Simultaneous Analysis of Lipid-Soluble Constituents of Erxian Decoction by GC–MS

Erxian Decoction (EXD), a traditional Chinese medicine formula mainly composed of six Chinese herbs, was originally developed for menopausal syndromes and had been practiced since the 1950s in China. Previous studies only focused on the water-soluble compounds involved in EXD by LC or TLC. This study analyzed the whole profile of the volatile constituents contained in EXD to supplement its quality evaluation method. Several EXD samples were extracted with chloroform and ethyl acetate, respectively, to get the lipid-soluble chloroform and ethyl acetate fractions and compared their gas chromatographic profiles by GC–MS. The EXD samples were hydrolyzed with hydrochloric acid in a water-bath at 100 °C, neutralized with 40% NaOH, and finally extracted with ethyl acetate and chloroform for the quantification of the total sarsasapogenins contained in EXD. A total of 56 compounds belonging to a variety of natural product categories such as aromatic phenols, terpenes, fatty acids, ketones, esters, and aldehydes, etc. were identified from the chloroform and ethyl acetate extracts by using the online EI–MS characterization. The GC–MS method showed a linear response for sarsasapogenin quantification with r = 0.994. The intra-day and inter-day variations of precision and accuracy of the assay were less than 5%. This developed GC–MS method could thus be successfully applied for the identification of lipid-soluble constituents derived from EXD, and also for the accurate quantification of the total sarsasapogenins contained in the acid hydrolyzed EXD samples.     

Identification of the absorptive constituents and their metabolites in vivo of Ziziphi Spinosae Semen by UPLC–ESI–Q-TOF–MS/MS

In this research, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC–ESI–Q-TOF–MS/MS) was used for detection and identification of the absorptive constituents and their metabolites in rat plasma, urine and feces following oral administration of Ziziphi Spinosae Semen alcohol extract. After structure elucidation, a total of 12 compounds in rat plasma, comprising seven prototypes and five metabolites, 28 compounds in urine, comprising 17 prototypes and 11 metabolites, and 23 compounds in feces, comrpising 17 prototypes and six metabolites, have been tentatively identified by comparison with standard compounds and reference literature information. To the best of our knowledge, this is the first comprehensive and systematical metabolic study on the seed. Mostly importantly, we propose that gastric acid could convert jujubosides into an absorbable form of ebelin lactone oligosaccharides, which may be responsible for the low bioavailability and specific bioactivities of these compounds. Additionally, we deduced that the absorption site of ebelin lactone oligosaccharides is located in the stomach, and that the ebelin lactone form of jujubosides may be more suitable for absorption than its hydrolysis product. Our investigation will be helpful to narrow the scope for potentially active ingredients of the seed, and pave the way for determination of the pharmacological mechanism of the seed.     

Analysis of plasma migration components in Patrinia villosa (Thunb.) Juss. effective parts by UPLC–Q-TOF–MS

Patrinia villosa (Thunb.) Juss. (PVJ) is described as pungent, bitter and slightly cold in Chinese medicine, and is associated with the large intestine, stomach and liver meridians. The preliminary experiments of our research team proved that PVJ total flavonoids have excellent inhibitory effects on liver cancer cells. The present experiment uses the UPLC–Q-TOF–MS technology and serum pharmacochemistry methods to analyze the chemical components in vitro and in vivo of PVJ antiliver tumors. A total of 14 chemical components were identified in the total flavonoids extract of PVJ, and it is mainly composed of flavonoids, flavonones, flavonols and phenolic acids. At the same time, seven prototypical components and seven metabolic components were detected in the drug-containing plasma. Hydrocaffeate and scutellarein are the phase I metabolites of caffeic acid and scutellarin, respectively. Sulfated apigenin, sulfated luteolin, sulfated kaempferol and methylated kaempferol are the II phase metabolites of apigenin, luteolin, kaempferol, respectively. The experiment provides a reference for the research and development of antitumor drug candidates, and provides a basis for revealing the bioactive components of PVJ and the antitumor mechanism.     

UPLC–ESI–Q-TOF–MS/MS analysis of anticancer fractions from Ophiocordyceps xuefengensis and Ophiocordyceps sinensis

Ophiocordyceps xuefengensis (O. xuefengensis), a new species of caterpillar fungus, has been identified as the sister taxon of Ophiocordyceps sinensis (O. sinensis). The aims of the present study are to evaluate the anticancer activity and to qualitatively analyze the potential bioactive chemical constituents of O. xuefengensis and O. sinensis, comparatively. An MTT assay was used to evaluate the in vitro anticancer activities of different fractions from O. xuefengensis and O. sinensis. The results show that ethyl acetate fractions of O. xuefengensis and O. sinensis have significant in vitro anticancer activity. These two bioactive fractions were analyzed by ultra-performance liquid chromatography–electrospray ionization with quadrupole–time of flight tandem mass spectrometry technology. A total of 82 compounds and 101 compounds were identified or tentatively characterized in the bioactive fractions of O. xuefengensis and O. sinensis, respectively. Among these compounds, 68 existed in both O. xuefengensis and O. sinensis. A total of 67 compounds were reported in O. xuefengensis and 8 compounds were reported in caterpillar fungus for the first time. This is the first detailed comparative analysis of the in vitro anticancer activity and chemical ingredients between O. xuefengensis and O. sinensis. The application of this work will provide reliable fundamental pharmacological substances for the use of O. xuefengensis by Yao people.     

Analysis of Para Red and Sudan Dyes in Egg Yolk by UPLC–MS–MS

Matrix solid-phase dispersion (MSPD) with alumina N as adsorbent has been used for extraction of para red, Sudan 1, Sudan 2, Sudan 3, and Sudan 4 dyes from egg yolk. The extracts were analyzed by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS–MS). Mean recovery for the five dyes ranged from 63.2 to 98.6%, with CV 0.55–10.00%. One sample was confirmed to contain 0.3 mg kg^?1 Sudan 4.     

Metabonomics analysis of semen euphorbiae and semen Euphorbiae Pulveratum using UPLC–Q-TOF/MS

Semen Euphorbiae (SE), the dry and mature seed of Euphorbia lathyris L., a common traditional Chinese medicine, has significant pharmacological activity. However, its toxicity limits its clinical application, and less toxic Semen Euphorbiae Pulveratum (SEP) is often used clinically. To explore the possible mechanism of SE frost-making and attenuation, this study used ultrahigh-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry to perform a comprehensive metabolomics analysis of serum and urine samples from rats treated with SE and SEP, and performed histopathological evaluation of liver, kidney and colon tissues. Meanwhile, the different metabolites were visualized through multivariate statistical analysis and the HMDB and KEGG databases were used to distinguish the differential metabolites of SE and SEP to reveal related metabolic pathways and their significance. In total, 32 potential biomarkers, 14 in serum and 18 in urine, were identified. The metabolic pathway analysis revealed that arachidonic acid metabolism, sphingolipid metabolism, tyrosine and tryptophan biosynthesis, the tricarboxylic acid cycle and seven other metabolic pathways were significantly altered. Importantly, compared with SE, SEP reduced the metabolic disorder related to endogenous components. The mechanism may be related to the regulation of lipid metabolism, intestinal flora metabolites, amino acid metabolism and energy metabolism. This study provided new insights into the possible mechanism of SE freezing and attenuation.     

Analysis of Biologically Active Constituents in Centella asiatica by Microwave-Assisted Extraction Combined with LC–MS

Centella asiatica (L.) Urban is a traditional herbal medicine used in Asiatic countries, and is commonly used to treat various wounds, leprosy, tuberculosis and lupus diseases. In this work, a new method based on microwave assisted extraction followed by liquid chromatography–diode array detection–electrospray ionization multistage tandem mass spectrometry analysis has been developed for the identification and quantification of biologically active constituents in C. asiatica, including asiatic acid, asiaticoside and madecassoside. The separation was performed on an Agilent Eclipse C₁₈ column (150 mm × 4.6 mm i.d., 5 μm) with gradient elution of acetonitrile and 0.1% aqueous acetic acid within 50 min. Detection was performed at 205 nm. The calibration curves showed good linearity (r ² > 0.9992). The limits of detection ranged from 1.2 to 1.6 μg mL^?1. The intra- and inter-day precision was less than 3% and the recovery of the assay was in the range of 95.4–106.8%. The method was successfully applied to the quantification of the three constituents in different samples of C. asiatica. The results indicated that the developed method could be considered to be a simple, rapid and reliable method for the quality evaluation of C. asiatica. The samples were also analyzed on a liquid chromatography–electrospray ionization–time-of-flight mass spectrometry system to confirm the identification results.     

SPMMTE and Q-TOF–UPLC–MS for monitoring of atenolol and atorvastatin in human plasma using pentafluoro phenyl column

Iron nanocomposite adsorbent was prepared by green technology with 90% yield. The prepared iron nanocomposite adsorbent was used in solid-phase micromembrane tip extraction (SPMMTE) sample preparation technique. Analysis of atenolol and atorvastatin was performed in human plasma using SPMMTE and Q-TOF–UPLC–MS methods. New generation Acquity UPLC HSS penta fluoro phenyl (2.1?×?75?mm²; 1.8?µm) column was used with acetonitrile–0.1% formic acid in water (50:50 v/v) as mobile phase. The flow rate was 0.2?mL?min^?1 with electrospray mass detection. The limits of detection were 0.2 and 0.4?ng active mass for atenolol and atorvastatin, while the limits of quantification were 1.0 and 2.0?ng active mass, respectively. The values of the retention times were 3.224 and 3.907 for atorvastatin and atenolol. The values of the separation and resolution factors were 1.31 and 1.71, respectively. The peaks were sharp with base lined separation within 4.2?min. The developed SPMMTE and Q-TOF–UPLC–MS methods were reproducible, fast, precise, robust, rugged, and economic for the analyses of atenolol and atorvastatin in human plasma. The reported methods can be applied for monitoring of the reported drugs at trace level.     

Multi-residue Methodologies for the Analysis of Non-polar Pesticides in Water and Sediment Matrices by GC–MS/MS

Chromatographia - A multi-residue method for the analysis of non-polar pesticides by GC–MS/MS in water and sediment matrices has been successfully developed, including 33 and 27 compounds,...     

Effects of voriconazole and fluconazole on the pharmacokinetics of almonertinib in rats by UPLC–MS/MS

Almonertinib was included in the first-line treatment of non-small cell lung cancer with EGFR T790M mutations by the Chinese Society of Clinical Oncology in 2021. Considering that immunocompromised lung cancer patients are prone to opportunistic fungal infections, and most triazole antifungal drugs are moderate or strong inhibitors of CYP3A4, this study was conducted to develop and validate an accurate and rapid ultra-performance liquid chromatography tandem mass spectrometry method for quantifying almonertinib in plasma and for investigating the pharmacokinetic changes of almonertinib caused by voriconazole and fluconazole in rats. After liquid–liquid extraction with tert-butyl methyl ether, an XSelect HSS T3 column (2.1 × 100 mm, 2.5 μm, Waters) was used for the chromatographic separation of almonertinib and sorafenib-D₃ (internal standard). The analytes were detected using an AB Sciex Triple Quad 5,500 mass spectrometer in the positive ionization mode. The method exhibited great linearity (0.5–200 ng/ml, r > 0.997) and stability under the established experimental conditions. All validation experiments were in accordance with the guidelines, and the results were all within the acceptable limits. This method was successfully applied to the researches of pharmacokinetics and drug interactions for almonertinib in rats. Voriconazole and fluconazole significantly altered the pharmacokinetic profiles of almonertinib and increased the systemic exposure of almonertinib in rats to different degrees, but further human trials should be conducted to validate the results.     

A Comparison of Three Methods of Extraction for the Determination of Polyphenols and Organic Acids in Tobacco by UPLC–MS–MS

Matrix solid phase dispersion (MSPD), ultrasonic extraction followed by a solid phase extraction (USE–SPE) and reflux extraction (REFLUX) were studied for the analysis of polyphenols and organic acids in tobacco. The analysis was by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC–MS–MS). The multi-mode support sorbent Zirconia/AA12S50 in MSPD is more suitable for the extraction of tobacco polyphenols than conventional silica or C18 silica. Although the matrix effect of USE–SPE is slightly stronger than MSPD and REFLUX for most target compounds, it gave higher extraction capacity, recoveries and sensitivity.     

Analysis of Linarin and Its Metabolites in Rat Urine by LC–MS/MS

Liquid chromatography–ion trap mass spectrometry was employed to investigate the metabolism of linarin in rats. Identification and structural elucidation of the metabolites were performed by comparing the differences in molecular masses, retention times, and full scan MS ⁿ spectra between linarin and its metabolites. Six metabolites (acacetin, apigenin, acacetin glucuronide, apigenin glucuronide, acacetin sulfate, apigenin sulfate) were detected in rat urine after oral administration of linarin at the dose of 50 mg kg^?1. Furthermore, a selective and sensitive liquid chromatography–triple quadruple mass spectrometry assay was developed and validated for the simultaneous determination of linarin and acacetin (the major metabolite of linarin) in rat urine. Chromatographic separation was carried out on a C₁₈ column, and mass spectrometric detection was performed using a triple-quadrupole mass spectrometer coupled with an electrospray ionization source in the positive ion mode. Quantitation of linarin and acacetin was performed using selected reaction monitoring of precursor–product ion transitions at m/z 593 → 285 for linarin, 285 → 242 for acacetin, and 303 → 153 for hesperitin (internal standard), respectively. The assay exhibited good linearity (r > 0.9900) for both linarin and acacetin. The intra- and inter-day precisions were <13.4 % and the accuracy was between ?8.1 and 3.1 %. The method was successfully applied to the urinary excretion study of linarin in rats after oral administration of linarin.     

Analysis of pyrethroid pesticides in Chinese vegetables and fruits by GC–MS/MS

A rapid and economical method using modified QuEChERS sample pretreatment coupled with high-sensitivity gas chromatography/triple quadrupole mass spectrometry was established to simultaneously determine ten pyrethroid pesticides in fruits and vegetables. All pesticides were detected within 20 min of one injection. Concurrent backflushing provided column protection, greatly facilitating instrument maintenance. For quantitation, matrix-matched calibration was used to compensate for signal-enhancement effects and to ensure the precision of the method. The limit of detection (LOD) was in the range of 0.3–4.9 μg/kg. The recovery rate was from 78.8 to 118.6%, with relative standard deviation (RSD) below 14.8%. The developed method is suitable for rapid and sensitive multi-residue analysis of pyrethroid pesticides in fruits and vegetables. It is good for users in professional institutions that implement safety controls for testing hundreds of agricultural product samples everyday.     

UPLC–MS Analysis for Simultaneous Determination of Eight Components in the ‘Ge-Gen-Qin-Lian’ Tablets

A novel and rapid ultra performance liquid chromatography–mass spectrometry method was developed and validated for simultaneous determination of eight constituents in ‘Ge-Gen-Qin-Lian’ tablets. A UPLC BEH C₁₈ column (2.1 × 100 mm, 1.7 μm) was utilized in this method, and a mixture of methanol, 0.1% formic acid, and 5 mmol L^?1 ammonium acetate was adopted in the gradient elution program. The mass spectrometer equipped with an electrospray ionization source was performed in the selected-ion reaction (SIR) mode. A good linearity correlation (R ² > 0.9991) was obtained over the investigated concentration ranges. Recoveries of this method for the eight components were from 96.00 to 100.6% (RSD % < 3.4%).     

Precursor Ion-Scan Mode-Based Screening Strategy for New Amorfrutin Derivatives from <Emphasis Type="Italic">Amorpha fruticosa</Emphasis> by UPLC–QqQ–MS and UPLC–Q-TOF–MS

This study presents a screening strategy for unknown compounds by coupling UPLC–QqQ–MS and UPLC–Q-TOF–MS analyses. Amorfrutins, potential PPARγ agonists in Amorpha fruticosa, were selected as the research target. First, MS/MS analysis of three amorfrutin references (AA, AB, and AC) by Q-TOF revealed the mass-spectrometric fragmentation patterns of amorfrutins, and m/z 225 was recognized as a characteristic and common fragment ion of amorfrutins. Then, unknown amorfrutins were screened in the fruits of A. fruticosa by UPLC–QqQ–MS operating in precursor ion-scan mode, and the product ion was set as m/z 225 for screening potential amorfrutins. According to the high-resolution MS–MS spectral data provided by UPLC–Q-TOF–MS, the molecular formula of potential amorfrutins and their respective characteristic fragments were calculated. Finally, three unknown amorfrutins, namely, amorfrutin D and two new amorfrutins, were identified. As far as we know, this is the first study to rapidly identify unknown compounds by combined application of QqQ–MS precursor ion scan and Q-TOF–MS formula calculation. This research strategy will also be effective for the discovery of other trace natural products and metabolites.     

New UPLC–MS/MS method for simultaneous determination of irbesartan and hydrochlorthiazide in human plasma

Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) is a preeminent analytical tool for rapid biomedical analysis with the objective of reducing analysis time and maintaining good efficiency. In this study a simple, rapid, sensitive and specific ultra-performance liquid chromatography–tandem mass spectrometry method was developed and validated for quantification of the angiotensin II receptor antagonist, irbesartan and hydrochlorthiazide in human plasma. After a simple protein precipitation using methanol and acetonitrile, irbesartan, hydrochlorthiazide and internal standard (IS) telmisartan were separated on Acquity UPLC BEH? C18 column (50 × 2.1 mm, i.d. 1.7 μm, Waters, USA) using a mobile phase consisting of acetonitrile:10 mM ammonium acetate:formic acid (85:15:0.1 % v/v/v) pumped at a flow rate of 0.3 mL/min and detected by tandem mass spectrometry with negative ion mode. The ion transitions recorded in multiple reaction monitoring mode were m/z 427.2 → 193.08 for irbesartan, m/z 295.93 → 268.90 for hydrochlorthiazide and m/z 513.2 → 287.14 for IS. The assay exhibited a linear dynamic range of 30–500 ng/mL for irbesartan and 1–500 ng/mL in human plasma with good correlation coefficient of (0.996) and (0.997) and with a limit of quantitation of 30  and 1 ng/mL for irbesartan and hydrochlorthiazide, respectively. The intra- and inter-assay precisions were satisfactory; the relative standard deviations did not exceed 10.13 % for irbesartan and 11.14 % for hydrochlorthiazide. The proposed UPLC–MS/MS method is simple, rapid and highly sensitive, and hence it could be reliable for pharmacokinetic and toxicokinetic study in both animals and humans.     

Simultaneous quantification of vortioxetine,fluoxetine and their metabolites in rat plasma by UPLC–MS/MS

In this study, a specific and quick ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was fully developed and validated for simultaneous measurement of the rat plasma levels of vortioxetine (VOR), Lu AA34443 (the major metabolite of VOR), fluoxetine and its metabolite norfluoxetine with diazepam as the internal standard (IS). After a simple protein precipitation with acetonitrile for sample preparation, the separation of the analytes were performed on an Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 μm) column, with acetonitrile and 0.1% formic acid in water as mobile phase by gradient elution. The detection was achieved on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via an electrospray ionization source. Good linearity was observed in the calibration curve for each analyte. The data of precision, accuracy, matrix effect, recovery and stability all conformed to the bioanalytical method validation of acceptance criteria of US Food and Drug Administration recommendations. The newly developed UPLC–MS/MS method allowed simultaneous quantification of VOR, fluoxetine and their metabolites for the first time and was successfully applied to a pharmacokinetic study in rats.     

Determination of acamprosate in human plasma by UPLC–MS/MS: Application to therapeutic drug monitoring

Acamprosate is a medication used to treat alcohol dependence. Therapeutic drug monitoring is important in drugs for the treatment of substance-related disorders. Therefore, in this study, a new selective, very simple and rapid ultra-performance liquid chromatography–tandem mass spectrometer method was developed for the therapeutic drug monitoring of acamprosate. The developed method allows the determination of acamprosate in human plasma. The method was validated in terms of selectivity and linearity, which was in the range of 100–1,200 ng/ml for acamprosate. Intra-assay and inter-assay accuracy and precision were within the acceptable limits of the Eueopean Medicines Agency guideline. The lower limit of quantitation was 100 ng/ml for acamprosate. The developed method was successfully applied for therapeutic drug monitoring in patient plasma samples.     

Determination of oleandrin and adynerin in rat plasma by UPLC–MS/MS and their pharmacokinetic study

Oleandrin and adynerin are the main toxic components of oleander, an evergreen shrub or a small tree of the oleander family, which belongs to the class of cardiac glycosides exhibiting delayed action. The pharmacokinetic differences of oleandrin and adynerin in rats were studied by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) under two different administration modes: oral (5 mg/kg) and sublingual intravenous injection (1 mg/kg). The chromatographic column was UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm), and the column temperature was set at 40 °C. The mobile phase was acetonitrile–water (containing 0.1 % formic acid), with gradient elution, the flow rate was 0.4 mL/min, and the elution time was 4 min. Electrospray (ESI) positive ion mode detection with multiple reaction monitoring mode (MRM) was used for quantitative analysis: oleandrin m/z 577 → 145, adynerin m/z 534 → 113, and internal standard m/z 237 → 135. The established UPLC–MS/MS method was successfully applied to the pharmacokinetics in rats after administering oleandrin and adynerin. The bioavailability of oleandrin and adynerin was found to be low, 7.0 % and 93.1 %; respectively.