12.74 μg/kg) were increased with the number of fermentation rounds. Methanethiol, 2-furfurylthiol, and 2-methyl-3-furanthiol increased during fermentation and distillation in the fifth round. Fermentation and distillation were important stages for their widespread production. After distillation, there were still a large number of volatile thiol compounds in the fermented grains. The thermal reaction was of great significance in the formation of these thiols.

Umifenovir is one of the most often prescribed antiviral medications for the prevention and treatment of COVID-19 and other viral infections. Herein, a UPLC-MS/MS method is developed through using ibrutinib as an internal standard (IS) for quantifying umifenovir in plasma samples. Both umifenovir and the IS were analytically separated on an Acquity BEH C18 column with a total run time of only 2.5 min. At a flow rate of 0.3 mLmin−1, acetonitrile:15 mM ammonium acetate (80:20) was employed as the mobile phase composition. Electrospray ionization in positive mode was used for ionization of the samples. Detection and quantification were performed in multiple reaction monitoring mode with parent-to-daughter ionization of 477.05 → 279.02 and 441.16 → 84.4 for umifenovir and the IS, respectively. The method was validated through following international guidelines for bioanalytical method validation, and all parameters were within the acceptable limits. Moreover, the eco-scale method using AGREE software was used for the evaluation of greenness, and results showed that the method is very environmentally friendly. The validated assay was successfully employed in the bioavailability assessment of a newly developed formulation of kneaded ternary umifenovir/β-cyclodextrin with 1% poloxamer 188 (KDB).

5-Fluorouracil (5-FU) is now used in eye drops for the management of conjunctival malignant melanoma, intraepithelial neoplasia, and corneal and conjunctival squamous cell carcinoma. The previously used methods for 5-FU quantification in AqH were time-consuming and less sensitive. Herein, a highly perceptive bioanalytical UPLC–MS/MS method was developed for the quantitative determination of 5-FU in the aqueous humor (AqH) of rabbits using allopurinol as the internal standard (IS). The 5-FU and IS were well separated in an Acquity™ HILIC column. Acetonitrile and 10 mM of ammonium acetate at 95:5 (v/v) were isocratically pumped at a 0.3 mL/min flow rate with a total runtime of 2.5 min. AqH samples were processed with a liquid–liquid extraction method in ethyl acetate. The 5-FU and IS were identified in the negative mode with electrospray ionization. The parent to daughter ion transitions for the 5-FU and IS occurred at m/z 128.92→41.68 and 134.80→64.10, respectively, as quantified using the multiple reaction monitoring mode. The developed method was validated with the ICH-Harmonized Guideline for Bioanalytical Method Validation, and the parameters were within acceptable limits. The calibration curve was linear at the 10.5–2000 ng/mL concentration range, with a correlation coefficient (R2) of 0.9946, and the lower limit of detection was 3.55 ng/mL. The developed and validated method was rapid, sensitive, accurate and robustly able to quantify 5-FU in rabbit AqH. The method was effectively applied to establish the ocular pharmacokinetics of 5-FU following the topical instillation of 5-FU-containing preparations in rabbits.

This study aimed to evaluate the absolute bioavailability of cyclosporine in cats by investigating the pharmacokinetic profile after intravenous and oral administration, respectively. Twenty-four clinically healthy cats were enrolled in this study and randomly divided into four groups, namely the intravenous group (3 mg/kg), low oral group (3.5 mg/kg), medium oral group (7 mg/kg), and high oral group (14 mg/kg). Whole blood was obtained at the scheduled time points after a single dose administration and cyclosporine was determined using ultra-performance liquid chromatography–tandem mass spectrometry technology (UPLC-MS/MS). Pharmacokinetic parameters were calculated using the WinNonlin 8.3.4 software via compartmental and non-compartmental models. As a result, the bioavailability values for the low, medium, and high oral groups were 14.64%, 36.98%, and 13.53%, respectively. The nonlinear pharmacokinetic profile was observed in the range from 3.5 mg/kg to 14 mg/kg in cats following oral administration. Whole blood concentrations taken 4 h after oral administration were better correlated with the area under the blood concentration–time curve AUC0–24 with a high regression coefficient (R2 = 0.896). This concentration would be a greater predictor in the following therapeutic drug monitoring. No adverse effect was observed in the whole study process.