Chromatography-triple Quadrupole Tandem Mass Spectrometry Research Articles

Transcriptomics and UHPLC-QQQ-MS analyses reveal the dysregulation of branched chain amino acids metabolism in renal fibrotic rats

The dysregulated levels of branched chain amino acids (BCAA) contribute to renal fibrosis in chronic kidney disease (CKD), yet specific analysis of BCAA contents and how they are regulated still remain unclear. It is therefore of great scientific interest to understand BCAA catabolism in CKD and develop a sensitive method for simultaneous determination of individual BCAA and their metabolites branched chain α-ketoacids (BCKA). In this work, the important role of BCAA metabolism that drives renal fibrosis in the process of CKD was first revealed by using transcriptomics. The key target genes controlling BCAA metabolism were then validated, that is, mRNA levels of BCKDHA and BCKDHB, the regulating rate-limiting enzymes during BCAA metabolism were abnormally reduced by quantitative PCR (qPCR), and a similar drop-off trend of protein expression of BCKDH, HIBCH and MCCC2 that are closely related to BCAA metabolism was also confirmed by western blotting. Furthermore, we established a novel strategy that simultaneously determines 6 individual BCAA and BCKA in serum and tissue. The method based on dansylhydrazine derivatization and ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-QQQ-MS) achieved to simultaneously determine the contents of BCAA and BCKA, which is efficient and stable. Compared with normal rats, levels of BCAA including leucine, isoleucine and valine in serum and kidney of CKD rats was decreased, while BCKA including α-ketoisocaproic acid, α-ketomethylvaleric acid and α-ketoisovaleric acid was increased. Together, these findings revealed the abnormality of BCAA metabolism in driving the course of kidney fibrosis and CKD. Our current study sheds new light on changes in BCAA metabolism during CKD, and may facilitate development of drugs to treat CKD and renal fibrosis.

Biosynthesis of Nanoparticles with Green Tea for Inhibition of β-Amyloid Fibrillation Coupled with Ligands Analysis.

Inhibition of amyloid β protein fragment (Aβ) aggregation is considered to be one of the most effective strategies for the treatment of Alzheimer's disease. (-)-Epigallocatechin-3-gallate (EGCG) has been found to be effective in this regard; however, owing to its low bioavailability, nanodelivery is recommended for practical applications. Compared to chemical reduction methods, biosynthesis avoids possible biotoxicity and cumbersome preparation processes. The interaction between EGCG and Aβ42 was simulated by molecular docking, and green tea-conjugated gold nanoparticles (GT-Au NPs) and EGCG-Au NPs were synthesized using EGCG-enriched green tea and EGCG solutions, respectively. Surface active molecules of the particles were identified and analyzed using various liquid chromatography-tandem triple quadrupole mass spectrometry methods. ThT fluorescence assay, circular dichroism, and TEM were used to investigate the effect of synthesized particles on the inhibition of Aβ42 aggregation. EGCG as well as apigenin, quercetin, baicalin, and glutathione were identified as capping ligands stabilized on the surface of GT-Au NPs. They more or less inhibited Aβ42 aggregation or promoted fibril disaggregation, with EGCG being the most effective, which bound to Aβ42 through hydrogen bonding, hydrophobic interactions, etc. resulting in 39.86% and 88.50% inhibition of aggregation and disaggregation effects, respectively. EGCG-Au NPs were not as effective as free EGCG, whereas multiple thiols and polyphenols in green tea accelerated and optimized heavy metal detoxification. The synthesized GT-Au NPs conferred the efficacy of diverse ligands to the particles, with inhibition of aggregation and disaggregation effects of 54.69% and 88.75%, respectively, while increasing the yield, enhancing water solubility, and decreasing cost. Biosynthesis of nanoparticles using green tea is a promising simple and economical drug-carrying approach to confer multiple pharmacophore molecules to Au NPs. This could be used to design new drug candidates to treat Alzheimer's disease.

Development and validation of a GC-MS/MS method for the determination of iodoacetic acid in biological samples.

Iodoacetic acid (IAA) is a halogenated disinfection by-product of growing concern due to its high cytotoxicity, genotoxicity, endocrine disruptor effects, and potential carcinogenicity. However, the data on distribution and excretion of IAA after ingestion by mammals are still scarce. Here, we developed a reliable and validated method for detecting IAA in biological specimens (plasma, urine, feces, liver, kidney, and tissues) based on modified QuEChERS sample preparation combined with gas chromatography-tandem triple quadrupole mass spectrometry (GC-MS/MS). The detection method for IAA exhibited satisfactory recovery rates (62.6-108.0%) with low relative standard deviations (RSD < 12.3%) and a low detection limit for all biological matrices ranging from 0.007 to 0.032ng/g. The study showed that the proposed method was reliable and reproducible for analyzing IAA in biological specimens. It was successfully used to detect IAA levels in biological samples from rats given gavage administration. The results indicated that IAA was found in various tissues and organs, including plasma, thyroid, the liver, the kidney, the spleen, gastrointestinal tract, and others, 6h after exposure. This study provides the first data on the in vivo distribution inand excretion of IAA by mammals following oral exposure.

Use of serum evaluation of contraceptive and ovarian hormones to assess reduced risk of pregnancy among women presenting for emergency contraception in a multicenter clinical trial

ObjectiveTo evaluate ovulation risk among women enrolling in an emergency contraception (EC) study by measuring contraceptive steroids and ovarian hormones. Study DesignWe used standard chemoluminescent assays to evaluate endogenous hormones [estradiol (E2), progesterone (P4), FSH, LH] and liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS/MS) to simultaneously analyze concentrations of ethinylestradiol (EE), dienogest (DNG), norelgestromin (NGMN), norethindrone (NET), gestodene (GSD), levonorgestrel (LNG), etonogestrel (ENG), segesterone acetate (NES), medroxyprogesterone acetate (MPA), and drospirenone (DRSP), in serum samples obtained at the time of enrollment in a recent study comparing oral ulipristal acetate and LNG EC in women with weight ≥ 80kg reporting no recent use of hormonal contraception. ResultsWe enrolled 532 and obtained a valid baseline blood sample from 520 women. Of these, 117 (22.5%) had detectable concentrations of a progestin [MPA (n=58, 11.2%), LNG (50, 9.6%), ENG (11, 2.1%), NET (5, 0.96%), NGMN (3, 0.06%), or DRSP (1, 0.02%)]. LNG was co-detected in all three participants with samples containing NGMN. Multiple progestins were detected in 8 other women: ENG/MPA (1); ENG/LNG (2); MPA/LNG (5). Samples from 55 (10.6%) had concentrations of one or more progestin considered above the minimum level for contraceptive [MPA ≥ 0.1ng/mL, n = 19; NGMN/LNG ≥ 0.2ng/mL, n = 31; ENG ≥ 0.09 ng/mL, n = 8; NET ≥ 0.35 ng/mL, n = 4]. We detected concentrations of serum P4 ≥ 3ng/mL, indicative of luteal phase (post-ovulation) status, in an additional 194 (37.3%) samples. ConclusionsMore than one third of enrolled in our clinical trial of oral emergency contraception had evidence of prior ovulation at the time of enrollment. Additionally, about 23% had evidence of recent use of hormonal contraception recent use of hormonal contraception. This results would have decreased the expected risk of pregnancy in the study. ImplicationsMany participants in a recent clinical trial of oral emergency contraception did not appear to be at risk for pregnancy or would not have benefited from intervention due to cycle timing. Investigators should consider the effects of these findings on expected pregnancy rates when determining sample size in future EC clinical trials, particularly when using non-inferiority designs or historical controls.Keywords

Detection of Extremely Low Level Ciguatoxins through Monitoring of Lithium Adduct Ions by Liquid Chromatography-Triple Quadrupole Tandem Mass Spectrometry.

Ciguatera poisoning (CP) is the most common type of marine biotoxin food poisoning worldwide, and it is caused by ciguatoxins (CTXs), thermostable polyether toxins produced by dinoflagellate Gambierdiscus and Fukuyoa spp. It is typically caused by the consumption of large fish high on the food chain that have accumulated CTXs in their flesh. CTXs in trace amounts are found in natural samples, and they mainly induce neurotoxic effects in consumers at concentrations as low as 0.2 µg/kg. The U.S. Food and Drug Administration has established CTX maximum permitted levels of 0.01 µg/kg for CTX1B and 0.1 µg/kg for C-CTX1 based on toxicological data. More than 20 variants of the CTX1B and CTX3C series have been identified, and the simultaneous detection of trace amounts of CTX analogs has recently been required. Previously published works using LC-MS/MS achieved the safety levels by monitoring the sodium adduct ions of CTXs ([M+Na]+ > [M+Na]+). In this study, we optimized a highly sensitive method for the detection of CTXs using the sodium or lithium adducts, [M+Na]+ or [M+Li]+, by adding alkali metals such as Na+ or Li+ to the mobile phase. This work demonstrates that CTXs can be successfully detected at the low concentrations recommended by the FDA with good chromatographic separation using LC-MS/MS. It also reports on the method's new analytical conditions and accuracy using [M+Li]+.

Simultaneous determination of seven artemisinin-related compounds in Artemisia annua by UPLC-QQQ-MS/MS

A variety of compounds in Artemisia annua were simultaneously determined to evaluate the quality of A. annua from multiple perspectives. A method based on ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLC-QQQ-MS/MS) was established for the simultaneous determination of seven compounds: amorpha-4,11-diene, artemisinic aldehyde, dihydroartemisinic acid, artemisinic acid, artemisinin B, artemisitene, and artemisinin, in A. annua. The content of the seven compounds in different tissues(roots, stems, leaves, and lateral branches) of A. annua were compared. The roots, stems, leaves, and lateral branches of four-month-old A. annua were collected and the content of seven artemisinin-related compounds in different tissues was determined. A multi-reaction monitoring(MRM) acquisition mode of UPLC-QQQ-MS/MS was used, with a positive ion mode of atmospheric pressure chemical ion source(APCI). Chromatographic separation was achieved on an Eclipse Plus RRHD C_(18) column(2.1 mm×50 mm, 1.8 μm). The gradient elution was performed with the mobile phase consisted of formic acid(0.1%)-ammonium formate(5 mmol·L~(-1))(A) and the methanol(B) gradient program of 0-8 min, 55%-100% B, 8-11 min, 100% B, and equilibrium for 3 min, the flow rate of 0.6 mL·min~(-1), the column temperature of 40 ℃, the injection volume of 5 μL, and the detection time of 8 min. Through methodological investigation, a method based on UPLC-QQQ-MS/MS was established for the simultaneous quantitative determination of seven representative compounds involved in the biosynthesis of artemisinin. The content of artemisinin in A. annua was higher than that of artemisinin B, and the content of artemisinin and dihydroartemisinic acid were high in all the tissues of A. annua. The content of the seven compounds varied considerably in different tissues, with the highest levels in the leaves and neither artemisinene nor artemisinic aldehyde was detected in the roots. In this study, a quantitative method based on UPLC-QQQ-MS/MS for the simultaneous determination of seven representative compounds involved in the biosynthesis of artemisinin was established, which was accurate, sensitive, and highly efficient, and can be used for determining the content of artemisinin-related compounds in A. annua, breeding new varieties, and controlling the quality of Chinese medicinal materials.

Toxicokinetics of MDMA and Its Metabolite MDA in Rats.

To investigate the toxicokinetic differences of 3,4-methylenedioxy-N-methylamphetamine (MDMA) and its metabolite 4,5-methylene dioxy amphetamine (MDA) in rats after single and continuous administration of MDMA, providing reference data for the forensic identification of MDMA. A total of 24 rats in the single administration group were randomly divided into 5, 10 and 20 mg/kg experimental groups and the control group, with 6 rats in each group. The experimental group was given intraperitoneal injection of MDMA, and the control group was given intraperitoneal injection of the same volume of normal saline as the experimental group. The amount of 0.5 mL blood was collected from the medial canthus 5 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h after administration. In the continuous administration group, 24 rats were randomly divided into the experimental group (18 rats) and the control group (6 rats). The experimental group was given MDMA 7 d by continuous intraperitoneal injection in increments of 5, 7, 9, 11, 13, 15, 17 mg/kg per day, respectively, while the control group was given the same volume of normal saline as the experimental group by intraperitoneal injection. On the eighth day, the experimental rats were randomly divided into 5, 10 and 20 mg/kg dose groups, with 6 rats in each group. MDMA was injected intraperitoneally, and the control group was injected intraperitoneally with the same volume of normal saline as the experimental group. On the eighth day, 0.5 mL of blood was taken from the medial canthus 5 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h after administration. Liquid chromatography-triple quadrupole tandem mass spectrometry was used to detect MDMA and MDA levels, and statistical software was employed for data analysis. In the single-administration group, peak concentrations of MDMA and MDA were reached at 5 min and 1 h after administration, respectively, with the largest detection time limit of 12 h. In the continuous administration group, peak concentrations were reached at 30 min and 1.5 h after administration, respectively, with the largest detection time limit of 10 h. Nonlinear fitting equations for the concentration ratio of MDMA and MDA in plasma and administration time in the single-administration group and continuous administration group were as follows: T=10.362C-1.183, R2=0.974 6; T=7.397 3C-0.694, R2=0.961 5 (T: injection time; C: concentration ratio of MDMA to MDA in plasma). The toxicokinetic data of MDMA and its metabolite MDA in rats, obtained through single and continuous administration, including peak concentration, peak time, detection time limit, and the relationship between concentration ratio and administration time, provide a theoretical and data foundation for relevant forensic identification.

Mitigation of multiple pesticide residues in green bell pepper using natural extracts as an emerging trend in pesticide decontamination using GC–MS/MS

AbstractThe main objective of this work is to assess the effect of natural extracts and its characterization for the mitigation of multiple pesticide residues in green bell pepper. In this study, four plant extracts such as Acacia concinna, Albizia amara, Tamarindus indica, and Azadirachta indica have been used to mitigate the six pesticides present in green bell pepper and its phytochemical constituents were analyzed using gas chromatography coupled to triple quadrupole tandem mass spectrometry (GC–MS/MS). In addition to that, the method for the quantification of six pesticide residues in green bell pepper using GC–MS/MS was validated according to SANTE/11312/2021 guidelines. The extracts of Acacia concinna, Albizia amara, Tamarindus indica, and Azadirachta indica were showed effective mitigation for selected pesticides. The phytochemical compounds present in plant extracts were correlated with its effect on the mitigation of multiple pesticide residues. The T. indica showed better pesticide mitigation efficiency when compared to A. indica, A. amara, and A. concinna, respectively. The plant extracts showed higher mitigation efficiency for organophosphorous pesticides than the synthetic pyrethroids. The nutritional and sensorial properties of green bell pepper were highly retained after pesticide decontamination using plant extracts. The results of this work indicated the application of plant extracts for the mitigation of multiple pesticides in green bell pepper by household and commercial processing units. This study addressed the balance between food safety and food quality.Practical ApplicationsPesticide contamination in vegetables is one of the major global food safety issues and it poses potent threat to human health. Pesticide decontamination is always a challenging task due to the unpredictability of types and levels of pesticides and the presences of multiple pesticides in contaminated vegetables. Washing of vegetables with water is the common procedure followed by both household and commercial units. However, washing of vegetables only with water is not enough to reduce the multiple pesticides residues to safe levels. The washing of vegetables with chemical solutions and other thermal and non‐thermal processing could result in toxic byproduct formation based on the types of pesticides present in contaminated sample. Hence, in this study, the plant extracts were characterized for its efficiency on the mitigation of multiple pesticide residues in vegetables has been analyzed by Gas Chromatography—Triple Quadrupole Tandem Mass Spectrometry and the method has been validated according to SANTE/11312/2021 guidelines. The phytochemical compounds of the plant extracts could be correlated with the effective multiple pesticide decontamination efficiency and the plant extracts were found to retain the physicochemical properties of vegetables during washing. The results of this study showed that the selected plant extracts could enhance the food safety without compromising food quality. The results of this study could be useful for the formulation of vegetables cleaning solutions for the effective mitigation of multiple pesticide residues.

Chemical profiling of Simiao pill and quantification of main effective constituents in it by ultra-high-performance liquid chromatography coupled with Q Exactive Orbitrap and triple quadrupole mass spectrometry.

Simiao pill is one of the most commonly used prescriptions in traditional Chinese medicine for the treatment of hyperuricemia and gout. However, methods based on more accurate and comprehensive qualitative and quantitative analyses of the active ingredients are not yet perfect due to limited methodology. This not only hinders the elucidation of the pharmacological mechanism of Simiao pill, but also its comprehensive clinical development and utilization. In this study, we employed ultra-high-performance liquid chromatography-Q Exactive Orbitrap-mass spectrometry technology to perform rapid analysis and identification of the chemical constituents in Simiao pill. A total of 101 chemical components were identified, including 26 alkaloids, 15 terpenoids, 11 flavonoids, eight steroids, six fatty acids, five limonoids, four saponins, five phenylpropanoids, and 21 other compounds. In addition, we established a new method by high-throughput ultra-high-performance liquid chromatography-Q Exactive Orbitrap-mass spectrometry combined with ultra-high-performance liquid chromatography-triple quadrupole-tandem mass spectrometry technology for quantification of 14 main active ingredients, such as adenosine (1), phellodendrine (2), mangnoflorine (3), β-ecdysterone (4), 25R-inokosterone (5), 25S-inokosterone (6), jatrorrhizine (7), palmatine (8), chikusetsu saponin IVa (9), limonin (10), atractylenolide III (11), atractylenolide I (12), obacunone (13), and atractylenolide II (14) in Simiao pill. This work laid a foundation for further analysis and quality control of effective components in Simiao pill.

The Natural Ficus carica L. (fig) Extract as an Effective Prophylactic Antibacterial Agent for Inflammation-Related Infections.

Klebsiella pneumoniae (K. pneumoniae) is a multidrug-resistance Gram-negative organism responsible for carbapenem-resistant infections. These challenges have inspired studies on the use of natural products as alternatives to conventional drugs. The aim of this study was to analyze the antibacterial and antioxidant effects of Ficus carica L. (fig) branch extracts and to perform in vivo animal experiments to better understand the absorption mechanisms of the antibacterial components during the digestion process after oral administration. The antibacterial components of the fig branch extracts were analyzed via gas chromatography-mass spectrometry (GC-MS). An in vivo animal study and liquid chromatography-triple quadrupole-tandem mass spectrometry (LC-QQQ-MS/MS) analyses were performed to analyze the deacetylation reactions of the fig extracts after oral administration in mice. Ultimately, the antibacterial effects of the fig extracts increased with the fractional distillation time. The fig extracts showed excellent antibacterial effects against K. pneumoniae, as well as Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa). The three antibacterial and antioxidant components of the fig extracts were revealed to be eugenol, acetyleugenol, and psoralen. Interestingly, in this study, we identified acetyleugenol in the phenolic compounds of the fig extract for the first time. Through in vivo animal testing, we observed the deacetylation reaction of acetyleugenol to eugenol in the fig extract as digestion proceeded in the internal organs of the mice after oral administration. The results of this study suggest the use of natural fig extract as an effective therapeutic and prophylactic antibacterial agent for inflammation-related infections with a wide variety of biomedical applications.

Protective effect and mechanism of Maiwei Yangfei Decoction on pulmonary fibrosis mice based on Nrf2 regulation of oxidative stress

This study explored the effect and mechanism of Maiwei Yangfei Decoction(MWYF) on pulmonary fibrosis(PF) mice. MWYF was prepared, and its main components were detected by ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLC-MS/MS). Male C57BL/6J mice were randomly divided into a control group, a model group, a pirfenidone(PFD) group, and low-, medium-, and high-dose MWYF groups, with 10 mice in each group. The PF model was induced in mice except for those in the control group by intratracheal instillation of bleomycin(BLM), and model mice were treated with saline or MWYF or PFD by gavage the next day. The water consumption, food intake, hair, and activity of mice were observed daily. The pathological changes in lung tissues were observed by hematoxylin-eosin(HE) staining, Masson staining, and CT scanning. The level of hydroxyproline(HYP) in lung tissues was detected by alkaline hydrolysis. Immunohistochemistry was used to observe the expression of collagen type Ⅲ(COL3) and fibronectin. The mRNA expression levels of α-smooth muscle actin(α-SMA), type Ⅰ collagen α1(COL1α1), COL3, and vimentin were detected by reverse transcription real-time fluorescence quantitative polymerase chain reaction(RT-qPCR). Superoxide dismutase(SOD) and malondialdehyde(MDA) kits were used to detect oxidative stress indicators in lung tissues and serum. The nuclear translocation of nuclear factor E2-related factor 2(Nrf2) protein was detected by immunofluorescence. The protein and mRNA expression levels of Nrf2, catalase(CAT), and heme oxygenase 1(HO-1) in lung tissues were detected by Western blot and RT-qPCR. Twelve chemical components were detected by UPLC-MS/MS. Animal experiments showed that MWYF could improve alveolar inflammation, collagen deposition, and fibrosis in PF mice, increase body weight of mice, and down-regulate the expression of fibrosis indexes such as HYP, α-SMA, COL1α1, COL3, fibronectin, and vimentin in lung tissues. In addition, MWYF could potentiate the activity of SOD in lung tissues and serum of PF mice, up-regulate the expression level of Nrf2, and promote its transfer to the nucleus, up-regulate the levels of downstream antioxidant target genes CAT and HO-1, and then reduce the accumulation of lipid metabolite MDA. In summary, MWYF can significantly improve the pathological damage and fibrosis of lung tissues in PF mice, and its mechanism may be related to the activation of the Nrf2 pathway to regulate oxidative stress.

Dissipation, residue and dietary risk assessment of difenoconazole in Rosa roxburghii

Rosa roxburghii is a medicinal and edible plant, which is favored by consumers due to its rich vitamin C content. Residues and potential health risks of difenoconazole in the R. roxburghii ecosystem has aroused a concern considering its extensive use for controlling the powdery mildew of R. roxburghii. In this study, the residue of difenoconazole in R. roxburghii and soil was extracted by acetonitrile, purified by primary secondary amine and detected by liquid chromatography-tandem triple quadrupole mass spectrometry. The average recoveries in R. roxburghii and soil matrix varied from 82.59% to 99.63%, with relative standard deviations (RSD) of 1.14%–8.23%. The limit of quantification (LOQ) and detection (LOD) of difenoconazole in R. roxburghii and soil samples were 0.01 mg/kg. The dissipation of difenoconazole followed well the first-order kinetic, with a half-life of 3.99–5.57 d in R. roxburghii and 4.94–6.23 d in soil, respectively. And the terminal residues were <0.01–2.181 mg/kg and 0.014–2.406 mg/kg, respectively. The chronic and acute risk quotient values of difenoconazole were respectively 0.42% and 4.1%, which suggests that the risk was acceptable and safe to consumers. This study provides a reference for the safe and reasonable use of difenoconazole in R. roxburghii production.

Mechanism difference of ginseng medicines with different natures on hyperthyroidism

Ethnopharmacological relevanceMedicinal nature is a unique index in traditional Chinese medicine (TCM) to measure the efficacy of Chinese medicines. Hyperthyroidism, qi-Yin deficiency syndrome in TCM was regarded as the classic heat syndrome category in Chinese medicine. Moreover, it is extensively employed to evaluate the medicinal nature of herbs within the framework of the ‘hot/cold’ nature theory. Ginseng, red and black ginseng (GS, RG, and BG, respectively), and ginseng leaves (GL) are qi-tonifying herbs with different medicinal natures and were just right used to examine the underlying mechanisms between the nature of ginseng medicines and their tonifying effect. Aim of the studyTo examine the different mechanisms between different natures of ginseng medicines and their qi-tonifying action by comparison of their effects on hyperthyroidism and elucidate the nature of BG. Materials and methodsThe hyperthyroidism model was induced through the oral administration of Euthyrox. Steroids belonging to hypothalamus pituitary adrenal (HPA) and hypothalamic pituitary gonadal (HPG) axes were determined with an ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS) method. Furthermore, the biochemical indexes associated with energy metabolism, including the serum cyclic nucleotide system and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signaling pathways were examined. Additionally, the gut microbiota was subjected to 16 S rDNA sequencing, followed by gas chromatography-mass spectrometry (GC-MS) analysis of short-chain fatty acids (SCFAs). ResultsGS, RG, and BG could treat hyperthyroidism rats by regulating the hypothalamus pituitary thyroid (HPT) axis. The RG also had a strong regulatory effect on the HPA and HPG axis. The BG has a regulatory effect on the HPG axis. GL had a strong regulatory effect on HPA, HPG, and HPT axes. GS had the ability to regulate Firmicutes/Bacteroides of the intestinal flora, and GS, RG, and BG ameliorated the decrease of SCFAs induced by hyperthyroidism. According to PCA, BG is similar to GS, indicating a mild-warm nature. ConclusionThe nature of BG was attributable to mild-warm and the GL with cold nature is best for the treatment of hyperthyroidism based on PCA analysis with all the bio-indices. In addition, the mechanism study indicated that the qi-tonifying effects of ginseng medicines are involved in HPT, HPA, and HPG axes to alleviate hyperthyroidism. In addition, the primary indexes reflected different natures of ginseng medicines are cyclic adenosine monophosphate (cAMP)/cyclic guanosine monophosphate (cGMP), AchE, and substance metabolism associated with Na+-K+-ATPase and PI3K/AKT signaling pathways, which are potential biomarkers for evaluation on the natures of qi-tonifying medicines.

Determination of oxylipins and their precursors in breast milk by solid phase extraction-ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry

Oxylipins and their precursors (long-chain polyunsaturated fatty acids, LCPUFAs) are key intercellular signaling molecules influencing the inflammatory response. Each oxylipin has pro- and/or anti-inflammatory effects, and the relative abundance of different oxylipins can alter the inflammatory balance, making it important to clarify the oxylipin profile of breast milk for optimal infant health. The extraction, identification, and simultaneous quantification of oxylipins in breast milk are challenging due to the structural similarity, limited stability, and the low endogenous concentration of oxylipins and the complex matrix of breast milk. This study aimed to develop a solid phase extraction-ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry (SPE-UPLC-MS/MS) method for the comprehensive and specific quantification of oxylipins and their precursors in breast milk. The LC conditions (including column, mobile phase, and gradient conditions) and SPE procedure (including SPE cartridges, elution solvent, and elution volume) were optimized to achieve accurate quantification and better analyte recovery. A single 18-minute chromatographic run allows for the quantification of 20 oxylipins and 5 PUFAs. The results showed good linearity (R2 > 0.99) over the concentration range of 2 to 100 ng/mL, with the instrument detection limits ranging from 0.01 to 0.90 ng/mL for oxylipins and 0.02 to 0.59 ng/mL for PUFAs. The method is rapid, sensitive, and reproducible (RSD ≤ 10%) and is suitable for the quantitative analysis of oxylipins and their precursors in infant formula samples.

Fungal microbiome related to mycotoxin contamination in medicinal and edible seed Semen Persicae

Medicinal and edible seed Semen Persicae is susceptible to mycotoxin and fungal contamination. However, the occurrence of mycotoxin contamination and fungal infection is still unclear. In this paper, ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry and high-throughput sequencing were conducted to determine the mycotoxin contents and fungal abundances of Semen Persicae. 42.86% of samples were positive for aflatoxin B1 (26.48–48.37 μg/kg) and 28.57% of samples were positive for aflatoxin B2 (1.47–4.82 μg/kg). Ochratoxin A and fumonisin B1 were only detected in one sample (91.02 and 34.61 μg/kg, respectively). Chao 1 and Shannon indices were significantly higher in the Dalian of Liaoning, Baotou of Innermongolia and Langfang of Hebei regions than in other groups. Ascomycota, Basidiomycota, Wallemia, Candica, Saccharomyces and Aspergillus were the predominant fungi and they were significantly region-specific. Simultaneously, the diversity, composition and co-occurrence network complexity in the mycotoxin-free group were significantly higher than those in the mycotoxin-contaminated group. Spearman correlation analysis showed aflatoxins, ochratoxin A and fumonisins contents were positively and significantly correlated with the abundances of Aspergillus, Rhodotorula, Wallemia and Candida. In conclusion, this study reported the prevalence of mycotoxin contamination and the great diversity of fungi associated with Semen Persicae for the first time, providing an early warning for subsequent potential mycotoxin biosynthesis.

Determination of glycidyl fatty acid esters in edible vegetable oils by HPLC-ESI-QQQ-MS/MS combined with cryo-degreasing tandem dispersive solid-phase extraction

Glycidyl fatty acid esters (GEs) have been identified as food contaminants that can form during the processing and heating of edible oils. Their widespread occurrence raises potential concerns for consumers. This study aimed to develop a simple and effective analytical method for molecular recognition using mass spectrometry fragmentation patterns. Furthermore, it sought to simultaneously and quantitatively determine five common GEs in highly complex fatty matrices (oils) using high-performance liquid chromatography-triple quadrupole tandem mass spectrometry (HPLC-ESI-QQQ-MS/MS). An extraction method using acetonitrile was used, and the purification of the extract was optimized using cryo-degreasing-tandem dispersive solid-phase extraction (d-SPE) to eliminate fatty interferents. The method validation yielded recoveries ranging from 81.17% to 109.28% and the method precision was < 10.00%. The limits of quantification ranged from 0.08 to 0.21 ng/g, which is below the current legal limits. This approach may play a crucial role in ensuring food safety.

Development and validation of an expanded panel of progestins using liquid chromatography-tandem triple quadrupole mass spectrometry to monitor protocol compliance in hormonal contraceptive pharmacokinetic/pharmacodynamic studies

We developed and validated the use of ultra-high-performance liquid chromatography–heated electrospray ionization-tandem triple quadrupole mass spectrometry to simultaneously analyze serum concentrations of ethinylestradiol, dienogest, norelgestromin, norethindrone, gestodene, levonorgestrel, etonogestrel, segesterone acetate, medroxyprogesterone acetate, and drospirenone. The calibration range for all targets was 0.009–10 ng/mL, with lower limit of quantification of 0.009 ng/mL for all analytes except gestodene (0.019 ng/mL). We used our assay to check compliance among participants in a clinical trial, confirmed the use of drospirenone in 11 of 13 study participants, and evidence of noncompliant progestins in 2 (levonorgestrel = 1, norethindrone = 1). We conclude that this approach provides an accurate method to check protocol compliance in contraceptive clinical trials. ImplicationsThe availability of a liquid chromatography-tandem triple quadrupole mass spectrometry multiprogestin analysis panel for simultaneous evaluation of the most common contraceptive steroids approved worldwide could improve monitoring of compliance and protocol adherence in clinical trials.

A hydrophobic deep eutectic solvent-based vortex-assisted liquid-liquid microextraction applied for doping control of aromatase inhibitors from equine urine

Aromatase inhibitors (AIs) can indirectly cause increased testosterone in animals, which leads to the improvement of the athletic ability of horses. For the protection of horses and the consideration of fair competition, AIs were listed as prohibited drugs by the Federation Equestre Internationale (FEI). There were several disadvantages using traditional pretreatment methods before analyzing these drugs from biological samples. A rapid and green pretreatment method has been developed by utilizing the hydrophobic deep eutectic solvent (DES)-based vortex-assisted liquid-liquid microextraction (DES-VALLME) followed by ultra-high performance liquid chromatography tandem triple-quadrupole mass spectrometry (UHPLC-MS/MS) technique for the efficient extraction and sensitive detection of AIs in equine urine samples. The combination of menthol and 4-fluorophenol in a molar ratio 1:4 was chosen as the optimum composition of DES for extracting AIs. Under the optimum conditions, only 80 μL of DES, 1 mL equine urine and 2 min were expended. An external standard calibration method was utilized for determination, and a linear relationship was achieved with a concentration range of 0.02–4000 ng mL−1 (r2 ≥ 0.9983). The limits of detection of the method based on a signal-to-noise ratio of 3 were 0.01–4 ng mL−1. The accuracy recoveries ranged from 94.9% to 113.4% within the intra-day and inter-day relative standard deviations of less than 9.1%. Compared with traditional extraction methods, the DES-VALLME method had the advantages of rapidity, simplicity, efficiency, low toxicity, and low cost. This method has potential and possessed brilliant prospects for doping control.

Qualitative and quantitative analysis of the chemical components in Yuquan capsules by using ultra-performance liquid chromatography-mass spectrometry.

The Yuquan capsules is a commonly used traditional Chinese Patent Medicine used for the treatment of diabetes mellitus. In this study, a high-throughput analytical method for identifying the chemical composition of Yuquan capsules was established for the first time by using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry. The data obtained were subjected to fragment analysis and this was combined with UNIFI processing of natural products. One-hundred sixteen compounds were characterized from Yuquan capsules. Twelve of the bioactive compounds were quantitatively analyzed by ultra-performance liquid chromatography-tandem triple quadrupole mass spectrometry. This study was undertaken to obtain a comprehensive chemical profile analysis as well as to evaluate the overall quality of Yuquan capsules. The results will provide a reference for the quality evaluation of different Yuquan preparations. In addition, the data will enable basic pharmacodynamic research into these extensively used capsules.

Simultaneous determination of nine phenolic compounds in imitation wild Dendrobium officinale samples using ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Dendrobium officinale Kimura et Migo (D. officinale), one of the nine everlasting types of grass, has gained increasing attention owing to its important roles in alternative medicines and drug discovery. Due to its natural resources being in danger of being extinct, imitation wild planting is becoming increasingly common. To assess the product's quality completely, an efficient ultrahigh performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) method was established to simultaneously quantify nine phenolic compounds in D. officinale samples. The extraction parameters, including solvent, solvent concentration, solid-liquid ratio, and extraction time, were systematically optimized with the single-factor test. The results demonstrated that extraction with a 1:200 solid-to-liquid ratio of 80% methanol for 1.5 h was the most efficient condition for the extraction of flavonoids. Satisfactory retention times and resolution of the nine analytes were acquired on the Thermo Scientific Hypersil GOLD column with multiple reaction monitoring in negative ion scanning mode. The method was validated to demonstrate its selectivity, linearity, precision, accuracy, and robustness. Thus, the verified UHPLC-QQQ-MS/MS method was successfully applied to the quantification of phenolic components present in D. officinale samples. The results indicated that the quantity and composition of phenolic components in D. officinale from various provenances were significantly different. This work provides a theoretical foundation for the cultivation and assessment of wild D. officinale quality.