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A pinanamine‐based analog, (1R,2R,3R,5S)‐N‐((3‐cyclopropylthiophen‐2‐yl)methyl)‐2,6,6‐trimethylbicyclo[3.1.1]heptan‐3‐amine (M090) was synthesized and demonstrated with anti‐influenza activity to overcome the multi‐drug resistance. Herein, a rapid and robust liquid chromatography‐tandem mass spectrometry method was developed and validated to quantify M090 in rat plasma. With simple protein precipitation by methanol, the separation of M090 was performed on a Waters BEH‐C18 column (2.1 × 100 mm, 1.7 μm) by gradient elution at 0.4 mL/min with mobile phases consisting of water containing 0.1 % formic acid (phase A) and methanol (phase B). M090 and clenbuterol (internal standard) were detected using multiple reaction monitoring in positive electrospray ionization mode with transitions of m/z 290.3→137.0 and m/z 277.0→203.0, respectively. The method was validated over a linear range of 1.0–2400 ng/mL with a regression coefficient of 0.9974 and no endogenous interference. The intra‐ and inter‐batch precisions were within 8.29% and accuracy ranged from 100.3% to 108.1% for M090. The validated method was utilized to evaluate the in vitro metabolic stability and in vivo pharmacokinetics of M090 in rats. [ABSTRACT FROM AUTHOR]

A reverse phase high-performance liquid chromatography (HPLC) method has been developed for the quantification of a typical drug Dasatinib (DST) and its related impurities in pharmaceuticals. Kinetex C18 (4.6 × 150 mm, 5 μm) column was used in the chromatographic separations, using buffer (1.36 g of KH2PO4 in 1000 mL of water, pH = 7.8; adjusted with diluted KOH solution) with solvent as acetonitrile and mode of elution as the gradient. The flow rate is 0.9 mL/min, column oven temperature as 45°C and the overall gradient run time as 65 min. The developed method was found to produce symmetric and good separation between the process-related and degradation impurities. Method optimization is achieved with photodiode array at 305 nm over the concentration range of 0.5 mg/mL and degradation studies were carried out under acidic, alkaline, oxidative, photolytic and thermal conditions to demonstrate the stability indicating capability of the method. Two major impurities were found in forced degradation studies in the HPLC analysis, the unknown, acid degradants were enriched and isolated by preparative HPLC, then characterized through high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy. The unknown acid degradation impurity was showing Exact Mass of 521.11, molecular formula C22H25Cl2N7O2S and its chemical name as 2-(5-chloro-6-(4-(2-hydroxyethyl) piperazin-1-yl)-2-methylpyrimidin-4-ylamino)-N-(2-chloro-6-methylphenyl) thiazole-5-carboxamide. Another impurity (oxidative degradant) found as known DST N-oxide Impurity-L and its chemical name as 4-(6-((5-((2-chloro-6-methylphenyl) carbamoyl) thiazol-2-yl) amino)-2-methylpyrimidin-4-yl)-1-(2-hydroxyethyl) piperazine 1-oxide. The analytical HPLC method was further validated as per ICH guidelines. [ABSTRACT FROM AUTHOR]

Rationale: Secondary hypertension is often caused by activation of complex multi‐organ endocrine systems, while renin activity indicated by angiotensins (Angs), aldosterone (ALD) and cortisol (COR) in such systems are generally accepted as its diagnostic markers. As antibody‐based methods cannot offer comparable quantification for these biomarkers, a liquid chromatography (LC)–tandem mass spectrometry (MS/MS)‐based approach was developed to quantify them simultaneously and accurately. Methods: Five different beads for magnetic solid‐phase extraction (MSPE) were evaluated towards their enrichment efficiency for these biomarkers. An LC system with optimized elution gradient and a triple‐quadrupole MS with tuned parameters were coupled to quantitatively monitor the extracted analytes. The method performance was further examined such as linearity, precision, stability, recovery rate and matrix effect. Based on the developed method, the abundance of Ang II, ALD and COR in plasma was measured and the quantification was compared with that derived from commercial ELISA kits. Results: As compared with other MSPEs, Angs, ALD and COR were highly enriched by the HLB magnetic beads with satisfactory recoveries. These analytes were simultaneously quantified by LC/MS/MS and all the method parameters for quantification were well matched with the requirements of clinical testing. Comparison of the quantitative results derived from ELISA and LC/MS/MS exhibited that the two methods offered basically comparable values with Pearson r values at 0.896, 0.895 and 0.835, respectively. The stability test for plasma Angs at room temperature indicated that the abundance of Ang II was relatively stable within 3 h, whereas that of Ang I and Ang 1–7 was time‐dependently changed. Conclusions: Coupling of HLB beads and LC/MS/MS thus enables simultaneous quantification of a set of biomarkers related to secondary hypertension. [ABSTRACT FROM AUTHOR]

Plasma proteomics holds immense potential for clinical research and biomarker discovery, serving as a non-invasive "liquid biopsy" for tissue sampling. Mass spectrometry (MS)-based proteomics, thanks to improvement in speed and robustness, emerges as an ideal technology for exploring the plasma proteome for its unbiased and highly specific protein identification and quantification. Despite its potential, plasma proteomics is still a challenge due to the vast dynamic range of protein abundance, hindering the detection of less abundant proteins. Different approaches can help overcome this challenge. Conventional depletion methods face limitations in cost, throughput, accuracy, and off-target depletion. Nanoparticle-based enrichment shows promise in compressing dynamic range, but cost remains a constraint. Enrichment strategies for extracellular vesicles (EVs) can enhance plasma proteome coverage dramatically, but current methods are still too laborious for large series. Neat plasma remains popular for its cost-effectiveness, time efficiency, and low volume requirement. We used a test set of 33 plasma samples for all evaluations. Samples were digested using S-Trap and analyzed on Evosep One and nanoElute coupled to a timsTOF Pro using different elution gradients and ion mobility ranges. Data were mainly analyzed using library-free searches using DIA-NN. This study explores ways to improve proteome coverage in neat plasma both in MS data acquisition and MS data analysis. We demonstrate the value of sampling smaller hydrophilic peptides, increasing chromatographic separation, and using library-free searches. Additionally, we introduce the EV boost approach, that leverages on the extracellular vesicle fraction to enhance protein identification in neat plasma samples. Globally, our optimized analysis workflow allows the quantification of over 1000 proteins in neat plasma with a 24SPD throughput. We believe that these considerations can be of help independently of the LC–MS platform used. [ABSTRACT FROM AUTHOR]

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

An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma. The rat plasma was treated with liquid-liquid extraction using ethyl acetate. The determination was performed on the UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 μm) with a mobile phase consisting of acetonitrile-water (containing 0.1% formic acid) and gradient elution at a flow rate of 0.4 mL/min. Electrospray (ESI) positive ion mode detection and multireaction monitoring (MRM) quantitative analysis were performed. A total of 36 rats were given tenacissoside G, tenacissoside H, and tenacissoside I, respectively, orally (5 mg/kg) and intravenously (1 mg/kg), with 6 rats in each group, to evaluate the pharmacokinetic difference of tenacissoside G, tenacissoside H, and tenacissoside I in rats. The calibration curves showed good linearity in the range of 5–2000 ng/mL, where r was greater than 0.99. The results of precision, accuracy, recovery, matrix effect, and stability met the requirements of biological sample detection methods. The established UPLC-MS/MS method was successfully applied to pharmacokinetic studies of tenacissoside G, tenacissoside H, and tenacissoside I, and the bioavailability was 22.9%, 89.8%, and 9.4%, respectively. [ABSTRACT FROM AUTHOR]

Direct oral anticoagulants are widely used in many indications to prevent thromboembolic events. Routine therapeutic monitoring is not required; however, there is increasing evidence suggesting the benefit of plasma level measurement in some situations. In addition, laboratory monitoring might help improve patient and drug non‐compliance and thus individualize therapy. In the present study, we developed a sensitive and high throughput ultra‐high‐performance liquid chromatography‐tandem mass spectrometry method for simultaneous quantification of apixaban, dabigatran, edoxaban, and rivaroxaban in human plasma. A one‐step extraction procedure in 96‐well formate for phospholipid and protein removal was used for sample pre‐treatment, and analytes were separated using gradient elution over 4.2 min. Analytes were detected on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode. The method was validated according to the European Medicine Agency guideline for the selectivity, linearity, and lower limit of detection, precision and accuracy, matrix effects, extraction recovery, carryover, dilution integrity, and stability over a concentration range of 3.0–1000 ng/ml for all analytes. The validated method was applied to real clinical samples of patients treated with one of the drugs. Therefore, we can conclude that our method is suitable for therapeutic drug monitoring of direct oral anticoagulants. [ABSTRACT FROM AUTHOR]

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

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

All regulatory organizations are paying close attention to the identification and measurement of genotoxic contaminants. Using conventional analytical techniques like high-performance liquid chromatography (HPLC) and gas chromatography to quantify probable genotoxic substances (PGIs) at the trace level is difficult (GC). Therefore, there is a necessity for advanced analytical techniques for the development of highly sensitive analytical procedures for the determination of trace-level PGIs in drug products and drug substances. This study's goal is to develop and evaluate an analytical technique for measuring allyl chloride, a possible genotoxic contaminant in gemfibrozil. For the detection of very low and trace levels of impurities, a gas chromatography with a triple quadrupole mass spectrometry detector (GC-MS/MS) approach was developed and validated. Using a column USP phase G27, a nonpolar and low bleed 5% diphenyl, 95% dimethylpolysiloxane, with dimensions of 30 m in length, 0.32 mm internal diameters, and 1.5 m film thickness, along with a flow rate of 2.0 mL/min and Helium (He) as a carrier gas, this method uses a thermal gradient elution program. The method was calibrated with a linearity range from 30% to 150% concentration with respect to the specification level and achieved a limit of detection (LOD) and limit of quantification (LOQ) were 0.005 ppm and 0.01 ppm, respectively, for allyl chloride. According to current ICH requirements, the method was validated, and it was discovered to be specific, exact, accurate, linear, sensitive, tough, robust, and stable. This method is suitable for determining allyl chloride in the regular analysis of Gemfibrozil. [ABSTRACT FROM AUTHOR]

Butachlor is an aromatic amide compound that plays a role as a herbicide, a xenobiotic, and an environmental contaminant. The aim of this work was to develop a highly selective and sensitive ultra‐performance liquid chromatography‐tandem mass spectrometry method based on the tandem mass spectrometry cubed technique to determine butachlor in a biological matrix. Butachlor and internal standard acetochlor were separated on a Waters Acquity ultra‐performance liquid chromatography BEH C18 column (2.1 × 50 mm, 1.7 μm) with gradient elution using 0.1% formic acid aqueous solution (A) and acetonitrile (B) as mobile phases. The transitions selected for tandem mass spectrometry cubed quantitative analysis in positive ion mode were: for butachlor, mass‐to‐charge ratio 312.2→238.1→162.1; for acetochlor, mass‐to‐charge ratio 270.1→224.0→148.1. The total running time for each sample was 5.5 min. The ultra‐performance liquid chromatography‐tandem mass spectrometry cubed method showed a linear relationship (R2 ≥ 0.995) in the concentration range of 0.5–100 ng/ml. The intra and interday accuracies are within the range of ‐10.6%–4.3% and precisions are between 4.48% and 13.14%. The novelty of the method is the use of tandem mass spectrometry cubed scanning mode, which improves selectivity and sensitivity. The results indicated that butachlor was cellular toxic. The safety of butachlor should be considered when it is used as a herbicide. [ABSTRACT FROM AUTHOR]

Alpha-methylthiofentanyl and thiofentanyl are psychoactive substances that have been abused in recent years, strongly threatening social and public security. Because they are rapidly metabolized in vivo, their determination presents a great challenge for forensic science. An automated method of online solid-phase extraction coupled with ultra-high performance liquid chromatography—tandem mass spectrometry was developed for the determination of alpha-methylthiofentanyl, thiofentanyl, and the metabolite norfentanyl in urine. The analytes were first enriched and purified by an Oasis HLB online SPE column and eluted on a Hypersil GOLD C18 analytical column. Chromatographic separation was achieved with mobile phase components of 2 mM ammonium formate and 0.1% formic acid in water and same composition in methanol in gradient elution mode. The limit of detection and lower limit of quantitation of the method for the three analytes were 0.2 and 2 pg/ml, respectively. Acceptable linearity was obtained from 2 to 800 pg/ml. The accuracy was in the range of 87.98–108.95%; and precision, calculated as the relative standard deviation, was less than 14.63%. The method recoveries were from 65.56 to 113.72%. This highly sensitive method was applied to study the detection window of the precursor compounds and metabolites in rat urine. Both alpha-methylthiofentanyl and thiofentanyl are metabolized to norfentanyl, which was first revealed in this study. With the aid of norfentanyl, the detection windows of alpha-methylthiofentanyl and thiofentanyl were extended to 18–26 days and 20–27 days, respectively. This study provides important auxiliary tools for the forensic identification of fentanyl analogs. [ABSTRACT FROM AUTHOR]

A method using HPLC with tandem mass spectrometry (HPLC-MS/MS) is proposed and validated for quantitative determination of ubiquinone and ubiquinol in human blood plasma. Sample preparation used precipitation of proteins by a 1:1 mixture of i-PrOH–EtOAc. The investigated substances were analyzed on a Shimadzu 8040 triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode for the main molecular ions of ubiquinone (m/z 880.7) and ubiquinol (m/z 882.7) using electrospray ionization (ESI) in positive- ion mode. Chromatographic separation used a Luna C18 column (150 × 4.6 mm, 5 μm) and gradient elution. Calibration curves showed a linear relationship in the concentration range 0.1 – 5 μg/mL. Validation of the proposed method confirmed its high sensitivity, specificity, accuracy, and precision. The method was successfully applied for determining ubiquinone and ubiquinol in blood plasma of conditionally healthy volunteers. [ABSTRACT FROM AUTHOR]

The total flavonoids of Desmodium styracifolium are the flavonoid extracts purified from Desmodii Styracifolii Herba, which has conventionally been used for treating urolithiasis in China. In this study, a sensitive and simple liquid chromatography‐tandem mass spectrometry method was developed to simultaneously determine five active components of the extracts in rat plasma. Chromatographic separation of the analytes (schaftoside, vicenin‐1, vicenin‐2, vicenin‐3, and isovitexin) was performed on an ACQUITY UPLC HSS T3 Column under gradient elution conditions. The calibration curves were linear over ranges from 0.5 to 100 ng/ml for schaftoside, vicenin‐1, vicenin‐2, and vicenin‐3, and 0.2–20 ng/ml for isovitexin. The relative standard deviation of intra‐ and inter‐day precisions were ≤6.8% and ≤8.3%, respectively, and the accuracies (relative error) were within ±7.6%. The recoveries of the analytes ranged between 97.3% and 100.3%, and the matrix effects ranged from 98.6% to 113.8%. The method was successfully applied to the pharmacokinetic studies of the five active ingredients of Desmodium styracifolium, for the first time, in both normal and urolithiasis model rats. Results revealed that the plasma levels of these components were significantly increased under the pathological state. This study provided valuable information facilitating the clinical investigation of this medicine. [ABSTRACT FROM AUTHOR]

• Measurement of intestinal permeability and absorptive capacity in the domestic cat. • Quantification of sugars in plasma using liquid chromatography-mass spectrometry. • Simultaneous analysis of lactulose, rhamnose, xylose, and 3-O-methylglucose (3-OMG) • Use of stable isotope labelled 13C standards for each target sugar. A novel method for quantifying the concentration of lactulose, rhamnose, xylose, and 3-O-methylglucose (3-OMG) in cat plasma using liquid chromatography-mass spectrometry (LC-MS) was developed. Domestic male cats (n = 13) were orally dosed with a solution containing the four sugars to test the permeability and absorptive capacity of their intestinal barrier. Plasma samples were taken 3 h later and were prepared with acetonitrile (ACN), dried under N 2, and reconstituted in 90 % ACN with 1 mM ammonium formate. Stable isotope labelled 13C standards for each analyte were used as internal standards. Chromatographic separation was conducted using a Phenomenex Luna NH2 column with a gradient elution system of deionized water and 90 % ACN with 1 mM ammonium formate at 300 µL/min for 13 min total analysis time. Recovery trials were conducted in triplicate over three days with RSD values (%) for each day ranging from 1.2 to 1.4 for lactulose, 5.4 – 6.0 for rhamnose, 3.3 – 5.5 for xylose, and 2.6 – 5.6 for 3-OMG. Inter-day variations for each analyte were not different (p > 0.05). Limit of detection and quantification were 0.2 and 0.7 µg/mL for lactulose, 0.8 and 2.4 µg/mL for rhamnose, 0.6 and 1.8 µg/mL for xylose, and 0.3 and 1.1 µg/mL for 3-OMG, respectively. Plasma sugar concentrations recovered from cats were above the limit of quantification and below the highest calibration standard, validating the use of this method to test intestinal permeability and absorptive capacity in cats. [ABSTRACT FROM AUTHOR]

• Rapid and simultaneous determination of 6 volatile fatty acids (VFAs) via HPLC-MS/MS. • The derivatisation method solves the problems of fatty acids in the detection process. • High throughput analysis of samples can be determined high sensitivity and accuracy. • VFAs can be accurately quantified using only microsamples (faeces, serum and urine). A stable isotope dilution-liquid chromatography tandem mass spectrometry method based on a low-temperature derivatization strategy with 3-nitrophenylhydrazine (3-NPH) was developed for the determination of six volatile fatty acids (VFAs) in serum, urine, and feces. Ice acetonitrile was used to precipitate proteins and extract the target analytes. The extract was derivatized with 3-NPH methanol solution at 4 °C. BEH C8 (1.7 μm, 2.1 × 100 mm) column was used for chromatographic separation, and acetonitrile–water (both containing 0.01 % formic acid) were used as the mobile phase with a gradient elution of 10 min. Electrospray ionization source (ESI) in negative ion multiple reaction monitoring (MRM) mode were used for analyte detection. The regression coefficients R2 of the calibration curves for the six VFAs were in the range of 0.9963–0.9994, and the LOQs were in the range of 0.02–0.5 μg mL−1, with the recoveries in the range of 85.3–104.3 %, and the intra- and inter-day precision in the range of 1.8–9.1 %. The method is simple, accurate and reliable, and has been applied in the sensitive determination of VFAs in complex biological samples. [ABSTRACT FROM AUTHOR]

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a highly selective and sensitive method for the quantification of kinase inhibitors, yet not widely available in clinical routine for therapeutic drug monitoring (TDM). To provide a more accessible alternative, a high-performance liquid chromatography method with ultraviolet/diode array detection (HPLC-UV/DAD) to quantify cabozantinib, dabrafenib, nilotinib and osimertinib, was developed and validated. Results were compared to LC-MS/MS. After liquid–liquid-extraction and reconstitution of the residue in 20 mM potassium dihydrogen phosphate (KH2PO4) (pH4.6), acetonitrile and methanol (50:25:25,v/v/v), chromatographic separation was achieved in 20.0 min using a Luna® C18(2)-HST column (100 × 2 mm, 2.5 μm), protected by a C18 guard column (4 × 2 mm) (column temperature: 30 °C, autosampler: 10 °C). Mobile phase A and B consisted of 20 mM KH2PO4 (pH4.9) and acetonitrile (9:1,v/v) and acetonitrile:20 mM KH2PO4 (pH4.9) (7:3,v/v), respectively. Gradient elution was performed at 200 µL/min. Analytes were quantified at 250, 280 and 330 nm, using sorafenib as internal standard. Calibration curves were linear (35–2,000 ng/mL). Method validation assays met requirements by U.S. Food and Drug Administration and European Medicines Agency. Compared to the more sensitive and specific LC-MS/MS, HPLC-UV/DAD showed a good correlation and a strong positive association (Kendall's tau 0.811¬–0.963, p < 0.05). Bland-Altman-plots revealed 100% (cabozantinib), 98.6% (dabrafenib), 98.6% (nilotinib) and 96.2% (osimertinib) of relative differences inside the limits of agreement. Regulatory agency criteria for sample reanalysis and cross validation were met (±20%-criterion:100% (cabozantinib), 94.3% (dabrafenib), 92% (nilotinib) and 84.6% (osimertinib). The developed HPLC-UV/DAD method is "fit-for-TDM" in clinical routine and serves as a genuine alternative to LC-MS/MS. [ABSTRACT FROM AUTHOR]

The long-term stability of drugs under normal laboratory storage conditions (−20°C) for years is important for research purposes, clinical re-evaluation, and also for forensic toxicology. To evaluate the stability of the analgesic opioid hydromorphone, 44 human frozen plasma samples of a former clinical trial were reanalyzed after at least three years. Blood samples were disposed using solid-phase extraction with an additional substitution of stable isotope labelled hydromorphone as an internal standard. Hydromorphone concentrations were determined by ultra-performance liquid chromatography (UPLC) with gradient elution, followed by tandem mass spectrometry with electrospray ionization. Calibration curves demonstrated linearity of the assay in the concentration range of 0.3–20 ng/mL hydromorphone. The limit of detection of the hydromorphone plasma concentration was 0.001 ng/mL, and the lower limit of quantification was 0.3 ng/mL. Intra- and interassay errors did not exceed 16%. The percentage deviation of the measured hydromorphone plasma concentrations between the reanalysis and the first analysis was −1.07% ± 14.8% (mean ± SD). These results demonstrate that hydromorphone concentration in human plasma was stable when the samples were frozen at −20°C over three years. This finding is of value for re-evaluations or delayed analyses for research purposes and in pharmacokinetic studies, such as in forensic medicine. [ABSTRACT FROM AUTHOR]

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

An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of senegenin and tenuifolin in mouse blood was developed. The pharmacokinetics of senegenin and tenuifolin in mice after intravenous (5 mg/kg) and oral (60 mg/kg) administration were studied, and the absolute bioavailability was calculated. A CORTECS T3 column was used, with a column temperature set at 40°C. The mobile phase was acetonitrile and 0.1% formic acid. Gradient elution was adopted, using a flow rate of 0.4 mL/min and an elution time of 4 min. Quantitative analysis was performed using electrospray ionization (ESI) with multiple reaction monitoring (MRM) in negative ion mode. Institute of Cancer Research (ICR) mice were bled from the tail vein after intravenous or oral administration of senegenin and tenuifolin. A UPLC-MS/MS method was established to determine the blood concentrations of each drug in mice, and the noncompartmental model was used to fit the pharmacokinetic parameters. Senegenin and tenuifolin showed a good linear relationship (r > 0.995) within a concentration range of 5–400 ng/mL in mouse blood. The intraday precision was <12%, the interday precision was <14%, and the accuracy was 87–109%. The recovery was >88%, and the matrix effect was 87–94%. The oral bioavailability of senegenin and tenuifolin in mice was 8.7% and 4.0%, respectively. The established UPLC-MS/MS method is suitable for pharmacokinetic studies of senegenin and tenuifolin in mice. [ABSTRACT FROM AUTHOR]

Tandem mass spectrometry (HPLC-MS/MS) was used to analyze biologically active compounds in extracts of the tuber and roots of Rhodiola rosea (family Crassulaceae) and in the pharmaceutical formulation Multiphytoadaptogen (MPA). Chromatography was run on Acquity UPLC BEH C18 columns with gradient elution. A TSQ Vantage ternary quadrupole mass spectrometer with electrospray ionization was used. MPA, like the Rhodiola rosea tuber/root extract, was found to contain salidroside, rosavin, rosarin, rosiridin, and rhodionin. These results can be used for standardization, identification, and quality control of phytocomplexes containing these Rhodiola rosea compounds to identify grounds for the mechanisms of the biological actions of MPA and studying its novel properties, taking account of the components identified. The PASS computer program was used for in silico evaluation of the spectra of the antitumor activities of these compounds. Antitumor effects were predicted with high probability for all five phytocomponents; the specific characteristic of the actions of individual compounds suggested that combinations might be used for treating a wide spectrum of malignant neoplasms. [ABSTRACT FROM AUTHOR]

Polyphenols are secondary metabolites of plants and used as effective antioxidants in dietary supplements, whose main sources are fruits, vegetables, and grains. To clarify the content and distribution of polyphenols in different fruit species samples accurately, a rapid and sensitive ultrahigh-pressure liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method combining dispersive liquid-liquid microextraction (DLLME) was developed for quantitative determination of fifteen polyphenol compounds in fruit juice. In this method, the targets were first extracted from 1 g of fruit juice sample using 10 mL of 80% ethanol solution by ultrasonic-assisted extraction (UAE). Then, 1.0 mL of UAE extracted solution, 60 μL of n-octanol and 2.0 mL of H2O were performed in the following DLLME procedure. A C18 reversed-phase column, ZORBAX SB (100 × 4.6 mm, 3.5 μm), was proposed under gradient elution with 0.1% formic acid aqueous solution and methanol mobile phases for the determination of 15 polyphenols, allowing us to obtain polyphenolic profiles in less than 23.0 min. Under the optimum conditions, the enrichment factors ranged from 162 to 194. The results showed that the 15 polyphenols had linear correlation coefficients (R2) more than 0.99. The limits of detection (LODs) were between 18.3 and 103.5 ng/g, and the average recoveries were between 96.9 and 116.3% with interday relative standard deviations (RSDs) ranging from 4.4 to 8.2% in all cases. The method was successfully applied to the analysis of real fruit juice samples and presented itself as a simple, rapid, practical, and environment-friendly technique. [ABSTRACT FROM AUTHOR]

Cinnarizine is a weak base, which can produce supersaturation and precipitation during gastrointestinal transit, affecting its absorption in vivo. Therefore, it is necessary to investigate whether the oral bioavailability of cinnarizine can be improved after co‐administration with precipitation inhibitors or not. In order to evaluate the pharmacokinetic behavior of cinnarizine in rats, a simple, rapid, sensitive, and environmentally friendly supercritical fluid chromatography‐tandem mass spectrometric method was established and validated. In this method, flunarizine, a structural analogue of cinnarizine, was selected as the internal standard, and cinnarizine was extracted from rat plasma using evaporation‐free liquid–liquid extraction method. The analytes were separated on a Torus 1‐AA column (3.0 mm × 100 mm, 1.7 μm) within 2.0 min, using a gradient elution procedure. The transitions of cinnarizine and flunarizine were m/z 369.1 → 167.1 and m/z 405.1 → 203.1, respectively. Cinnarizine showed good linear correlation in the range of 1–500 ng/ml with a lower limit of quantification of 1 ng/ml. The intra‐ and interday precision and accuracy of all quality control samples were within ±15%. This high‐throughput, accurate, sensitive, and reproducible method has been successfully applied to study the effects of the precipitation inhibitor cinnarizine on the pharmacokinetics in rats. [ABSTRACT FROM AUTHOR]

• 11 free fatty acids were simultaneously determined by LC-MS/MS. • Microvolume serum pretreated by two-step derivatisation strategy. • High throughput analysis of samples can be achieved. • Method was developed and validated with good results. A stable isotope dilution–liquid chromatography–tandem mass spectrometry method based on a derivatisation strategy involving an N,N'-carbonylimidazole solution (CDI) with 4-(dimethylamino)-benzenemethanamine was developed for the determination of 11 free fatty acids (FFAs) in human blood samples. Serum samples were subjected to liquid‒liquid extraction and centrifuged, and the supernatant was collected for a two-step derivatisation reaction with a CDI and 4-(dimethylamino)-aniline acetonitrile solution. The derivatised solution was separated on a ACQUITY UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) column with a mobile phase consisting of water–acetonitrile in gradient elution and then detected by tandem mass spectrometry using electrospray ionisation (ESI) and multiple reaction monitoring (MRM) in positive ion mode and quantified using the isotope internal standard method. The effects of the derivatisation reaction time, temperature and concentration of derivatisation reagents on the response values of the analytes were investigated. The optimal conditions were as follows: 1.0 mg mL−1 CDI acetonitrile solution at 25 °C for 25 min, followed by a reaction with a 1.0 mg mL−1 4-(dimethylamino)-benzenemethanamine acetonitrile solution at 70 °C for 30 min. Under the optimal conditions, the limits of detection (LODs) of the 11 FFAs were in the range of 3.0–14.0 ng mL−1; the limits of quantification (LOQs) were in the range of 8.0–45.0 ng mL−1; and the mean recoveries ranged from 83.4 to 112.8%, with intraday and interday precisions ranging from 0.7 to 9.1% and 3.7–9.5%, respectively. The experimental method is simple in terms of the pretreatment operation, accurate and reliable, and can be applied to the sensitive determination of FFAs in human blood samples. [ABSTRACT FROM AUTHOR]

To investigate the pharmacokinetics of 2, 3, 8-trimethylellagic (TMEA) in rats in vivo and determine the possible effects of the pathological conditions and compatibility, a rapid and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for quantitative determination was developed. TMEA and Artemetin (internal standard, IS) were separated on an Acquity Shim-pack GIST column with a total running time of 7 min using gradient elution at a flow rate of 0.3 mL/min. The intraday and interday relative standard deviations were <9.50%, and the relative error of accuracy was between −5.70% and 2.96%. The calibration curve of TMEA demonstrated good linearity with r2 = 0.9996, with the average recovery changing from 94.77% to 102.47% and the matrix effect from 93.16% to 100.15%. Compared with the normal group, the area under the plasma concentration-time curve from time 0 to the last time of quantifiable concentration (AUC(0 − t)), area under the plasma concentration-time curve from time 0 extrapolated to infinite time (AUC(0 − ∞)), and the maximum concentration (Cmax) of TMEA increased, whereas the time of maximum concentration (Tmax) and apparent clearance (CL/F) remarkably decreased in the TMEA group. With significantly reduced CL/F, AUC(0 − t), AUC(0 − ∞), and Cmax for TMEA were increased approximately one time after combining with 3, 7-Di-O-methylducheside A (DOMA). AUC(0 − t) and Cmax for TMEA in the 2, 3, 8-trimethylellagic-3, 8-dimethoxyellagic acid-2-oxyglucoside (TMEA-DMAG) group were significantly lower than that in the TMEA group with clearly prolonged Tmax and increased CL/F. These findings indicate that the changes in the pharmacokinetic parameters of TMEA may be caused by pathological and combination conditions. [ABSTRACT FROM AUTHOR]

Caffeine is a widely consumed psychostimulant with several mechanisms of action and various positive and negative effects on organisms. Caffeine undergoes extensive hepatic metabolism to form main metabolites such as theobromine, theophylline, paraxanthine, and 1,3,7‐trimethyluric acid. However, interspecies diversities have been observed in caffeine metabolism. In the present study, we developed a sensitive and straightforward ultra‐high‐performance liquid chromatography‐tandem mass spectrometry method to quantify caffeine and its primary metabolites, namely theobromine, theophylline, paraxanthine, and 1,3,7‐trimethyluric acid in rat plasma. After extraction of analytes using micro solid‐phase extraction plate, analytes were separated by elution gradient on the Acquity UPLC HSS T3 (50 × 2.1 mm, 1.8 μm) column over 4 min. The detection was done on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring modes using a positive electrospray ionization interface. The method was successfully validated according to the European Medicine Agency guideline over a concentration range of 5–1500 ng/ml for caffeine, 5–1200 ng/mL for theobromine, and 2.5–1200 ng/mL for theophylline, paraxanthine, and 1,3,7‐trimethyluric acid. The developed method was applied to analyze samples from animal experiments focusing on the metabolism and effects of caffeine and caffeine‐containing beverages. [ABSTRACT FROM AUTHOR]

Rationale: α-Amanitin is a highly toxic peptide widely found in species of poisonous mushrooms. The matrix effect has been a major obstacle for accurate determination of α-amanitin in plasma samples by liquid chromatography/tandem mass spectrometry (LC/MS/MS). In this study, the strategy to eliminate the matrix effect of α-amanitin with a one-step dilution approach after deproteinization was applied. Methods: Rat plasma samples were processed by protein precipitation with methanol followed by a nine-fold dilution with pure water. The matrix effect value of α-amanitin was 19.7%-22.2% by protein precipitation and then changed to 87.5%-88.7% after dilution. α-Amanitin and the internal standard (roxithromycin) were analyzed on an ACQUITY UPLC® BEH C18 (50 mm x 2.1 mm, 1.7 μm) column within 3.0 min by gradient elution. Results: The linear ranges were 0.90-600 ng/mL with a correlation coefficient r >0.9958. A lower limit of quantification (LLOQ) of 0.90 ng/mL was achieved using only 50 μL of rat plasma. The intra- and inter-day precisions for the analyte ranged from 3.2% to 7.5% and 3.1% to 7.1%, respectively, and the accuracy ranged from -5.3% to -8.0%. Conclusions: The matrix effect of α-amanitin was reduced by sample dilution after plasma deproteinization. A reliable LC/MS/MS method for the determination of α-amanitin in rat plasma was developed. This method was successfully applied for a toxicokinetic study of rats after intravenous injection of α-amanitin with a subacute toxicity dose at 0.10 mg/kg. [ABSTRACT FROM AUTHOR]

A selective RP-HPLC method for separation and determination of potential-related impurities (process related and degradants) of Ibrutinib drug substance has been developed and validated. The separation was accomplished on a X-Bridge C18, (150 x 4.6 mm, 3.5 μm) column connected to a photodiode array detector using 10 mM potassium dihydrogen phosphate with 0.025% of trifluoroacetic acid (pH ~ 5.5 adjusted with KOH solution) and acetonitrile in a ratio of 85:15 respectively as mobile phase A, and 10 mM potassium dihydrogen phosphate with 0.07% of trifluoroacetic acid (pH ~ 5.5 adjusted with KOH solution) and acetonitrile in a ratio of 30:70 respectively as mobile phase B, under gradient elution. The flow rate and detection wavelength were 1.0 mL/min and 220 nm, respectively. Quality by design approach using design expert software was strategically designed to optimize the critical chromatographic parameters like column temperature, flow rate and mobile phase B, pH variation in the mobile phase to achieve the separation of process impurities and thermal degradants. Two unknown impurities found in IBT thermal stability condition at more than 0.1% in HPLC analysis were enriched and isolated by preparative HPLC and structure was confirmed by 1H NMR, 13C NMR, mass spectroscopy and FT-IR spectroscopy. This method can be used for the quality control of both drug substance and drug product. The performance of the method was validated according to the International Conference on Harmonization guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, ruggedness and robustness. [ABSTRACT FROM AUTHOR]

Ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has become the main method for the detection and analysis of food additives because of its good separation, high selectivity, and high sensitivity. The aim of this study was to establish an UHPLC-MS/MS method that can quickly and accurately measure the content of carrageenan in livestock and poultry meat. Chromatographic separation was performed on an ACQUITY UPLC BEH HILIC C18 column (2.1 mm × 50 mm, 1.7 μm) using a gradient elution with methanol and 0.1% (v/v) formic acid in water as a mobile phase. The quantitative analysis was executed using a triple quadrupole mass spectrometer in which electrospray ionization, multiple reaction monitoring, and negative mode were operated. The retention time was about 1.3 min for carrageenan. The carrageenan content showed a good linear relationship from 0.05 to 1.00 g/kg. The limit of detection (LOD) was 0.06 g/kg, and the limit of quantification (LOQ) was 0.18 g/kg. The standards were spiked at three levels (low, medium, and high) and were analyzed in six replicates. The recovery values of carrageenan in pork, beef, lamb, chicken, and duck meat were 82.06–111.55%, 85.43–112.50%, 89.55–116.00%, 83.80–102.15%, and 82.41–110.90%, respectively. The relative standard deviations (RSDs) were all lower than 7.51%. The developed method shows a high recovery rate and good precision and can be used for the rapid detection of carrageenan in livestock and poultry meat. [ABSTRACT FROM AUTHOR]

Accurate quantitation of aldosterone is clinically important in standardized testing for primary aldosteronism. The results are often variable when performed by clinical immunoassays. To standardize and ensure the accuracy of clinical systems, reference measurement procedures (RMPs) with higher metrological order are required. A simple and reliable isotope dilution LC-IDMS/MS-based measurement procedure for human plasma aldosterone has been developed. This method involved plasma spiked with a deuterium-labelled internal standard, equilibrated for 0.5 h, and extracted by liquid-liquid extraction (LLE) without derivatization. Aldosterone and its structural analogues were baseline separated with a C18-packed UHPLC column with gradient elution within 7 min. The signal intensity variability and measurement imprecision were reduced by bracketing calibration during plasma aldosterone value assignment. The limit of detection (LoD) was 19.4 pmol/L with a signal-to-noise ratio (S/N) > 3. The lowest limit of quantification (LLoQ) was 27.7 pmol/L (S/N > 10 and CV < 10.0%). LLE was performed with 1 mL of n-hexane/ethyl acetate (3:2, v/v), and the extraction recovery was determined to be 92.15 ± 3.54%. The imprecisions were ≤ 3.18% for samples at 124.8, 867.0, and 2628.5 pmol/L. The recoveries were 98.11–101.61%. The relative bias between this candidate RMP and the established RMP was 2.76–1.89%. The linearity response ranged from 27.7 to 2774.4 pmol/L with R2 = 0.999. The method performance met the requirements of RMPs (≤ 5% total CV and ≤ 3% bias). Furthermore, the developed method was applied to evaluate immunoassays through 41 patient sample comparisons. The calibration and measurement capability (CMC) of this method were also evaluated by measuring these samples. The candidate RMP can serve as an accurate reference baseline for routine methods and can be used for value assignment for reference materials. [ABSTRACT FROM AUTHOR]

Haloperidol, butyrophenone derivative, is a typical antipsychotic drug used in the treatment of schizophrenia, manic phase of bipolar disorder, and acute psychomotor agitations. According to the recent guidelines for therapeutic drug monitoring, it is strongly recommended to measure plasma level during the therapy with haloperidol. The objective of this study was to develop and validate a simple liquid chromatography–tandem mass spectrometry-based method to quantitate haloperidol in human plasma. After one-step extraction procedure using OSTROTM plate, gradient elution on Acquity UPLC BEH C18 (50 ×2.1mm, 1.7μm) column over 3.2 min was performed. The detection was conducted on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode in positive ionization mode with transitions at m/z 376.29 → 165.14 and m/z 380.28 → 169.17 for haloperidol and haloperidol-d4 (used as an internal standard), respectively. The method was fully validated to cover wide concentration range of 0.05–80 ng/mL in human plasma and meets the criteria for the selectivity, linearity and lower limit of detection, precision and accuracy, matrix effect, extraction recovery, carryover, dilution integrity and stability. The extraction recovery was nearly 100%, and no significant matrix effects were observed. Therefore, the method is applicable to routine therapeutic drug monitoring in patients' plasma. [ABSTRACT FROM AUTHOR]

Combination therapy is a common approach for clinical treatment of type 2 diabetes mellitus, especially for patients with poor monotherapy. Meta-analysis suggested that omarigliptin, a long-acting DPP-4 inhibitor, combined with pioglitazone might improve the side effects of pioglitazone. However, little is known about the pharmacokinetic properties after a coadministration. In this study, a rapid and reliable method for the simultaneous determination of the pioglitazone and omarigliptin in rat plasma by UHPLC-MS/MS was established and validated for the first time. An exsil mono C18 column (2.0 × 50 mm, 3 μm) was used to separate the analytes and the column temperature was kept at 30°C. Sitagliptin was selected as the internal standard. 0.02% formic acid aqueous solution (A) and methanol-acetonitrile (B) were used as mobile phases with gradient elution at a flow rate of 0.3 mL/min. The elution procedure was as follows: 20%B (0–0.1 min), 80%B (0.1–0.3 min), 80%B (0.3–2.0 min), and 20%B (2.1–3.0 min). A multiple reaction monitor (MRM) was used under positive ionization mode with electrospray ion source to detect pioglitazone (357.1 ⟶ 134.1), omarigliptin (399.2 ⟶ 153.0), and sitagliptin (408.2 ⟶ 235.0). The linear ranges of pioglitazone and omarigliptin were 5–2000 ng/mL and 10–4000 ng/mL, respectively. Good linear relationships were exhibited in the corresponding linear ranges (r ≥ 0.9944). The bioanalytical method was validated, and the selectivity, linearity, sensitivity, accuracy, precision, stability, recovery, and matrix effect were acceptable. The validated method was then successfully applied to pharmacokinetic study of pioglitazone combined with omarigliptin in rats. Results suggested that the combination of the two drugs had little effect on the pharmacokinetic parameters of each other in rats. [ABSTRACT FROM AUTHOR]

Rationale: Proxalutamide is a novel drug for the treatment of prostate cancer. However, to date, there are almost no reports on the pharmacokinetics of proxalutamide in vivo. This study developed a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to determine the concentrations of proxalutamide in biological samples for pharmacokinetic studies. Methods: Chromatographic separation was achieved on a Kromasil 100-5C8 column followed by gradient elution using a Shimadzu HPLC system. MS was performed in positive ion electrospray ionization mode using a SCIEX API 4000 triple quadrupole system. A simple and rapid one-step protein precipitation method was used for sample processing, and a low sample volume of 10 μL was used for processing and analysis. Results: The method was validated to show good selectivity, sensitivity, precision, and accuracy. Good linearity (r² > 0.99) was observed for rat plasma (range: 2--5000 ng/mL) and rat tissue homogenates (range: 2-2000 ng/mL). The extraction recovery was above 98%, and no significant matrix effect was observed. This method was successfully applied to investigate the pharmacokinetics and tissue distribution of proxalutamide in rats. Conclusions: A rapid and sensitive LC/MS/MS method was developed and validated to determine the quantity of proxalutamide in rat plasma and tissue homogenates and to further study the pharmacokinetic parameters of proxalutamide in a rat model. The results showed that proxalutamide had good oral bioavailability and wide tissue distribution in vivo. [ABSTRACT FROM AUTHOR]

Purpose: To determine the metabolism of caffeic acid in rats. Methods: Sprague-Dawley rats were intragastrically administered caffeic acid in saline suspension, and biological samples collected. After sample pretreatment by solid phase extraction, ultra-high performance liquid chromatography combined with quadrupole-time of flight mass spectrometry system (UHPLC-Q-TOF-MS/MS) was established to rapidly screen and characterize caffeic acid metabolites in rats. Waters HSS T3 UPLC chromatographic column (2.1 mm × 100 mm, 1.7 µm) was applied for the gradient elution with aqueous solution of formic acid (A)-acetonitrile (B). Mass spectral data for the biological samples in electrospray positive and negative ion modes were collected and analyzed by SCIEX OS 1.3 workstation. Results: Based on their precise molecular weights and multistage mass spectrometry cleavage information, caffeic acid and 21 metabolites in vivo were identified. The results demonstrate that the biotransformation of caffeic acid in vivo was mainly achieved via hydrogenation, hydroxylation, methylation, sulfonation, glucuronidation, acetylation, and composite reactions. Results: Based on their precise molecular weights and multistage mass spectrometry cleavage information, caffeic acid and 21 metabolites in vivo were identified. The results demonstrate that the biotransformation of caffeic acid in vivo was mainly achieved via hydrogenation, hydroxylation, methylation, sulfonation, glucuronidation, acetylation, and composite reactions. [ABSTRACT FROM AUTHOR]

• A supercritical fluid-based method for extraction and analysis of indole alkaloids. • A more specific two-step SFE method towards alkaloids of complex natural plants origin. • C8SCX as adsorbent in SFE provide additional electrostatic attraction towards alkaloids. • An SFC-MS/MS method was utilized for analysis of indole alkaloids within 8 min. The development of an approach based on simultaneous supercritical fluid extraction-sample cleanup, followed by supercritical fluid chromatography/tandem mass spectrometry (SFE-SFC-MS/MS) was as a tool for the extraction, separation and characterization of indole alkaloids of Uncaria rhynchophylla. A two-step SFE method was designed. A mixture of the U. rhynchophylla sample and an adsorbent named C18SCX with the ratio of 1:1 (w/w) was placed into an extraction cell. The extraction temperature was 40 °C and the pressure was 25 Mpa. In the first step, 10 % EtOH as the co-solvent was used to extract for 60 min, which was considered as a cleanup process to remove non-alkaloid components. In the second step, 0.1 % DEA was added to 10 % EtOH and it extracted for 60 min to obtain the desired extract. By introducing an additional adsorbent, the specificity of SFE towards alkaloids was greatly improved. An SFC-MS/MS method was then utilized for analysis of the SFE extract. Using 2-EP as stationary phase with the gradient elution of 0–10 min, 5–25 % EtOH (+0.05 % DEA) in CO 2 , column temperature 40 °C, and back pressure 13.8 Mpa, 10 peaks were separated within 8 min. Further MS/MS analysis confirmed that nine of the 10 peaks in the SFE extract were indole alkaloids. This study developed a supercritical fluid-based method specifically towards extraction and analysis of alkaloids, which is helpful to the study of alkaline compounds in complex samples. [ABSTRACT FROM AUTHOR]

In our present study, the standard chemicals of triacetin were purified by reverse-phase and normal-phase semi-preparative high-performance liquid chromatography (HPLC), and 1H NMR and 13C NMR were employed to determine the purity and structure of triacetin. Moreover, a simple and rapid HPLC-photodiode array (PDA) method was developed to determine the contents of triacetin in 30 batches from different suppliers. The chromatographic separation was performed on a Phenomenex Gemini-NX C18 column (250 × 4.6 mm, 5 μm) using a gradient elution system of water and acetonitrile (contained 0.1% of formic acid) solution with a flow rate of 1.0 mL/min at 30°C at 210 nm. Sample preparation method is rapid and energy efficient, and the obtained sample have a good purity. Validation shows good specificity, linearity (R 2 = 0.9995), precision, stability, repeatability (% RSD < 2.80) and the average recovery (99.72%) of triacetin. The content of triacetin in most samples is concentrated in 94–97%. This developed approach is simple, rapid, accurate and can be used to quickly determine the purity and the content of triacetin in plasticizers and filter plugs. [ABSTRACT FROM AUTHOR]

We proposed a method for determining the presence of trace amounts of adducts of isopropylmethylphosphonic acid (IMPA) and cyclohexylmethylphosphonic acid (CMPA), biomarkers of exposure to sarin and cyclosarin in a body, with a tripeptide (Tyr-Thr-Lys). The method is based on plasma trypsinolysis with the formation of stable adducts IMPA–Tyr-Thr-Lys and СMPA–Tyr-Thr-Lys and their detection by high-performance liquid chromatography (HPLC) with high-resolution tandem mass spectrometry detection. The following conditions for the sample preparation of human blood plasma exposed to sarin and cyclosarin were optimized: aliquot portion volume, enzymatic hydrolysis conditions, mass spectrometric detection (selection of optimal pairs of ion reactions and detection modes), and gradient elution programs in the HPLC separation of the test mixture. The limit of detection for sarin and cyclosarin in human blood plasma was 5 ng mL–1. The proposed approach was tested in the analysis of blood plasma samples submitted to analysis in the framework of the Fourth Official Biomedical Test of the Organization for the Prohibition of Chemical Weapons; the results showed good determination specificity. [ABSTRACT FROM AUTHOR]

Objectives: Hydroxychloroquine (HCQ) is an anti-malarial and immunomodulatory drug reported to inhibit the Corona virus, SARS-CoV-2, in vitro. At present there is insufficient evidence from clinical trials to determine the safety and efficacy of HCQ as a treatment for COVID-19. However, since the World Health Organisation declared COVID-19 a pandemic in March 2020, the US Food and Drug Administration issued an Emergency Use Authorisation to allow HCQ and Chloroquine (CQ) to be distributed and used for certain hospitalised patients with COVID-19 and numerous clinical trials are underway around the world, including the UK based RECOVERY trial, with over 1000 volunteers. The validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of HCQ and two of its major metabolites, desethylchloroquine (DCQ) and di-desethylchloroquine (DDCQ), in whole blood is described. Methods: Blood samples were deproteinised using acetonitrile. HCQ, DCQ and DDCQ were chromatographically separated on a biphenyl column with gradient elution, at a flow rate of 500 μL/min. The analysis time was 8 min. Results: For each analyte linear calibration curves were obtained over the concentration range 50-2000 μg/L, the lower limit of quantification (LLOQ) was 13 μg/L, the inter-assay relative standard deviation (RSD) was <10% at 25, 800 and 1750 μg/L and mean recoveries were 80, 81, 78 and 62% for HCQ, d4-HCQ, DCQ and DDCQ, respectively. Conclusion: This method has acceptable analytical performance and is applicable to the therapeutic monitoring of HCQ, evaluating the pharmacokinetics of HCQ in COVID-19 patients and supporting clinical trials. [ABSTRACT FROM AUTHOR]

The purpose of this study was to develop a method for simultaneous analysis of aucubin, catalpol, and geniposide, which are representative iridoid glycoside constituents of Rehmannia glutinosa, in rat plasma, urine, and feces using hydrophilic interaction ultra high‐performance liquid chromatography with tandem mass spectrometry. The three components were separated using 10 mmol/L aqueous ammonium formate containing 0.01% (v/v) formic acid and acetonitrile as a mobile phase by gradient elution at a flow rate of 0.2 mL/min, equipped with a Kinetex® HILIC column (50 × 2.1 mm, 2.6 μm). Quantitation of this analysis was performed on a triple quadrupole mass spectrometer employing electrospray ionization and operated in multiple reaction monitoring mode. The chromatograms showed high resolution, sensitivity, and selectivity with no interference with plasma constituents. In all three iridoid glycosides, both the intra‐ and interbatch precisions (coefficient of variation %) were less than 4.81%. The accuracy was 96.56–103.55% for aucubin, 95.23–106.21% for catalpol, and 94.50–104.16% for geniposide. The developed analytical method satisfied the criteria of international guidance and was successfully applied to pharmacokinetic studies including oral bioavailability of aucubin, catalpol, and geniposide, and their urinary and fecal excretion ratios after oral or intravenous administration to rats. The new method was also applied to measure plasma protein binding ratios in vitro. [ABSTRACT FROM AUTHOR]

In this study, an efficient and sensitive assay for the detection of 42 polar neurochemicals, including neurotransmitters, amino acids, and biogenic amines, was established by combining reversed‐phase liquid chromatography tandem mass spectrometry with chemical derivatization. An optimally designed benzoyl chloride derivatization was easily conducted in a one‐pot reaction and stable neurochemical derivatives were obtained under mild conditions within 5 min (except for acetylcholine and melatonin). Derivatization also enabled the introduction of heavy labeling of the analytes through the use of labeled derivatization agents. Chromatography separation was performed on an HSS T3 column within 15 min by gradient elution. Multiple reaction monitoring acquisition mode enabled quantitation of neurochemicals with limits of detection of 0.05 to 11.63 nM and lower limits of quantitation of 0.09 to 46.50 nM in rat serum. The assay was well validated in terms of linearity and extraction recovery. Furthermore, the instrumental precision, specificity, matrix effect, accuracy, precision, stability, dilution effect, and carry‐over effect were also validated. Finally, the overall efficacy of the assay was experimentally tested using serum from six Sprague–Dawley rats. The results demonstrated that the developed method is effective for broad targeted analysis of 42 neurochemicals in serum. [ABSTRACT FROM AUTHOR]

Chimonanthi Radix (CR) is widely used in the treatment of influenza in China. Extensive studies revealed that the major bioactive constituents of CR were coumarins. However, pharmacokinetic study of coumarins in CR has not been fully studied. The purpose of this study was to establish a convenient and effective high-performance liquid chromatography–tandem mass spectrometry method that was used to simultaneously determine scopoletin, scopolin and isofraxidin in rat plasma after oral administration of CR extract using xanthotoxin as the internal standard. The chromatographic separation was carried out on a COSMOCORE C18 column (100 × 2 mm, 2.6 μm), using gradient elution with the mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B). Three coumarins and IS were quantified by positive ion electrospray ionization in multiple reaction monitoring mode. The method was fully validated in terms of specificity, accuracy, precision (intra- and inter-day), matrix effect, recovery as well as the stability of the analytes under various conditions. The results could provide further research foundation for anti-influenza mechanism of three coumarins in CR. [ABSTRACT FROM AUTHOR]

Artemether; a sesquiterpene lactone is widely used for the treatment of malaria as artemisinin-based combination therapy (ACT). The present work involves the development and validation of sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method for quantification of artemether (ART) in polymeric nanoparticles. ART was transformed to α, β-unsaturated decalones by pre-column acid treatment to enhance the sensitivity of chromophoric group lacking ART for quantification by HPLC-UV. Waters Spherisorb® 5 µm ODS(C18) column (4.6*250 mm) with gradient elution by mobile phase comprising of ACN and PBS (10 mM; pH 6.0) was used to separate acid-treated ART. The analysis was carried at λmax of 253 nm with 20 min and 20 µL run time and injection volume, respectively. The method was found to be linear in the concentration range of 0.5–10 µg mL−1 with 0.09 µg mL−1 and 0.27 µg mL−1 as LOD and LOQ respectively. Further, the method was also found to be specific for ART in presence of blank polymeric nanoparticles, accurate (% average recovery rate 101.7 ± 1.68%), precise (RSD <2%), and robust. The method was successfully used to determine % entrapment efficiency and in vitro release of ART-loaded polymeric nanoparticles with HPLC using a UV-visible detector. [ABSTRACT FROM AUTHOR]

A sensitive, reproducible reverse-phased high performance liquid chromatography electrospray tandem mass spectrometry (HPLCESI- MS/MS) method with simple sample preparation was developed for the simultaneous determination of a wide range of ceramides, diacylglycerols (DAGs) in cultured cells. Chromatographic separation of the compounds was achieved in a 14-minute run using a C8 column with a gradient elution by methanol and 10 mM ammonium acetate buffer as mobile phase at a flow rate of 0.5 ml/min. Various ceramides, DAGs were detected with a triple quadrupol system in multiple reaction monitoring mode, which is based on a soft positive electrospray ionization. The usual sample preparation process was shortened by the application of pure methanol for the extraction instead of the widely used methanol/chloroform mixture. C17:0 ceramide which does not occur in the cell samples, was used as an internal standard. The sample preparation process was optimized and the methodology was tested on a human hepatocarcinoma cell culture. Our results clearly showed accumulation of some ceramides and DAGs in the cells treated with BSA-conjugated palmitate for 8 hours. Since both ceramides and DAGs are important lipid intermediates and signal messengers, alteration in their cellular levels have major impact on cell functions, and thus our novel analytic method can be widely used in lipotoxicity research. The presented technique can be further developed to measure other intermediates of ceramide synthesis and other derivatives of DAGs as well. [ABSTRACT FROM AUTHOR]

The new ultra-high performance liquid chromatography method with tandem mass spectrometry detection (UHPLC-MS/MS) has been optimized to allow fast, selective, and high-throughput analysis of two Candida albicans quorum sensing molecules (QSM), farnesol and tyrosol. The problem of the presence of the interference in the samples and system was successfully solved by careful optimization of chromatographic conditions. Charged hybrid stationary phase modified with pentafluorophenyl group and optimized gradient elution provided adequate separation selectivity and peak shapes. The impurity was identified as dibutyl phthalate and had the same m/z ions as farnesol leading to an important interference on selected reaction monitoring channel. Two different types of biological matrices originating from vaginal fluid, supernatant and sediment, were analysed. Micro-solid phase extraction in pipette tips was optimized for the selective isolation of QSM from the supernatant. The insufficient retention of farnesol on the extraction sorbent was improved when 1% of organic solvent was added prior to extraction, while the retention of tyrosol was only possible when using combined C8 and polymer sorbent type. Strong retention of farnesol had to be solved by increasing elution solvent strength and volume up to 600 μL. However, this approach did not allow the pretreatment of sediment samples due to the sorbent clogging. Therefore, our previously developed protein precipitation method was modified and validated to analyse the sediments. New developed UHPLC-MS/MS method provided suitable accuracy and precision for the determination of QSM in vaginal fluid while using only 50 μL sample volume and two different sample preparation methods. [ABSTRACT FROM AUTHOR]

Chaihu-shugan-san (CHSGS) has been widely used in China to treat depression and gastrointestinal diseases for thousands of years, but little is known about its pharmacokinetic properties. The purpose of our study is to develop a reliable and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to detect five components in beagle plasma and study their pharmacokinetic after oral administration of CHSGS in beagles. An Agilent C18 column (2.1 × 150 mm, 3.5 μm) was used to separate the analytes, and the column temperature was maintained at 40°C. A gradient elution procedure was used with solvent A (acetonitrile) and solvent B (0.1% formic acid, aqueous) as mobile phases. The elution procedure was 60% B—10% B (0–3 min) and 10% B—60% B (3.1–4 min). The flow rate was 0.3 mL/min, and the total measurement time was 4 min. Within the determined range, the standard calibration curves of the five analytes had a satisfactory linear relationship (r2 ≥ 0.9923). The recovery rate (n = 6) of the five analytes was between 85.42% and 90.85%, and the matrix effects (n = 6) were between 94.52% and 103.91%. These results show that the validated method could be successfully applied to study the pharmacokinetic in beagles after a single dose of CHSGS. [ABSTRACT FROM AUTHOR]

Acute renal failure caused by Lianbizhi® injection can be attributed to andrographolide sodium bisulphite (ASB) and its main related substance RS413. For the safety evaluation of ASB and RS413, the pharmacokinetics of both compounds in rats was investigated through a liquid chromatography-tandem mass spectrometry method. After a simple protein precipitation with methanol, ASB and RS413 with dehydroandrographolide as an internal standard in plasma were separated on a C18 column using gradient elution with methanol and water and detected in the negative selected reaction monitoring mode. The intra- and inter-day accuracies of the method varied from 96 to 107% over the concentration range of 10–1000 ng/mL. Precisions were within 7.8% and the extraction recoveries ranged between 71–99% without obvious matrix effects. The method was applied for the simultaneous quantification of ASB and RS413 in rats treated intravenously with ASB and RS413 at the dose of 80 mg/kg for the first time. [ABSTRACT FROM AUTHOR]

Vanillylmandelic acid (VMA) is one of the most important catecholamine metabolites, and it is usually used to aid in diagnosis of pheochromocytoma and paraganglioma. A new digital control microextraction by packed sorbent (MEPS) procedure coupled to liquid chromatography-mass spectrometry (LC-MS/MS) method has been developed to determine VMA in human urine. We evaluated important parameters influencing MEPS efficiency, including stationary phase, extracting cycles, and sample dilution. In optimized MEPS conditions, Only 10 μL of sample volume and 3 min preparation time for one sample were needed. Chromatographic separation was achieved with a hydrophilic interaction liquid chromatography (HILIC) column using gradient elution. VMA was detected using multiple reaction monitoring (MRM) with an electrospray source operating in negative ion mode. The method was validated for linearity, limit of quantification, accuracy, imprecision, matrix effect, and interference. Linearity was 0.5–100 μg/mL for VMA. Intra-assay, inter-assay, and total imprecision were less than 9.6%. Interferences precluding quantitation of VMA in dilute-and-shoot approach were reduced significantly using a MEPS approach. Method comparison of LC-MS/MS and homogeneous enzyme immunoassay was performed, and the reference interval was established. The developed MEPS-LC-MS/MS method certainly contributes to method robustness and makes it suitable for measurement of urinary VMA in routine clinical biochemistry laboratories. [ABSTRACT FROM AUTHOR]

A multiple columns and detections liquid chromatography system, including size exclusion chromatography (SEC) and reversed phase liquid chromatography (RPLC), for the analysis of macromolecules and micromolecules in ginseng root and leaf was developed. The columns were connected by two switching valves. Macromolecules were separated on a SEC column (TSK gel SuperMultipore PW-H column, 6 mm× 150 mm, 8 μm) by isocratic elution of 50 mM ammonium acetate aqueous solution, 0.3 mL/min of flow rate and detected by evaporative light scattering detection (ELSD). Micromolecules were analyzed on a Poroshell RP column (Agilent Poroshell 120 SB-Aq column, 4.6 mm × 50 mm, 2.7 µm) with gradient elution of water and acetonitrile, 0.6 mL/min of flow rate and detected by ultraviolet detection (UV). As a result, in the macromolecules chromatogram of ginseng root sample showed two main peaks while only one major peak for ginseng leaf. For micromolecules analysis, 17 compounds (3 nucleosides + 14 saponins) and 17 compounds (3 nucleosides + 1 flavonoid + 13 saponins) were found in ginseng root and leaf, respectively. The developed method is helpful for the quality evaluation of ginseng root and leaf. [ABSTRACT FROM AUTHOR]

Rationale: A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated to determine levodopa, carbidopa, entacapone, and corresponding six related substances - levodopa impurity B, levodopa impurity C, methyldopa, methylcarbidopa, entacapone impurity C, and entacapone impurity A - in film-coated tablets for the first time. Methods: Chromatographic separation was achieved with a gradient elution by using a C18 column, a mobile phase containing 0.5% formic acid in water and 0.5% formic acid in methanol. The mobile phase flow rate was 0.5 mL min-1. The UV detector was set at 280 nm and the triple quadrupole mass spectrometer was used in multiple reaction monitoring (MRM) mode. Results: The limit of detection (LOD) and limit of quantification (LOQ) results were 1.3 and 3.94 ng mL-1 for levodopa impurity B; 5.26 and 15.9 ng mL-1 for levodopa impurity C; 0.833 and 2.53 ng mL-1 for methyldopa; 3.31 and 10.0 ng mL-1 for methylcarbidopa; 1.67 and 5.06 ng mL-1 for entacapone impurity C; and 0.61 and 1.86 ng mL-1 for entacapone impurity A. Conclusions: The method was rapid, linear, accurate, and reproducible. The LC/MS/MS method that was developed to determine the related substances and assay of levodopa, carbidopa, and entacapone can be used to evaluate the quality of regular samples in the pharmaceutical industry. It can be also used to test the stability of samples. [ABSTRACT FROM AUTHOR]

Oroxylin A, obtained from the root of Scutellaria baicalensis Georgi, is a flavonoid with antitumor and other pharmacological activities. Our previous studies showed for the first time that it is mainly metabolized to oroxylin A sodium sulfonate by sulfotransferase enzymes in beagle dogs. In this study, rapid, universal, selective, and robust ultra‐high‐performance liquid chromatography–tandem mass spectrometry methods were established and fully validated to quantitatively detect oroxylin A, oroxylin A 7‐O‐glucuronide, and oroxylin A sodium sulfonate in beagle dog plasma. The quantitative analysis for oroxylin A sodium sulfonate was reported for the first time. Plasma samples were processed with acetonitrile, a universal protein precipitant. Gradient elution was performed to resolve carryover effects and to achieve separation efficiency and sufficient chromatographic retention. The linear relationships of oroxylin A, oroxylin A 7‐O‐glucuronide, and oroxylin A sodium sulfonate in plasma were in the range of 2.0–500.0, 5.0–500.0, and 1.881–940.5 ng/mL, respectively. The assay method was successfully applied to pharmacokinetic study. This is the first paper that reveals the pharmacokinetic profile of oroxylin A, oroxylin A 7‐O‐glucuronide, and oroxylin A sodium sulfonate after single‐dose intravenous and oral administration of Oroxylin A in beagle dogs. [ABSTRACT FROM AUTHOR]