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Oligosaccharides constitute fundamental components in numerous traditional Chinese medicines (TCMs). Conventional chromatographic methods for natural product analysis are not suitable for oligosaccharides due to their large polarity and structural similarity. Herein, an ultra-high performance liquid chromatography with charged aerosol detector (UHPLC-CAD) method was developed for the profiling of oligosaccharides using 9 neutral (DP3-DP11) reference oligosaccharides. Various factors, including columns, mobile phase, elution conditions, flow rate, and column temperature were systematically examined. Optimal separation was achieved using an Amide column with gradient elution within 18 min, at 0.5 mL/min flow rate and 30°C column temperature. Moreover, an ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) method was also optimized to provide structural information. The developed method was applied to detect oligosaccharides in several TCMs, including Morindae Officinalis Radix (MOR), Ziziphi Spinosae Semen (ZSS), Menthae Haplocalycis Herba (MHH) and Chrysanthemi Indici Flos (CIF), revealing 9 and 16 oligosaccharides being uncovered from MHH and CIF respectively for the first time. This study presents a versatile UHPLC-CAD and UHPLC-Q-TOF/MS method with the potential for advancing oligosaccharides discovery and contributing to the quality analysis of TCMs. • A universal method for profiling oligosaccharides in TCMs was developed. • The relative content of oligosaccharides in TCMs was investigated by UHPLC-CAD. • Nine and sixteen oligosaccharides were uncovered from MHH and CIF, respectively. [ABSTRACT FROM AUTHOR]

Antimicrobials, particularly antibiotics, are among the most common classes of drugs reported as substandard and falsified (SF) in developing countries. Therefore, it is important to develop simple and affordable analytical methods for the quality control of antimicrobial medicines. In this study, a liquid chromatographic method with ultraviolet detection (LC-UV) was developed and validated for the screening and quantification of 13 antimicrobial medicines and one beta-lactamase inhibitor in pharmaceutical formulations. LC separation was carried out on a Kinetex C18 column (150 mm × 4.6 mm, 2.6 µm) with gradient elution. The mobile phase consisted of mixtures of acetonitrile-water-10 mM phosphate buffer pH 3.5 at ratios of 3:92:5, v/v/v for mobile phase A and 50:45:5, v/v/v for mobile phase B with a flow rate of 0.5 mL/min. The screening method was intended for confirmation of the identity of the actives and validated for specificity and robustness, whereas the quantification method (using only a different detection wavelength) was further validated in terms of linearity, accuracy, sensitivity and precision (repeatability, intermediate precision). For all compounds, the method was found to be linear (r2 > 0.999), precise (%RSD < 1%), accurate (% recovery of 98–102%), sensitive, specific and robust. The developed LC method was successfully applied for the identification and assay of 12 antimicrobial samples from Ethiopia. Among the 12 samples analyzed, one (8.3%) product was confirmed to be falsified. [Display omitted] • Novel LC-UV method for screening and quantification of antimicrobial medicines. • Antimicrobial drugs mostly prone to counterfeiting were included in this study. • Successfully validated according to the ICH guidelines. • Method was applied to analyse suspicious samples. [ABSTRACT FROM AUTHOR]

Alternative blood sampling strategy can enhance the application of therapeutic drug monitoring (TDM), then improve precision therapy and medication compliance. In developing nations, alternative sampling strategy that allows self-sampling and room temperature transport is especially important. This study validates the use of dried blood spot (DBS) and dried plasma spot (DPS) sampling along with liquid chromatography–tandem mass spectrometry (LC–MS/MS) for analyzing seven common antiepileptic drugs (AEDs) (phenytoin, lamotrigine, levetiracetam, topiramate, carbamazepine, oxcarbazepine and its active metabolite 10,11-dihydro-10-hydroxy carbamazepine) and evaluates their applicability to clinical practice. Following simple protein precipitation with acetonitrile, the AEDs were separated on a C18 column by gradient elution with a mobile phase consisting of acetonitrile–water–0.1% formic acid at a flow rate of 0.65 mL/min. The method provided linear analysis over the tested concentration ranges, with a total run time of 7 min. Intra- and inter-assay precision for all quality controls were ≤12% with accuracies of 85.9%–113%. The average extraction efficiencies were 69.0%–92.4% for DBS and 65.9%–96.5% for DPS, and no significant matrix effects were observed. The AEDs were stable in all samples for seven days at room temprature and 40°C. There was good correlation between the dry and wet plasma concentrations with greater accuracy for DPS compared to DBS indicating that alternative sampling strategy using DBS and DPS are suitable for monitoring the concentrations of AEDs with satisfied performance and logistical advantages. • Development and validation of a LC–MS/MS method for the quantification of seven antiepileptic drugs in dried microsamples. • Demonstration of the method's applicability in clinical practice by comparing the results with traditional plasma analyses. • An affirmation that the antiepileptic drug concentrations in dried samples are highly consistent with plasma levels. Dried plasma spot-based analyses showcasing higher accuracy than dried blood spot-based analyses. [ABSTRACT FROM AUTHOR]

BGT-002, a new type of ATP-citrate lyase inhibitor, is a promising therapeutic for treatment of hypercholesterolemia. After an oral administration of BGT-002 to subjects, it underwent extensive metabolism and an acyl monoglucuronide (ZM326E-M2) on 1- carboxylic acid group was the major circulating metabolite. In this study, an LC-MS/MS method was developed and validated for the simultaneous determination of BGT-002 and ZM326E-M2 in plasma and the evaluation of their pharmacokinetic characteristics in humans. After extraction from the plasma by acetonitrile-induced protein precipitation, the analytes were separated on a Waters ACQUITY UPLC® BEH C 18 column using acetonitrile and 2 mM ammonium acetate containing 0.1% formic acid as the mobile phase for gradient elution. Negative electrospray ionization was performed using multiple reaction monitoring (MRM) of m/z 501.3→325.4 for ZM326E-M2 and m/z 507.3→331.2 for D 6 -ZM326E-M2, and pseudo-MRM of m/z 325.3→325.3 for BGT-002 and m/z 331.3→331.3 for D 6 -ZM326E, respectively. The method was validated with respect to accuracy, precision, linearity, stability, selectivity, matrix effect, and recovery. The analytical range in human plasma was linear over a concentration range of 0.0500–50.0 μg/mL for BGT-002 and 0.0100–10.0 μg/mL for ZM326E-M2. The pharmacokinetic results showed that after a single oral administration of 100 mg BGT-002, the parent drug was rapidly absorbed with a mean time to peak concentration (t max) of 1.13 h, compared with BGT-002, the t max (4.00 h) of ZM326E-M2 was significantly delayed. The peak concentration and plasma exposure of ZM326E-M2 were about 14.1% and 19.5% of the parent drug, suggesting that attention should be paid to the safety and efficacy of ZM326E-M2 in clinical research. • BGT-002 is a new type of ATP-citrate lyase inhibitor developed for hypercholesterolemia therapy • First LC-MS/MS method for the simultaneous quantification of BGT-002 and its acyl glucuronide metabolite in human plasma. • The developed method was successfully applied to a clinical pharmacokinetic study. [ABSTRACT FROM AUTHOR]

Multicomponent drugs are medications that combine two or more active pharmaceutical ingredients in a single dosage form. These dosage forms improve the patient compliance, reduce the risk of drug interactions, and simplify dosing regimens. However, quality control of these multicomponent dosage forms can be challenging, especially if the final product contains four or more ingredients that are active (comprise stabilizers, preservatives, excipients, and other components). This problem can be more pronounced if the excipients can interfere with the analysis. In this work, a stability indicating assay method was developed and validated (according to the ICH International Guidelines) for the simultaneous determination of hydroquinone (HQ), tretinoin (TRT), hydrocortisone (HCA), butylated hydroxytoluene (BHT), methyl paraben (MP) and propyl paraben (PP) in commercially available pharmaceutical creams. The proposed method is based on gradient elution using X-Bridge C18 (150 × 4.6 mm, 5 µm) column with a flow rate of 1 mL/min. The linear ranges (μg/mL) were 240–560 for HQ, 24–56 for MP, 132–308 for HCA, 6–14 for PP, 12–28 for BHT, 6.6–15 for TRT. During the validation process, the intra- and interday precision and trueness (evaluated as recovery) were found to be below 2.0% and between 100–102%, respectively. System suitability tests (SST) allow validating the herein proposed procedure specifically for pharmaceutical and industrial applications. SST test shows that the reported procedure fulfill with the Guidelines, allowing excellent separation of the analytes with very sensitive, accurate (precise and true) and reproducible quantitation of each analytes. The method was successfully applied in forced degradation studies of the six analytes. Specifically, acid degradation slightly affected HCA and BHT (91% recovery), while alkaline degradation drastically reduced HCA recovery (5.5%) and moderately affected BHT (85%). Photodegradation primarily influenced TRT quantity, and oxidative degradation intensified the BHT peak (130%). [Display omitted] • Validated HPLC method for the quantitation of hydroquinone, tretinoin, hydrocortisone, butylated hydroxytoluene and parabens. • Innovative analytical approach for multicomponent formulations. • Practical application for drug quality control in cream dosage form. • First tool to successfully quantify up to 6 components simultaneously in a topical formulation. • AGREEprep and BAGI evaluation of the proposed method. [ABSTRACT FROM AUTHOR]

The P. heterophylla and its adulterants were identified by HPLC-CAD fingerprint of sucrose and oligosaccharides in P. heterophylla. The improved quantitative analysis of multi-components with a single marker (iQAMS) was further established for simultaneous determinations of sucrose and oligosaccharides in P. heterophylla. The HPLC-CAD fingerprint and similarity coefficients between P. heterophylla and its adulterants showed significant differences. The relative errors (REs) between iQAMS method and external standard method (ESM) were below 3.00%, but significant difference was shown between iQAMS (different marker for whole program with gradient elution) and QAMS (one marker for whole program with gradient elution), indicating that QAMS method should be improved, especially for gradient elution which influence the response of analytes. The accuracy, precision, reproducibility, and stability of this method were validated which exhibited satisfactory results, indicating that iQAMS method could be used for quantitative analysis of sucrose and oligosaccharides in P. heterophylla instead of ESM. The iQAMS combined with HPLC-CAD fingerprint could be used to determine the content of each oligosaccharide, and it can be used for quality control of P. heterophylla. • P. heterophylla and its adulterants were firstly rapid identification by HPLC-CAD fingerprint. • A novel method, HPLC-CAD combined with improved QAMS, was developed for P. heterophylla analysis. • Total contents of sucrose and oligosaccharides were quantified by improved QAMS with PHO DP3–1 and PHO DP4. • The developed QAMS can improve the accuracy of HPLC quantification with gradient elution in the absence of reference standards. [ABSTRACT FROM AUTHOR]

Unecritinib (TQ-B3101) is a selective tyrosine kinase receptor inhibitor. In the study, in vitro metabolic experiments revealed that the hydrolysis of TQ-B3101 was mainly catalyzed by carboxylesterase 2 (CES2), followed by CES1. Next, a sensitive and reliable LC-MS/MS method was established for the simultaneous determination of TQ-B3101 and its metabolite crizotinib in rat plasma. To prevent in vitro hydrolysis of TQ-B3101, sodium fluoride, the CESs inhibitor at a concentration of 2 M , was immediately added after whole blood collection. Plasma samples were extracted by acetonitrile-induced protein precipitation method, and chromatographically separated on a Gemini C 18 column (50 mm × 2.0 mm i.d., 5 μm) using gradient elution with a mobile phase of 0.1% formic acid and 5 mmol/L ammonium acetate with 0.1% formic acid. The retention times for TQ-B3101 and crizotinib were 2.61 and 2.38 min, respectively. The analytes were detected with tandem mass spectrometer by positive electrospray ionization, using the ion transitions at m/z 492.3 → 302.3 for TQ-B3101, m/z 450.3 → 260.3 for crizotinib, and m/z 494.0 → 394.3 for imatinib (internal standard). Method validation was conducted in the linear range of 1.00–800 ng/mL for the two analytes. The precision, accuracy and stabilities all met the acceptance criteria. The pharmacokinetic study indicated that TQ-B3101 was rapidly hydrolyzed to crizotinib with the elimination half-life of 1.11 h after a single gavage administration of 27 mg/kg to Sprague-Dawley rats, and the plasma exposure of TQ-B3101 was only 2.98% of that of crizotinib. • TQ-B3101 is an amide prodrug of crizotinib, a selective tyrosine kinase receptor inhibitor. • First LC-MS/MS method for the simultaneous quantification of TQ-B3101 and crizotinib in rat plasma. • The developed method was successfully applied to a pharmacokinetic study. [ABSTRACT FROM AUTHOR]

Arginase is an enzyme responsible for converting arginine, a semi-essential amino acid, to ornithine and urea. Arginine depletion suppresses immunity via multiple mechanisms including inhibition of T-cell and NK cell proliferation and activity. Arginase inhibition is therefore an attractive mechanism to potentially reverse immune suppression and thus has been explored as a therapy for oncology and respiratory indications. Small molecules targeting arginase present significant bioanalytical challenges for in vitro and in vivo characterization as inhibitors of arginase are typically hydrophilic in nature. The resulting low or negative LogD characteristics are incompatible with common analytical methods such as RP-ESI-MS/MS. Accordingly, a sensitive, high-throughput bioanalytical method was developed by incorporating benzoyl chloride derivatization to increase the hydrophobic characteristics of these polar analytes. Samples were separated by reversed phase chromatography on a Waters XBridge BEH C18 3.5 μm, 30 × 3 mm column using gradient elution. The mass spec was operated in positive mode using electrospray ionization. The m/z 434.1→176.1, 439.4→181.2, 334.9→150.0 and 339.9→150.0 for AZD0011, AZD0011 IS, AZD0011-PL and AZD0011-PL IS respectively were used for quantitation. The linear calibration range of the assay was 1.00–10,000 ng/mL with QC values of 5, 50 and 500 ng/mL. The qualified method presented herein exhibits a novel, robust analytical performance and was successfully applied to evaluate the in vivo ADME properties of boronic acid-based arginase inhibitor prodrug AZD0011 and its active payload AZD0011-PL. [Display omitted] • Arginase inhibition as a potential mechanism to reverse immune suppression. • Water loss from parent mass observed when using APCI, but not with ESI. • Prodrug was utilized to increase bioavailability utilizing PEPT1 transporter. • Derivatization with benzoyl chloride to facilitate reversed phase chromatography of polar compounds. • LC-MS/MS method qualification of prodrug and parent compounds. [ABSTRACT FROM AUTHOR]

A sensitive, reproducible, robust, high-throughput ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous quantification of fexofenadine and olmesartan in human serum. Samples (50 µL) undergo protein precipitation prior to UPLC-MS/MS analysis. The analytes were separated using an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 µm) at a flow rate of 0.5 mL/min using a gradient elution with a total run time of 4 min. The analytes were detected in positive ion mode and selected reaction monitoring (SRM) was used for quantitation. The standard curve concentration range was 1.0–500.0 ng/mL for both analytes and each analyte showed excellent linearity with correlation coefficients (R2 > 0.99). The intra- and inter-day accuracy and precision were ±15% for each analyte, and excellent recovery was demonstrated (93–98%) for both analytes. The method is well suited for high-throughput quantitative determination of fexofenadine and olmesartan simultaneously and was successfully applied to an in vivo pharmacokinetic and transporter phenotyping study in humans. • An assay was validated for fexofenadine and olmesartan quantitation in human serum. • Excellent linearity was achieved for both drugs between 1.0 and 500.0 ng/mL. • A simplified sample preparation procedure allowed for high-throughput analysis. • The method was successfully applied to a pharmacokinetic study in humans. [ABSTRACT FROM AUTHOR]

Technical advances in the field of quality analysis allow an increasingly deeper look into the impurity profile of drugs. The ability to detect unexpected impurities in addition to known impurities ensures the supply of high-quality drugs and can prevent recalls due to the detection of harmful unexpected impurities, as has happened recently with the N -nitrosamine and azido impurities in losartan (LOS) drug products. In the present study, the LC-MS/HRMS approach described by Backer et al. was applied to an even more complex system, being the investigation of 35 LOS drug products and combination preparations purchased in 2018 and 2022 in German pharmacies. The film-coated tablets were analysed by means of four LC-MS/HRMS method variants. For the separation a Zorbax RR StableBond C18 column (3.0 ×100 mm, particle size of 3.5 µm, pore size of 80 Å), a gradient elution and for mass spectrometric detection a qTOF mass spectrometer with electrospray ionization in positive and negative mode was used. An information-dependent acquisition method was applied for the acquisition of high-resolution mass spectrometry data. The combination of an untargeted and a targeted screening approach revealed the finding of eight impurities in total. Beside the five LOS related compounds, LOS impurity F, J, K, L, M, and related compound D from amlodipine besilate, LOS azide and an unknown derivative thereof were detected. Identification and structure elucidation, respectively, were successfully performed using in silico fragmentation. Differences in the impurity profiles of drug products from 2018 and 2022 could be observed. This study shows that broad screening approaches like this are applicable to the analysis of drug products and can be an important enhancement of the quality assurance of medicinal products. [Display omitted] • Analysis of losartan finished dosage forms by means of HRMS. • Structural elucidation by means of MS and MS/MS data. • Verification of proposed structural formulae by in silico fragmentation. • Comparison of the impurity profiles of losartan tablets purchased in 2018 and 2022. • Development of a procedure which provides a broad look into the impurity profile of drugs. [ABSTRACT FROM AUTHOR]

Areca nuts have been used as a traditional Chinese medicine (TCM) for thousands of years. Recent studies have shown that it exhibits good pharmacological activity and toxicity. In this study, the pharmacokinetics of five major components of areca nut extract in rats were investigated using a highly sensitive ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) method. Arecoline, arecaidine, guvacoline, guvacine, and catechin were separated and quantified accurately using gradient elution with mobile phases of (A) water containing 0.1 % formic acid-10 mM ammonium formate, and (B) methanol. The constituents were detected under a timing switch between the positive and negative ion modes using multiple reaction monitoring (MRM). Each calibration curve had a high R2 value of >0.99. The method accuracies ranged −7.09–11.05 % and precision values were less than 14.36 %. The recovery, matrix effect, selectivity, stability, and carry-over of the method were in accordance with the relevant requirements. It was successfully applied for the investigation of the pharmacokinetics of these five constituents after oral administration of areca nut extract. Pharmacokinetic results indirectly indicated a metabolic relationship between the four areca nut alkaloids in rats. For further clarification of its pharmacodynamic basis, this study provided a theoretical reference. • Sensitive detection of five major components in areca nut using UPLC-MS/MS method. • The pharmacokinetics study of five major components of areca nut extract in rats. • The results indicated a metabolic relationship between the four alkaloids in rats. [ABSTRACT FROM AUTHOR]

Niraparib is a potent and orally bioavailable inhibitor of poly (ADP-ribose) polymerase (PARP) with high specificity for isoforms 1 and 2. It has been approved by the U.S. Food and Drug Administration for ovarian cancer maintenance therapy and is currently under development for various cancers, including glioblastoma. To assess central nervous system (CNS) penetration of niraparib in glioblastoma patients, a novel bioanalytical method was developed to measure total and unbound niraparib levels in human brain tumor tissue and cerebrospinal fluid (CSF). The method was validated using plasma as a surrogate matrix over the concentration range of 1–10,000 nM on an LC-MS/MS system. The MS/MS detection was conducted in positive electrospray ionization mode, while chromatography was performed using a Kinetex™ PS C18 column with a total 3.5-minute gradient elution run time. The maximum coefficient of variation for both intra- and inter-day precision was 10.6%, with accuracy ranging from 92.8% – 118.5% across all matrices. Niraparib was stable in human brain homogenate for at least 6 hours at room temperature (RT) and 32 days at −20°C, as well as in stock and working solutions for at least 21 hours (RT) and 278 days (4°C). Equilibrium dialysis experiments revealed the fractions unbound of 0.05 and 0.16 for niraparib in human brain and plasma, respectively. The validated method is currently employed to assess niraparib levels in human glioblastoma tissue, CSF, and plasma in an ongoing trial on newly diagnosed glioblastoma and recurrent IDH1/2(+) ATRX mutant glioma patients (NCT05076513). Initial results of calculated total (K p) and unbound (K p,uu) tumor-to-plasma partition coefficients indicate significant brain penetration ability of niraparib in glioblastoma patients. [Display omitted] • The LC-MS/MS method was validated to measure niraparib levels in human plasma, brain tumor, and CSF. • Unbound fractions of niraparib were established in human plasma, brain, and brain tumor. • The total and unbound levels of niraparib in glioblastoma patients have been evaluated. [ABSTRACT FROM AUTHOR]

An increasing number of drug research institutions consider genotoxic impurity research a core task in drug research and development. Peroxides in drugs may directly or indirectly attack and damage cell DNA, posing a potential carcinogenic risk to the human body. Currently, the literature only studies hydroperoxide impurities, and benzyl peroxide has not been studied yet. In this study, an effective method for ultra-performance liquid chromatography mass spectrometry (UPLC MS/MS) was established and verified to detect and quantify the potential genotoxic impurity (PGI), empagliflozin benzyl peroxide, in metformin–empagliflozin combination formulations, which has not been reported thus far. A Waters ACQUITY UPLC HSS T3 (3.0 ×100 mm, 1.8 µm) column was used to achieve chromatographic separation with gradient elution. The mass spectrometry conditions were optimized using electrospray ionization in the negative mode. Following the International Conference of Harmonization (ICH) guidelines, this methodology can quantify PGIs at 1.35 ng/mL (5.4 ppm) in samples. This validated method exhibited good linearity over a concentration range of 5.4 to 36 ppm, and the accuracy of this method was in the range of 83.2–95.0% for empagliflozin benzyl peroxide. This approach fills the gap in the detection method for benzyl peroxide impurities in metformin hydrochloride and empagliflozin tablets, providing technical support for the quality control of the drug. • A potentially new genotoxic impurity, benzyl peroxide, was identified. • Impurities in metformin–empagliflozin combination formulations were detected. • Hydroperoxide is unstable and produces reactive free radicals that attack DNA. • A method was developed and validated for trace–level quantification of impurities [ABSTRACT FROM AUTHOR]

DO-2 is a highly selective MNNG HOS transforming (MET) inhibitor. This deuterated drug is thought to diminish the formation of the Aldehyde Oxidase 1 inactive metabolite M3. For various reasons, quantification of DO-2 and its metabolites M3 and DO-5 is highly relevant. In this study, we present an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to quantify DO-2, M3 and DO-5. Rolipram served as the internal standard. Aliquots of 25 µL were mixed with 100 µL internal standard consisting of 10 ng/mL rolipram in acetonitrile. Separation of the analytes was achieved on an Acquity UPLC ® HSS T3 column, utilizing gradient elution with water/formic acid and acetonitrile/formic acid at a flow-rate of 0.400 mL/min. Calibration curves were linear in the range of 1.00 – 1000 ng/mL for DO-2 and DO-5, and 2.00 – 2000 ng/mL for M3 in human plasma. The within-run and between-run precisions of DO-2, DO-5 and M3, also at the level of the LLQ, were within 12.1%, while the accuracy ranged from 89.5 to 108.7%. All values for accuracy, within-run and between-run precisions met the criteria set by the Food and Drug Administration. The method was effectively employed in the analysis of samples obtained from a clinical trial. [Display omitted] • We describe the first UPLC-MS/MS method for the analysis of DO-2 and its metabolites DO-5 and M3 in human plasma. • The assay met all of the current requirements of bioanalytical method validation. • Clinical applicability of the assay was demonstrated for pharmacokinetic analysis in clinical pharmacokinetic studies. [ABSTRACT FROM AUTHOR]

Recalls of medicinal products can cause supply bottlenecks. This is often due to the findings of unexpected impurities that pose a health risk to patients. A recent example is losartan potassium which was contaminated with azido-impurities. The choice of the analytical method determines which substances can be detected and thus controlled. In this study a combination of an untargeted screening approach for impurities and a targeted evaluation of high-resolution mass spectrometry data was applied to search for impurities not described so far, leaving out a precise quantification. Six losartan potassium samples were studied regarding known and unknown impurities and hence highlight the applicability and capability of the approach. For separation a Zorbax RR StableBond C18 column (3.0 ×100 mm, particle size of 3.5 µm, pore size of 80 Å), a gradient elution and an electrospray ionization in positive and negative mode for mass spectrometric detection was used. An information-dependent acquisition method was applied for the measurement of losartan potassium samples. The untargeted data evaluation using general unknown comparative screening revealed the presence of N -methyl-2-pyrrolidone (NMP) and another impurity from synthesis. The identity of NMP was corroborated by a spiking experiment and the amount was estimated by means of standard addition. A targeted data evaluation by generating extracted ion chromatograms resulted in finding of four additional impurities. Combined approaches like this are needed to detect and respond to changes in the quality of drugs precociously. [Display omitted] • Impurity profiling of losartan potassium with targeted and untargeted approaches. • Structural elucidation by means of high resolution mass spectrometry data. • Detection and identification of impurities not structurally related to losartan. • Procedure that can help to avoid expensive drug recalls. [ABSTRACT FROM AUTHOR]

The eburnamine-vincamine alkaloids exhibit a range of pharmacological activities. There is a limited understanding of the pharmacokinetics and pharmacodynamics of vindeburnol, a synthetic derivative of this chemical class of alkaloids. A fast and reliable UPLC-HRMS method was developed and validated to quantify vindeburnol in Soviet Chinchilla rabbit plasma from pharmacokinetics studies. An ultra-performance liquid chromatography system equipped with a Waters Acquity UPLC HSS T3 column was used for chromatographic separation by gradient elution with 0.1% (v/v) formic acid in water and acetonitrile. An Impact II QqTOF high-resolution mass spectrometer equipped with an Apollo II electrospray ionization source was used for analysis in positive mode; the ions [M+H]+ m/z 269.1648 ± 0.003 and m/ z 351.2067 ± 0.003 were monitored for vindeburnol and internal standard (vinpocetine), respectively. Preliminary metabolite profiling was also performed, and the pharmacokinetics of the identified metabolites were evaluated. The mean retention times for vindeburnol and vinpocetine were 2.0 and 3.5 min. The UPLC–HRMS method was validated with accuracy and precision within the 15% acceptance limit (8.2% and 11.0%, respectively). The mean extraction recovery value of vindeburnol from rabbit plasma was 77%. Pharmacokinetic evaluation of vindeburnol revealed that the compound is distributed rapidly with a short elimination half-life. Vindeburnol undergoes extensive first-pass metabolism and is metabolized into hydroxyvindeburnol and vindeburnol glucuronide. • A UPLC-HRMS method for the quantitative determination of vindeburnol was developed and validated. • The method was applied to study the pharmacokinetics of vindeburnol in rabbits. • A preliminary study of vindeburnol metabolism in rabbits was carried out. [ABSTRACT FROM AUTHOR]

Forced degradation studies provide rapid access to degradation products (DPs), where structural characterization and assessment of their potential toxicity are vital for pharmaceutical safety and regulatory compliance. As per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q1A R(2) guidelines, a forced degradation study of Bictegravir (BIC), a USFDA-approved drug for HIV wild type, in hydrolytic conditions (acid, base, and neutral) revealed the formation of six DPs in RP-HPLC (Reverse Phase- High-Performance Liquid Chromatography) gradient elution program using a C18 (4.6 × 250 mm, 5 µm) column. DP-1, 2, and 3 were characterized using liquid chromatography-tandem mass spectrometry (LC-MS/MS), whereas DP-4, 5, and 6 posed difficulties in characterization due to their isomeric nature. Using characteristic NOEs (Nuclear Overhauser Effect) from 2D ROESY NMR (Nuclear Magnetic Resonance) studies, we have elucidated diastereomeric DP-4/5 and isomeric DP-6/BIC configurational structures. Furthermore, in silico toxicity studies for the six degradation products were predicted for toxicity endpoints by employing DEREK, SARAH, and Pro Tox-II application tools. The DP-1 (methanamine) and DP-3 (carboxylic acid) resulting from acid-catalyzed hydrolysis, were predicted to have potential carcinogenic and mutagenic properties. These findings contribute significantly to our understanding of BIC's stability and safety profile in pharmaceutical development and underscore the rigorous characterization of stereoisomers by NMR that were further utilized for toxicity prediction. [Display omitted] • Bictegravir was subjected to hydrolytic stress according to ICH Q1A (R2) guidelines. • The DPs were separated and characterized in LC-PDA-Q/TOF-MS/MS and three of them are found to be isomers • 2D NMR studies were performed to elucidate a pair of R & S isomers and a functional isomer. • The degradation pathway is proposed and the in silico toxicity was assessed for the DPs. [ABSTRACT FROM AUTHOR]

Clorsulon is a potent anthelmintic agent and is widely used for the treatment and control of parasites including adult liver flukes in cattle and sheep. A rapid ion-paired reversed phase ultraperformance liquid chromatography (IP-UPLC) method has been developed and validated for determination of Clorsulon and its related substances in bulk drug substance batches of Clorsulon with a short octadecyl column. Analytes were eluted by a gradient elution on a Acquity UPLC® BEH C18 column (50 mm × 2.1 mm i.d., 1.7 µm particle size). Column temperature was maintained at 55 °C and all analytes were monitored by UV detection at 268 nm. Mobile phase-A constitutes 3 mM tetrabutylammonium hydroxide in H 2 O and mobile phase-B constitutes acetonitrile/methanol (50/50, v/v), respectively. All analytes of interest were adequately separated within 5 min. The analytes were quantitated against an external reference standard of Clorsulon. The chemical structures of degradation products of Clorsulon were proposed based on two-dimensional UPLC-MS data. The IP-UPLC method has been successfully validated and demonstrated to be accurate, sensitive, robust, and specific. • First reported stability-indicating UPLC method to analyze Clorsulon and its related substances. • The new developed method can easily be implemented for routine or non-routine analysis of Clorsulon API samples • Sample analysis time reduced of new method by 65% compared to the current USP HPLC method • The chemical structures of Clorsulon degradation products of Clorsulon are proposed based on two-dimensional UPLC-MS data [ABSTRACT FROM AUTHOR]

Trifluridine (FTD) and tipiracil (TPI) hydrochloride tablets (TAS-102) were used for the treatment of patients with metastatic rectal cancer that was resistant to conventional chemotherapy drugs. In this study, a rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed and fully validated for the simultaneous determination of TPI, FTD, and the metabolite 5-trifluoromethyluracil (FTY) of FTD in human plasma. The plasma samples were prepared by protein precipitation. The chromatography separation was performed using ACE Excel 3 AQ (100 × 2.1 mm i.d., 1.7 µm, ACE, England) column protected by a security guard cartridge (4.0 × 2.0 mm i.d., 5 µm, Phenomenex, USA) with a gradient elution of 0.05% acetic acid in water and methanol at a flow rate of 0.35 mL/min. The MS/MS analysis was performed by using multiple reaction monitoring with the segmented polarity (positive for TPI: m/z 243.1→183.0, and negative for FTD: m/z 295.1→252.0 and FTY: m/z 178.9→158.9) electrospray ionization mode. The segmented polarity mode was designed to achieve two advantages: better sensitivity and simultaneous determination of the analytes with different ion polarities. The calibration ranges were as follows: 1.00–250 ng/ for TPI, 8.00–8000 ng/mL for FTD and 5.00–1250 ng/mL for FTY. The selectivity, accuracy, precision, matrix effect, recovery, carryover, dilution integrity and stability test results meet ICH acceptance criteria. The method was evaluated using the RGB model and successfully applied to a clinical study in patients with solid tumors. For TPI, FTD and FTY, the maximum plasma concentration was 137–147 ng/mL, 6160–6240 ng/mL and 724–725 ng/mL, respectively; the plasma elimination half-life was 1.69–1.78 h, 1.70 h, and 3.09–3.14 h, respectively, after an oral administration of 60 mg TAS-102. [Display omitted] • A sensitive LC-MS/MS method was established for pharmacokinetics of nucleoside drugs. • Simultaneous determination of tipiracil, trifluridine and 5-trifluoromethyluracil. • MS response of analytes can be improved by using segmented polarity. • The PK parameters were obtained in Chinese patients after the administration. [ABSTRACT FROM AUTHOR]

In the present study, a reliable LC-QTOF-MS method was developed and employed for the separation and characterization of process-related substances and forced degradation products of Lurasidone hydrochloride. The chromatographic separation was carried out using an Agilent Poroshell 120 Bonus-RP C18 column (100 mm × 4.6 mm, 2.7 µm) and a mobile phase consisting of a gradient elution of 10 mM ammonium formate solution and methanol. The degradation studies followed the guidelines outlined in ICH Q1A (R2). It was observed that Lurasidone hydrochloride exhibited instability under photolytic, alkaline, and oxidative stress conditions, while remaining relatively stable under acidic and thermal stresses. Through positive ESI-QTOF mass spectrometric analysis, fourteen related compounds in total, including both process-related and stress degradation products, were identified based on the accurate masses of parent and product ions and calculated elemental compositions. Amongst these substances, nine had not been previously reported, and their formation mechanisms were speculated. The process-related substances were further confirmed by NMR spectra determination, and suggestions were proposed to eliminate them. This study highlights the potential for monitoring and controlling related substances during the manufacturing processes, providing valuable insights for process optimization and quality control of Lurasidone hydrochloride. • LC/QTOF/MS analysis was utilized to characterize both process-related substances and the degradants of Lurasidone. • A total of fourteen, including nine previously unreported lurasidone-related substances, were successfully identified. • The mass fragmentation pathways for Lurasidone and its related substances were accurately elucidated. • Strategies for monitoring and controlling manufacturing-related substances were suggested. [ABSTRACT FROM AUTHOR]

Ricobendazole hydrochloride is an active ingredient of a veterinary antiparasitic drug. The aim of this study was to investigate the degradation of ricobendazole hydrochloride under stress and stability testing conditions, for which we developed and validated the first stability indicating, specific, precise, accurate, and robust assay and related substances UPLC methods. The Acquity UPLC BEH C 18 column was used for the related substances and assay analyses of ricobendazole hydrochloride, and the analyses were performed at 25 °C sample and 30 °C column temperatures with a 2 µL injection volume. In both methods, a mixture of water and methanol (60:40, v/v) was used as the diluent, mobile phase A was a phosphate buffer (50 mM potassium dihydrogen phosphate solution, pH 3.2 ± 0.05, adjusted with 10% o-phosphoric acid), and mobile phase B was a mixture of mobile phase A and acetonitrile (50:50, v/v). For the analysis of related substances, a gradient elution system was used at a flow rate of 0.4 mL/min for 35 min with a detection wavelength of 220 nm, while for the assay analysis; a gradient elution system was used at a flow rate of 0.3 mL/min for 15 min with a detection wavelength of 290 nm. The calibration curves showed excellent linearity with high R-squared (R2) values for each compound, ricobendazole (0.9998, 0.249 – 3.740 µg/mL), ricobendazole amine (0.9998, 0.255 – 3.819 µg/mL), albendazole (0.9998, 0.255 – 7.646 µg/mL), and albendazole sulfone (1.0000, 0.251 – 15.090 µg/mL) indicating a strong correlation between the concentrations of the compounds and their respective peak areas in the UPLC analysis. The method showed excellent accuracy with relative standard deviation values of less than 2.5%. The stress and photostability studies showed that ricobendazole hydrochloride was insensitive to daylight and UV radiation and showed significant degradation at elevated temperature (85 °C, 9 days) and under all hydrolysis and oxidation conditions. The major impurity was ricobendazole amine under thermal and hydrolysis conditions, while albendazole sulfone was the major oxidative impurity. [Display omitted] • New stability-indicating UPLC methods were developed for ricobendazole HCl. • Both assay and related substances methods were validated according to ICH Q2(R1). • Stress-testing and stability studies of ricobendazole HCl were carried out. • The major impurity was ricobendazole amine formed under thermal and hydrolysis. • The major oxidation impurity was albendazole sulfone formed when treated with H 2 O 2. [ABSTRACT FROM AUTHOR]

In December 2021, the U.S. Food and Drug Administration (FDA) granted emergency authorization for Paxlovid® as an antiviral treatment for COVID-19. Paxlovid® is composed of two tablets, nirmatrelvir and ritonavir. Dose adjustment is necessary in cases of renal insufficiency. The aim of present study is to establish a LC–HRMS method for simultaneous determination of nirmatrelvir/ritonavir in human serum for therapeutic drug monitoring. Internal standard saquinavir was added in 25 μL human serum samples, and then the samples were precipitated with methanol. The analytes were separated by gradient elution on a C18 column, using a mobile phase of 0.1 % formic acid-water and methanol, at a flow rate of 0.4 mL/min. The injection volume was 2 μL, and the analysis time was 5 min. The determination of the analytes was performed by electrospray ionization in positive mode by full mass monitoring. The detected ions of nirmatrelvir, ritonavir and saquinavir were m/z 500.24792, 721.32004 and 671.39155, respectively. The linear concentration range for nirmatrelvir was 78.13–20000 ng mL−1, for ritonavir was 15.63–4000 ng mL−1 (r2＞0.9900). The accuracy ranged from 87.45 %∼104.63 %, and the intra-day and inter-day precision RSD was <15 %. The recovery of nirmatrelvir ranged from 98.72 %∼109.83 %, and that of ritonavir was 95.41∼112.36 %. The matrix effect of nirmatrelvir was 88.31∼97.73 %, and that of ritonavir was 85.17∼103.05 %. This method was used to measure the trough concentrations of nirmatrelvir/ritonavir in 17 patients. The trough concentration of nirmatrelvir was 1331.7–8352.5 ng/mL, and that of ritonavir was 53.4–1325.5 ng mL−1, with large individual differences. The method is simple, sensitive, specific, and reproducible, and can be used for monitoring the blood concentration and pharmacokinetic study of nirmatrelvir/ritonavir in COVID-19 patients. • This is a rapid LC–HRMS method for detection of nirmatrelvir/ritonavir in serum. • The nirmatrelvir trough concentration was 1331.7–8352.5 ng/mL in COVID-19 patients. • The trough concentration of ritonavir was 53.4–1325.5 ng/mL in COVID-19 patients. • This method can be used for pharmacokinetic study in COVID-19 patients furtherly. [ABSTRACT FROM AUTHOR]

Capillary microsampling (CMS) is a technique that can significantly reduce the blood collection volume compared to conventional sampling methods, and thus is much preferred for studies in rats and mice. BIIB131 (SMTP-7) is a novel thrombolytic drug candidate currently under Phase 2 clinical development for the treatment of acute ischemic stroke. To support the safety studies in rats, an accurate and reliable CMS LC–MS/MS assay for the quantification of BIIB131 in rat plasma was developed and validated. This method utilized stable-isotope labeled [13C 5 15N 2 ]-BIIB131 as the internal standard. The samples were extracted using acid-assisted liquid–liquid extraction with methyl tert-butyl ether (MTBE) and formic acid. The chromatographic separation was achieved on an ACE Excel 3 Super C18 analytical column (2.1 mm × 50 mm, 3.0 µm) using a gradient elution. The mass spectrometric detection of BIIB131 and its internal standard was achieved using positive ion electrospray multiple reaction monitoring (MRM). The standard curve ranged from 0.50 to 300 ng/mL for BIIB131 and was fitted to a 1/x2 weighted linear regression model. For regular QCs, the intra-assay precision was 1.7–6.1 % CV, the inter-assay precision was 2.7–11.0 % CV, and the intra-assay and inter-assay accuracy (%Bias) were –20.0–10.6 % and –7.8–6.3 %, respectively. For CMS QCs, the intra-assay and inter-assay precision were 2.2–13.6 % and 6.7–12.9 % CV, and the intra-assay and inter-assay accuracy (%Bias) were –13.2–15.0 % and –7.8–4.2 %, respectively. The validated CMS LC–MS/MS method has been successfully applied to a safety study in rats. • A novel capillary microsampling LC–MS/MS method for quantitation of BIIB131 in rat plasma. • Acid-assisted LLE to minimize matrix effect and improve extraction recovery. • First reported bioanalytical method for SMTP family compounds. • The validated method has been successfully applied to a safety study in rats. [ABSTRACT FROM AUTHOR]

A rapid, simple, and robust LC–MS/MS method was developed and validated for the quantitation of colistin and colistin methanesulfonate (CMS) in human plasma. The method also prevented overestimation of colistin concentration by establishing the stability of CMS under sample preparation conditions, including blood and plasma storage conditions. Polymyxin B1 was used as an internal standard, and positive-ion electrospray ionization in multiple reaction monitoring mode was used for quantification. Chromatographic separation was achieved using a Zorbax eclipse C18 column (3.5 µm, 2.1 mm i.d. × 100 mm), with a flow rate of 0.5 mL/min, 5 μL injection volume, and gradient elution with a mixture of acetonitrile-water (containing 0.1 % trifluoroacetic acid). The method had a quantifiable range of 0.043–8.61 and 0.057–11.39 μg/mL for colistin A and B in human plasma, respectively, under a total runtime of 6.0 min. Further, it demonstrated appropriate extraction efficiency, no significant interference from co-eluting endogenous compounds, and satisfactory intraday and interday precision and accuracy. The proposed procedure for sample preparation successfully addressed the issue of CMS instability, consequently diminishing the probability of overestimating the concentration of colistin. Therefore, this simple and robust LC-MS/MS method for CMS and colistin quantification in human plasma is a valuable tool for clinicians to accurately monitor colistin treatment in patients with infections caused by multidrug-resistant (MDR) Gram-negative bacteria. • Simple and robust LC-MS/MS detection method for CMS and colistin in human plasma. • Extraction process that stabilizes CMS through protein precipitation, and dilution with deionized water. • Analysis of total CMS by converting CMS to colistin using a thermal hydrolysis method. • Prevent overestimation of colistin level and establish the stability of CMS in both blood and plasma during storage. • Useful for TDM in patients undergoing colistin therapy, and suitable for large-scale pharmacokinetic studies. [ABSTRACT FROM AUTHOR]

• UHPLC-MS/MS determination of ethyl-glucuronide (EtG) in hair samples has been developed and validated. • Rapid analysis of EtG in 25 mg of hair with a simple supported liquid extraction (SPE) allowing high-throughput testing. • Good quantification limit (5 pg/mg) and analytical recoveries (>81.0%). • Good comparison with previous proficiency tests results. To improve the reproducibility, suitability and speed of hair testing for Ethylglucuronide (EtG), an ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated together with a supported liquid extraction (SLE) EtG from the keratin matrix. EtG was analyzed using reversed phase chromatography with gradient elution and detection with tandem mass spectrometry operated in multiple reaction monitoring (MRM) mode via negative electrospray ionization (ESI). The method showed good linearity from limit of quantification (LOQ) to 100 pg/mg hair (r2 0.996 ± 0.004). Recovery of the analyte was always higher than 80%, whereas intra- and inter-assay precision were always better than 15%. The developed method was applied to the analysis of more than 200 samples with medico-legal and epidemiological purposes ranging from non-detection of the analyte to 88.1 pg/mg and its robustness was proved by reanalysis of six different proficiency testing samples from the Society of hair testing obtaining a Z-score always less than 2. [ABSTRACT FROM AUTHOR]

• A fully validated LC–MS/MS method was developed, allowing for first time simultaneous determination of IRB and ROS. • The proposed method succeeded in studying pharmacokinetic and drug-drug interaction. • IRB affected ROS Pharmacokinetic parameters and vice versa. • The proposed method provides a mean for the dose monitoring of IRB and ROS. The synergistic vascular protective effect of statins and angiotensin receptor blockers (ARBs) is well known, however, the pharmacokinetic interaction among these classes is yet to be understood and the necessity of developing analytical methods for their determination in vivo is gradually increased. Herein, first chromatographic separation coupled tandem mass spectrometric was developed and fully validated for simultaneous measurement of rosuvastatin (ROS) and irbesartan (IRB) in rat plasma after oral administration. The two analytes were extracted from plasma sample using acetonitrile-induced protein precipitation then separated on an Agilent Eclipse Plus ODS (4.6 × 100 mm, 3.5 μm) column by gradient elution using 6 mM ammonium formate/0.1% formic acid and ACN at a flow rate 0.4 mL min−1. Multiple reaction monitoring in positive ion mode was used for quantification of precursor to production at m/z 492.1 → 206.9 for IRB, 482.1 → 258.1 for ROS, and 409.2 → 238.2 for the internal standard, amlodipine (AML). Linearity was obeyed in the range of 1–10000 ng mL−1 and 1–5000 ng mL−1 with detection limits (S/N of 3) of 0.05 and 0.07 ng mL−1 for IRB and ROS, respectively. The current method was validated in terms of selectivity, recovery, accuracy, precision, matrix effects, and stability as per US-FDA bioanalytical guidelines. The application of our method reported her is the first to study pharmacokinetic interaction of IRB and ROS in rat plasma after a single oral dose. The area under the concentration-time curve (AUC), peak plasma concentrations (C max), half-life time (t 1/2), and volume of distribution (V d) of ROS and IRB were affected when the two drugs were co-administering. The current study provided a valuable tool for studying drug-drug interaction and might be useful for therapeutic drug monitoring and bioequivalence studies. [ABSTRACT FROM AUTHOR]

• Antiretroviral drug, Dolutegravir found to be unstable under acidic and oxidative conditions. • Two degradants were identified by extensive analysis of UPLC-MS, HRMS and NMR spectra. • In vitro cytotoxicity potential of degrandants were evaluated and found to be safe. Dolutegravir was approved by USFDA, Canada and European regulatory authorities as antiretroviral medication. In this article, DLG forced degradation studies as per the International Council for Harmonization (ICH) prescribed stress conditions was conducted and the resulting degradants were fully characterized. DLG was stable in basic, thermal and photolytic stress conditions, whereas DLG was found to unstable in acidic and oxidative conditions. One degradant each from acid and peroxide treated solutions was resolved on LC–MS and labelled as DP-1 and DP-2 with RT 1.80 min and 1.41 min, respectively. DP-1 and DP-2 were isolated by preparative HPLC with C18 column using gradient elution method. Subsequently DP-1 and DP-2 peaks were subjected to HRMS for accurate mass. Molecular mass of DP-1 and DP-2 were m/z 420.1379 (positive mode) and m/z 214.0319 (negative mode), respectively. Further, DP-1 & DP-2 were subjected to NMR spectroscopic analysis (including 2D) for structural confirmation. DP-1 was identified as N -(2,4-difluorobenzyl)-9-hydroxy-2-(4-hydroxybutan-2-yl)-1,8-dioxo-2,8-dihydro-1H-pyrido[1,2- a ]pyrazine-7-carboxamide and it is earlier reported by Gudisela et al. [19] as DLG process impurity. DP-2 was identified as 2-(2,4difluorobenzylamino)-2-oxoacetic acid which is novel DLG degradant and not reported earlier to the best of our knowledge. DLG along its forced degradation products were found to be non-cytotoxic in in vitro assay conditions using HepG2 cells. [ABSTRACT FROM AUTHOR]

• Validated UPLC-PDA method for estimation of five bioactive markers in D. erecta. • First report on uncertainty measurement of phenylethanoid glycosides in D. erecta. • Uncertainty estimation done using validation data by EURACHEM/CITAC CG4 protocol. • Highest content of acteoside reported in D. erecta compared to other plants. Duranta erecta L.is a valuable herb used in traditional system of medicine in India and China for the treatment of malaria and abscesses. The presence of phenylethanoid (e.g., acteoside as major bioactive) in addition to iridoid and flavonoids glycosides are mainly responsible for reported pharmacological properties. Although D. erecta is widely used in many Asian countries, the quality assurance of raw herb and its derived products are still in question, due to the lack of validated protocol. Current Indian guidelines for quality assurance of phytopharmaceutical product demands accurate determination of minimum four chemicals. Moreover, there is no chromatographic method to simultaneously analyse acteoside, isoacteoside, durantoside I, quercetin, and methylapigenin-7-O-β-D-glucopyranuronate in D. erecta. The present study establishes a systematic approach for the quality evaluation of D. erecta with efficient and validated UPLC-PDA method that simultaneously analyses the five bioactive marker chemicals in D. erecta. A fast, sensitive, and reliable ultraperformance liquid chromatography-photodiode array (UPLC–PDA) method was developed for the quantification five marker compounds-acteoside, isoacteoside, durantoside-I, quercetin, and methylapigenin-7-O-β-D-glucopyranuronate in D. erecta. The optimized UPLC conditions on Reverse phase column Phenomenex Luna® (C 18 , 2.5 μm, 2.0 × 100 mm)) and binary gradient elution resulted in best separation for all five targeted phytoconstituents. Validation characteristics viz. linearity (r2 >0.999), accuracy (%RSD < 2.0), precision (%RSD, 1.62–2.59), recovery (99–101%), and sensitivity (LOD- 0.27-0.40 μg mL−1; LOQ- 0.90–1.35 μg mL−1) were satisfying the ICH criteria. Uncertainty in the measurement was also estimated using method validation data and other sources to fulfil the technical requirement of ISO 17025:2017. It is the first validated method that provides the simultaneous and accurate analysis of five bioactive phytoconstituents of D. erecta in short time with defined traceability and accuracy profile. Further, it delivers a holistic quality analysis of raw medicinal herb and its preparation. [ABSTRACT FROM AUTHOR]

• E0703 is a novel radioprotective agent, which will cause great concern in the field of radiation medicine. • An UPLC-MS/MS method was firstly developed for simultaneous quantitation of E0703 and its oxidative metabolite M1 in human plasma. • The LLOQs of the method were 0.1 ng/mL for E0703 and 0.2 ng /mL for M1 and the total analysis time was shortened to 2.5 min per sample. • The validated method was successfully applied to pharmacokinetic study in human. A rapid, sensitive and reliable bioanalytical method was firstly developed and validated based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS), for simultaneous quantitation of the novel radioprotective compound E0703 and its oxidative metabolite M1 in human plasma. Plasma samples were deproteinated with acetonitrile containing the internal standard IS1229 as precipitant and separated on a short CAPCELL PAK C18 IF2 column (2.0 mm × 20 mm, 2 μm) by gradient elution using acetonitrile (containing 0.1% formic acid) and water (containing 0.1% formic acid) with a run time of 2.5 min per sample. MS detection was carried out on a triple quadrupole mass spectrometer (Xevo TQ-S) coupled with electrospray ionization in positive multiple reaction monitoring (MRM) mode. The method was linear over the concentration ranges of 0.100–50.0 ng/mL for E0703 and 0.200–100 ng/mL for M1, with correlation coefficient (r2) values ≥0.993. A full validation of this method was performed, and all results were within acceptable limits. The novel assay was sensitive enough to monitor E0703 and M1 levels in human plasma, and was successfully applied to a clinical pharmacokinetic study of healthy Chinese subjects after a single oral administration of 30 mg E0703 tablets. In conclusion, the validated method is accurate, sensitive and high-throughput, and can be successfully implemented for subsequent clinical pharmacokinetic studies of E0703 and M1. [ABSTRACT FROM AUTHOR]

• Simultaneous determination of bimatoprost chemical purity and assay. • Determination of bimatoprost and nine impurities within 15 min. using a new, highly selective and sensitive UHPLC UV method. • The developed method fulfilled all validation acceptance criteria. • The applicability of the method for the standard quality control as well as for stability testing. A simple, rapid and accurate ultra-high performance liquid chromatographic (UHPLC) method with a UV detection for the determination of the chemical purity and assay of bimatoprost (BT-1) was developed. The chromatographic separation was achieved with the use of an Acquity BEH C8, 150 × 2.1 mm, 1.7 μm reversed phase analytical column. The mobile phase consisted of 0.01% H 3 PO 4 : acetonitrile (initial conditions 80 : 20, v/v) was passed through the column at the flow rate of 0.7 mL min−1. The separation was carried out in the gradient elution mode. The presented method allows to separate ten potential impurities of BT-1. The full validation according to the ICH Q2 (R1) guidelines was carried out for five of the potential impurities while limit tests were performed for four BT-1 related substances. The performed validation tests proved the suitability of the method for its intended purposes. An additional LC/MS method was utilized for the identification of the unknown impurities in bimatoprost as well as the degradation impurities generated during the forced degradation studies. [ABSTRACT FROM AUTHOR]

• Drug disposition in children is influenced by the physiological changes associated with growth and maturation. • Dosage adaptations to the different pediatric age groups are required. • A highly sensitive UPLC-MS/MS method for the simultaneous determination of seven antibiotics was developed. • The method requires low serum sample volume (50 μL). • The method was successful applied to support routine monitoring and pediatric PK studies, including in neonates. A rapid and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay was developed for quantification of 7 antibiotics in low sample volumes (50 μL): amoxicillin, azithromycin, cefotaxime, ciprofloxacin, meropenem, metronidazole and piperacillin, for both routine monitoring and pharmacokinetic studies. After protein precipitation by acetonitrile, the antibiotics were separated on an Acquity UPLC HSS T3 column (run time, 4 min). The mobile phase consisted of a mixture of (A) ammonium acetate (pH 2.4; 5 mM) and (B) acetonitrile acidified with 0.1% formic acid, delivered at 500 μl/min in a gradient elution mode. Total time run was 2.75 min. Ions were detected in the turbo-ion-spray-positive and multiple-reaction-monitoring modes. The assay was accurate and reproductible for the quantification of the seven antibiotics in serum samples over large concentration ranges. [ABSTRACT FROM AUTHOR]

• First reported quantitative assay for the determination of galunisertib in human plasma. • Fast and elegant HPLC-MS/MS method for quantification of galunisertib in human plasma. • Application of a simple chromatographic method for quantification of galunisertib in small volumes of mouse plasma. Galunisertib is an anti-cancer drug currently evaluated in phase I and II clinical trials. This study describes the development and validation of a bioanalytical assay to quantify galunisertib in human plasma using HPLC-MS/MS. Stable isotope labelled galunisertib was added as internal standard and the analyte and internal standard were extracted from the matrix by protein precipitation using acetonitrile-methanol (50:50, v/v). Final extracts were injected onto a C18 column, gradient elution was applied for chromatographic separation and detection was performed using a triple quadrupole mass spectrometer operating in the positive ion mode. The assay was linear over the range 0.05–10 ng/mL, with acceptable accuracy (bias ranging from −6.1 to 3.1%) and precision (below 5.7% C.V.) values. The applicability of the assay was demonstrated in a pharmacokinetic experiment in mice. [ABSTRACT FROM AUTHOR]

• Development and validation of an absolute quantification assay for human CYP450 s. • Targeted proteomic assay by HPLC-MS/MS based on quantification of proteotypic peptides. • Determination of interindividual variability in drug metabolism by human CYP450 s. • Evaluation of CYP450 content in human liver and intestine microsomes. The cytochrome P450 (CYP450) superfamily constitutes the major enzymatic system involved in drug metabolism. CYP450s are highly expressed in the liver and other tissues and limited data on absolute characterization of CYP450s in extra hepatic organs, such as the small intestine, are available. Our objective was to develop and validate an absolute quantification assay by HPLC-MS/MS-based targeted proteomics allowing the simultaneous quantification of fourteen major human CYP450 isoforms (CYP1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7 and 4F2) in human liver and intestine microsomes. Absolute protein quantification was performed using two proteotypic peptides for each of the fourteen CYP450s. Peptides were obtained after a tryptic digestion of microsomes and samples were analyzed by high performance liquid chromatography with heated electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS). Chromatographic separation was performed on a Biobasic-8 analytical column (5 μm 100 x 1 mm) with a gradient elution using acetonitrile and water both fortified with 0.1% formic acid (flow rate: 75 μL/min). Calibration curves were linear over a wide range of concentrations (0.1–50 nM) and the assay met all requirements of sensitivity, linearity, precision, accuracy and matrix effect. Strong correlations were observed between the two proteotypic peptides for each isoenzyme, corroborating the strength of this method. Twelve CYP450s were detected in commercially available human liver microsomes while seven CYP450s were detected in human intestine microsomes. To our knowledge, this is the most sensitive (0.1 nM) and the first most extensively validated assay that can be applied to the absolute quantification of CYP450s in human tissues. [ABSTRACT FROM AUTHOR]

• UPLC–MS/MS method for simultaneous analysis of DEP and MEP in rat biological samples. • Successfully applied to characterize PK, tissue distribution and metabolism of DEP in rats. • The first study for simultaneous analysis of DEP and MEP in rat biological samples. • Useful for developing PBPK models for DEP and its metabolite in future exposure study. This study describes the development of a novel and sensitive UPLC-MS/MS method for simultaneous determination of diethyl phthalate (DEP) and its major metabolite, monoethyl phthalate (MEP), in rat plasma, urine, and 11 different tissues collected from a toxicokinetic study. Analytes were separated using 0.1% (v/v) aqueous formic acid and acetonitrile containing 0.1% (v/v) formic acid as a mobile phase by gradient elution at a flow rate of 0.25 mL/min with a KINETEX core-shell C 18 column (50 × 2.1 mm, 1.7 μm). Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique operated in multiple reaction monitoring. The assay achieved lower limit of quantification for 0.04 ng/mL of DEP and 0.1 ng/mL of MEP in plasma, urine, and all tissues. The disposition of DEP was characterized by short half-life (1.30–1.34 h) and a high clearance (11.76 ± 0.08 L/h/kg). It was rapidly metabolized to MEP. Its levels consistently exceeded DEP levels. The tissue distribution of DEP and MEP of liver, kidney, gastro-intestinal tract, spleen, testis, lung, brain, muscle, thymus, heart, and adipose was determined at 24 h after drug administration. For DEP, the tissue to plasma partition coefficient was the highest in the kidney (16.72), followed by that in the liver (10.04), spleen (1.35), and adipose (1.18). For MEP, the tissue to plasma partition coefficient was the highest in the liver (2.18). It was less than unity for all other tissues. The developed analytical method satisfied the criteria of international guidance. It could be successfully applied to toxicokinetic studies of DEP after oral and intravenous administration of DEP to rats. In addition, findings of this study may be useful to evaluate exposure and toxic potential of DEP and its metabolite in risk assessment. [ABSTRACT FROM AUTHOR]

• Rapid and highly sensitive LC/MS-MS detection method for histamine in a leukocyte suspension. • Pre-column derivatization with PITC increase the ionization efficiency of histamine in ESI/MRM mode. • Require only a small amount of whole blood (2 mL) and leukocyte suspension (0.1 mL). • Methods successfully validated and applied to the diagnosis and research of respiratory or cutaneous allergic diseases. The basophil histamine release test (HRT) is an important in vitro diagnostic assay to evaluate immunoglobin E (IgE)-mediated allergic responses. In this study, a bioanalytical LC–MS/MS method was developed and validated to quantify histamine in the leukocyte suspension from human peripheral blood. The method used pre-column derivatization with phenylisothiocyanate (PITC) and the resulting phenylthiocarbamyl (PTC) histamine was analyzed by positive-ion electrospray ionization using the multiple reaction monitoring mode. Chromatographic separation was achieved using an Imtakt-HT C18 column (2.1 mm × 50 mm, 3.0 μm), with a flow rate of 0.35 mL/min, 2 μL injection, and gradient elution with a mixture of acetonitrile-2 mM ammonium acetate buffer (both containing 0.1% formic acid). The total runtime of the method was 3.0 min including equilibration time. The method had a lower limit of detection of 0.1 ng/mL, and the quantifiable range was 0.1–100 ng/mL in the leukocyte suspension. The intra-day and inter-day precision and accuracy results were within the acceptable limits. It was established that histamine quantification should be performed within 2 h of preparing the leukocyte suspension, and freezing and thawing should be avoided. This method was successfully applied to the diagnosis and evaluation of the pathophysiologic mechanism of respiratory or cutaneous allergic diseases. [ABSTRACT FROM AUTHOR]

Sulfasalazine has been identified as a candidate molecule to be investigated as an intervention to treat preterm pre-eclampsia during pregnancy. However, placental exposure of sulfasalazine and its systemically absorbed metabolite, sulfapyridine, is unknown. A robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously quantitate these analytes in human placenta with an application to a pilot clinical trial. The placental tissue was homogenised using a water:methanol (1:1, v/v) mixture, followed by sample extraction using both protein precipitation and solid phase extraction. Sulfasalazine-d4 and sulfapyridine-d4 were used as internal standards. An Agilent Poroshell EC-C18 (3.0 ×100 mm, 2.7 µm) column was used for chromatographic separation, with gradient elution employed at a flow rate of 0.450 mL/min over a total run time of seven minutes. The mobile phases consisted of water with 0.1% formic acid (mobile phase A) and acetonitrile:methanol (90:10, v/v) with 0.1% formic acid (mobile phase B). A Shimadzu-8040 mass spectrometer was operated in multiple reaction monitoring (MRM) mode using positive electrospray ionisation (ESI). For both analytes, the assay was validated over the range 30–30,000 ng/mL, or 150–150,000 ng/g. During inter-day validations (n = 18), the average accuracies of quality controls ranged from 101.6% to 112.7% with corresponding precisions of 4.4–6.7% for sulfasalazine, and from 97.4% to 108.4%, with corresponding precisions of 3.7–10.0% for sulfapyridine. No significant matrix effects were observed, and the method proved to be sensitive and specific for both analytes. This study presents the first validated analytical method for quantifying sulfasalazine and sulfapyridine in human placenta as part of a pilot clinical trial to generate preliminary data on its pharmacokinetics and efficacy as in intervention for preterm pre-eclampsia. • A novel LC-MS/MS method was developed to simultaneously quantitate sulfasalazine and its metabolite in human placenta. • The method uses protein precipitation followed by solid phase extraction of the analytes from homogenised tissue. • The method allowed for the quantitation of these analytes in placentas of pre-eclamptic patients at delivery. • The method creates a platform for the investigation of placental penetration of drugs as interventions for pre-eclampsia. [ABSTRACT FROM AUTHOR]

We have successfully developed and validated a bioanalytical assay using liquid chromatography tandem mass spectrometry to simultaneously quantify the first approved KRASG12C inhibitor sotorasib and its major circulating metabolite (M24) in various mouse matrices. M24 was synthesized in-house via low-pH hydrolysis. We utilized a fast and efficient protein precipitation method in a 96-well plate format to extract both analytes from biological matrices. Erlotinib was selected as the internal standard in this assay. Gradient elution using methanol and 0.1 % formic acid in water (v/v) was applied on an Acquity UPLC BEH C18 column to separate all analytes. Sotorasib, M24, and erlotinib were detected with a triple quadrupole mass spectrometer in positive electrospray ionization in multiple reaction monitoring mode. During the validation and sample quantification, a linear calibration range was observed for both sotorasib and M24 in a range of 4 – 4000 nM and 1 – 1000 nM, respectively. The %bias and %CV (both intra- and inter-day) for all tested levels in all investigated matrices were lower than 15 % as required by the guidelines. Sotorasib had a rather short room temperature stability in mouse plasma for up to 8 h compared to M24 which was stable up to 16 h at room temperature. This method has been successfully applied to measure sotorasib and M24 in several mouse matrices from three different mouse strains. We can conclude that the plasma exposure of sotorasib in mice is limited via human CYP3A4- and mouse Cyp3a-mediated metabolism of sotorasib into M24. [Display omitted] • Validated LC-MS/MS method for both sotorasib and M24 in eight mouse matrices. • First validated quantitative method for a metabolite of sotorasib. • Sotorasib plasma exposure is limited by its metabolism into M24 by Cyp3a & Cyp3A4. [ABSTRACT FROM AUTHOR]

Dacarbazine (DTIC) is a chemotherapeutic drug currently used for the systemic treatment of melanomas. Considering the easy access to these tumors, a topical route of drug administration could provide a more comfortable and less toxic treatment. However, DTIC quantification aiming at the design of topical formulations is challenging, pondering all the interferents present in the drug samples recovered from the skin. Hence, this work intended to validate a selective chromatographic method for DTIC determination in skin permeation studies. A reversed-phase C 18 column was used as a stationary phase, and gradient elution of a mobile phase consisting of methanol and pH 6.5 sodium phosphate monohydrate buffer (0.01 mol/L) at a flow rate of 1.0 mL/min was implemented. DTIC was detected at 364 nm. The method was selective against skin interferents, linear (r = 0.9995) in a concentration range of 1.0–15.0 μg/mL, precise with an overall variation coefficient lower than 3.8%, accurate achieving recovery from the skin layers within 91–112%, and sensitive for the proposed application (detection limit = 0.10 μg/ mL, quantification limit = 0.30 μg/mL). Furthermore, the analytical method was successfully tested in in vitro skin permeation studies. In conclusion, the developed method is appropriate for DTIC analysis from the skin sample matrix. • HPLC method for dacarbazine determination in skin samples to be used in permeation studies. • The method was selective against skin interferents, with linearity, precision, and accuracy. • The method also showed high recovery capacity of dacarbazine from skin matrices. • The analytical method complies with the international validation guidelines of ICH and FDA. [ABSTRACT FROM AUTHOR]

This article describes the first simple, fast, time-saving, and cost-effective UHPLC method that was developed and validated for simultaneous quantification of abiraterone acetate, its four degradation products, and six specified process impurities in bulk and tablet form. Moreover, when coupled with a mass spectrometer detector, the proposed method provides additional advantages for confirmation of peak and correct identification based on molecular weight. The eleven peaks were separated on a Water Acquity BEH C18, (150 mm length, 2.1 mm internal diameter, 1.7 µm particle size) column maintained at a 50.0 °C temperature. Using 0.05% formic acid in 10 mM ammonium formate, acetonitrile, and methanol as mobile phases in gradient elution at a flow rate of 0.40 mL/min. provides excellent separation at 260 nm. The linearity curves of all analytes showed promising results with a correlation coefficient of 0.999 with a lower limit of detection and quantification. A forced degradation study on solid abiraterone acetate proved its specificity with improvements and significance. This proposed method provides improved separation with a lower flow rate, which offers faster analysis, reduces wastage and cost, and specifies the greener advantages compared to reported methods. The outcome of the specificity, linearity, precision, and trueness as per ICH guidelines proved that the proposed method is fast, time-saving, and cost-effective for the intended purpose. [Display omitted] • MS-compatible UHPLC method established for qualitative and quantitative analysis. • Helpful for quantification of active, its four degradants, and six process impurities. • Determination of eleven analytes with improved speed, separation, and sensitivity. • Successfully applied the method for batches of API and tablets of abiraterone acetate. • Correct identification and confirmation of all analytes based on molecular weight. [ABSTRACT FROM AUTHOR]

A novel simple, specific, sensitive, accurate and precise reversed phase high performance liquid chromatographic method (RP-HPLC/UV) was developed and validated for the simultaneous estimation of Glycopyrronium bromide (GLY), Indacaterol acetate (IND) and Mometasone furoate (MOF) in pure form, in laboratory prepared mixtures and in pharmaceutical dosage form. Experimental design methodology was applied by using Plackett–Burman and face-centered composite designs to achieve the best resolution with minimum experimental trials. The designed model was statistically analyzed, graphically presented by surface plots and the relationships between coefficients of the derived polynomial equations were interpreted. Chromatographic separation was achieved on Inertsil ODS C 18 column (250 ×4.6 mm, 5 µm) at ambient temperature using a mobile phase composed of methanol: 0.1% glacial acetic acid (pH4) in a gradient elution at a flow rate 1 mL /min. UV detection was carried out at 233 nm. Response was found to be linear in the concentration range of 20–120 µg /mL with regression coefficient (r2 = 0.999) for GLY, 50–300 µg /mL with regression coefficient (r2 = 0.9995) for IND and 50–300 µg /mL with regression coefficient (r2 = 0.9998) for MOF. The method was validated as per ICH guidelines and satisfactory results were achieved. The method was successfully applied for the analysis of the cited drugs in their fixed dose combination (FDC) pharmaceutical formulation. Statistical comparison between the results obtained by the proposed method and the reference methods for GLY, IND and MOF showed no significant difference. The developed method could be implemented in quality control aspects of the cited drugs. Four green metrics were used to evaluate the new RP-HPLC/UV method's greenness and compare it to other published techniques. [Display omitted] • RP-HPLC/UV for estimation of Glycopyrronium, Indacaterol & Mometasone in inhaler. • Experimental designs applied using Plackett–Burman & face-centered composite designs. • The proposed method was fully validated according to ICH guidelines. • The proposed method recorded highest AGREE & ESA scores among compared methods. [ABSTRACT FROM AUTHOR]

Monosaccharides are essential for maintaining the normal physiological functions of living organisms. Under disease states, metabolic disorders in vivo will inevitably affect the levels of monosaccharides, which brings the possibility of monosaccharides as a biomarker of some diseases. In this study, a method was developed and validated for simultaneously determining 10 monosaccharides (glucose, galactose, mannose, rhamnose, fucose, xylose, iduronic acid, glucuronic acid, N -acetylgalactosamine and N -acetylglucosamine) in SD rat plasma using liquid chromatography-tandem mass spectrometry. The method employed 1-phenyl-3-methyl-5-pyrazolone (PMP) as a derivatization reagent, considerably improved the chromatographic retention and ionization efficiency of monosaccharides. After protein precipitation of plasma samples, monosaccharides and isotope internal standards were derivatized and liquid-liquid extraction was performed to remove excess PMP. To achieve the baseline separation of several isomers, the resulting derivatives were chromatographed on a Bridged ethyl hybrid (BEH) Phenyl column using gradient elution with a total run time of 8 min. The method was linear within the range of 0.0100–5.00 μg/mL for rhamnose, 0.0500–25.0 μg/mL for fucose, xylose, iduronic acid, glucuronic acid, N -acetylgalactosamine and N -acetylglucosamine, 1.00–500 μg/mL for galactose, 10.0–5000 μg/mL for mannose, and 50.0–25,000 μg/mL for glucose. And the accuracy and precision verification of surrogate matrix samples and plasma samples met the required criteria. The method has been used successfully to study the effect of hepatic insufficiency on monosaccharide levels in rats. It was found that the concentration of glucuronic acid in SD rat plasma was abnormally increased in rats with liver injury. • Simultaneously determine 10 monosaccharides in rat plasma. • PMP precolumn derivatization and gradient elution to obtain the resolution of isomers. • Investigating the effect of hepatic insufficiency on monosaccharide plasma levels in rats. • Glucuronic acid may be a biological index in the evaluation of liver injury and prognosis. [ABSTRACT FROM AUTHOR]

Pomegranate extracts standardized to punicalagins are a rich source of ellagitannins including ellagic acid (EA). Recent evidence suggests that gut microbiota-derived urolithin (Uro) metabolites of ellagitannins are pharmacologically active. Studies have evaluated the pharmacokinetics of EA, however, little is known about the disposition of urolithin metabolites (urolithin A (UA) and B (UB)). To address this gap, we developed and applied a novel ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the characterization of EA and Uro oral pharmacokinetics in humans. Subjects (10/cohort) received a single oral dose (250 or 1000 mg) of pomegranate extract (Pomella® extract) standardized to contain not less than 30 % punicalagins, < 5 % EA, and not less than 50 % polyphenols. Plasma samples, collected over 48 h, were treated with β-glucuronidase and sulfatase to permit comparison between unconjugated and conjugated forms of EA, UA and UB. EA and urolithins were separated by gradient elution (acetonitrile/water, 0.1 % formic acid) using a C 18 column connected to a triple quadrupole mass spectrometer operating in the negative mode. Conjugated EA exposure was ∼5–8-fold higher than unconjugated EA for both dose groups. Conjugated UA was readily detectable beginning ∼8 h post-dosing, however, unconjugated UA was detectable in only a few subjects. Neither form of UB was detected. Together these data indicate EA is rapidly absorbed and conjugated following oral administration of Pomella® extract. Moreover, UA's delayed appearance in the blood, primarily in the conjugated form, is consistent with gut microbiota-mediated metabolism of EA to UA, which is then rapidly converted to its conjugated form. • Quantification of pomegranate's polyphenol and its metabolites in plasma. • Conjugated ellagic acid (EA) exposure was higher than unconjugated EA. • Eight of ten subjects showed urolithin A (UA), and none showed urolithin B (UB). • UA conjugates were detectable up to 48 h post-administration. • UA formed through the gut microbiota-mediated transformation of EA. [ABSTRACT FROM AUTHOR]

Highlights • An LC/MS/MS method for the quantification of GW9508 in rat plasma. • Pharmacokinetics of GW9508 was investigated. • Oral bioavailability was 54.88%. • Metabolites of GW9508 were characterized by LC-Q-Exactive-Orbitrap-MS. • Hydroxylation and glucuronidation were the primary metabolic pathways. Abstract In this study, a simple, fast and sensitive LC/MS/MS method was developed and validated for the determination of GW9508 in rat plasma. The sample was precipitated with acetonitrile and subsequently separated on ZORBAX Eclipse XDB C 18 column (50 mm × 2.1 mm, 5 μm). Mobile phase was composed of 0.1% formic acid in water and acetonitrile with gradient elution, at a flow rate of 0.4 mL/min. The analyte and internal standard were quantitatively monitored with precursor-to-product transitions of m/z 348.2→183.1 and m/z 397.2→260.2, respectively. The linearity of the assay was evident in the range of 1–1000 ng/mL with correlation coefficient more than 0.998. The validation parameters were all within the acceptable limits. The validated method has been successfully applied to the pharmacokinetics study of GW9508 in rat plasma, and our results demonstrated that GW9508 showed low clearance, moderate half-life and ideal bioavailability (54.88%). Furthermore, metabolites stemmed from rat plasma, rat hepatocytes and human hepatocytes were analyzed by an LC-Q-Exactive-Orbitrap-MS assay, resulting in the identification of seven metabolites based on the accurate mass and fragment ions. Acylglucuronide conjugate (M6) was found as the most abundant metabolite in all tested matrices. The metabolic pathways were proposed as hydroxylation and glucuronidation. This study provided an overview of disposition of GW9508, which is highly instructive for better understanding the effectiveness and toxicity of this drug. [ABSTRACT FROM AUTHOR]

Highlights • We developed a HPLC-MS method for analysis of therapeutic agents for LF treatment. • The method was validated as per ICH and FDA guidelines. • The method was successfully applied to pharmacokinetic and biodistribution studies. Abstract A sensitive and relatively fast, cost-effective high-performance liquid chromatographic method coupled with mass spectrometer (HPLC-MS) is herein reported for the first time for a simultaneous quantification of plasma and organs concentration of three therapeutic agents that are widely used in treatment of lymphatic filariasis (LF), namely, doxycycline (DOX), diethylcarbamazine (DEC) and albendazole (ABZ) metabolites. The method was developed and validated as per ICH and FDA guidelines and successfully employed to quantify DOX, DEC and ABZ metabolites (albendazole sulfoxide (ABZ-OX) and albendazole sulfone (ABZ-ON)) in the plasma and organs of Sprague Dawley rats after oral concomitant administration of the above mentioned therapeutic agents. Importantly, a simple, one-step protein precipitation and extraction method was used to extract the four compounds efficiently with a recovery in the range of 79.88 ± 5.02%–90.71 ± 5.13%, 85.72 ± 7.22%–93.17 ± 5.55%, 94.38 ± 7.35%–101.00 ± 8.88% and 94.38 ± 7.35%–99.87 ± 10.22% in plasma and organs for DOX, DEC, ABZ-OZ and ABZ-ON, respectively. Separation of all analytes was performed on a Xselect CSH™ C18 HPLC column (Waters, 3.0 x 150 mm, 3.5 μm particle size) with gradient elution employing a mobile phase consisting of 0.1% v/v formic acid in water and methanol with a run time of 20 min. Quantification was carried out employing a single, quadruple MS detector operated with single ion monitoring (SIM) mode and the ion transitions at m / z of 445.4, 200.2, 282.3 and 298.3 for DOX, DEC, ABZ-OX and ABZ-ON respectively. The MS response for plasma samples was linear across the concentration range of 2.5–2500 ng/mL for DOX, 0.5–500 ng/mL for DEC, 1–1000 ng/mL for ABZ-OX and ABZ-ON with a correlation coefficient (r2) ≥ 0.998. The method was selective, precise and accurate. This method allowed us to get an insight into the pharmacokinetics and biodistribution of the three therapeutic agents after simultaneous oral administration to Sprague Dawley rats. This bioanalytical method could provide a reliable, reproducible and excellent tool for routine therapeutic drug monitoring of the above mentioned therapeutic agents and also support other clinical pharmacokinetic-based studies. [ABSTRACT FROM AUTHOR]

Highlights • A rapid, flexible, and accurate method was developed for analyzing five ginsenosides. • Rh1, Rg1, Rg2, and Re were isocratically eluted in 10 min by HPLC using NH 2 -based column. • Rg1 & Re/Rg2 & Rf eluted in 35 min with good resolution, respectively. • The NH 2 -based column showed similarity to inverse size exclusion chromatography. • The method can be extended to predict the retention/elution order of ginsenosides. Abstract An efficient and flexible protocol was developed for simultaneous detection of the ginsenosides Rg1, Re, Rf, Rh1, and Rg2 in ginseng extract. In the analysis of white ginseng that contains no ginsenoside Rf, separation of the remaining ginsenosides was achieved within just 10 min under isocratic chromatographic conditions. For the analysis of red ginseng that contains ginsenoside Rf as a characteristic constituent, the gradient elution conditions were optimized. The method is based on high-pressure liquid chromatography employing a Shiseido UG 80 Capcell Pak NH 2 (4.6 mm I.D. × 250 mm, 5 μm)column and isocratic elution using acetonitrile (A) and water(B) in a ratio of 76: 24 (v/v), The optimal gradient elution conditions are as follows: 0–3 min, 89% A, 3–25 min, 89–84% A, 25–30 min, 84–82% A, 30–35 min, 82-76% A, then returning to 89% solvent A in 5 min. The flow rate was 0.80 mL min−1. The column temperature was set at 25℃ and the detection wavelength was at 203 nm. The working concentration ranges for ginsenoside Re, Rh1, Rg2, Rg1, and Rf were 0.23−1450.0 mg• L−1, 0.05−1130.0 mg• L−1, 0.11−687.0 mg• L−1, 0.051−1325 mg• L−1, and 0.55−800.0 mg• L−1, respectively. The method was validated for linearity, precision, and accuracy. And the further confirmation of five ginsenosides was conducted by QTOF-MS. Analysis of raw extracts of ginseng, white ginseng, and red ginseng for the five components showed satisfactory recovery. [ABSTRACT FROM AUTHOR]

Highlights • Although the toxicity of KPC on normal rats was stronger than KA, KPC exhibited better efficacy to malignant ascites with little side effects. • Novel UPLC-MS/MS methods for determination of kansuiphorin C and kansuinin A in rat feces were established. • The quicker accumulative excretion rate of KPC in rat feces than KA within 48 h indicating the potential different interactions with microbiota. Abstract Malignant ascites (MA) is one of the severe complications of gastrointestinal tumors, affecting the patients' survival time and quality of life. Euphorbia kansui is a commonly used toxic Chinese herbal medicine for malignant ascites. Our previous study showed that the biological and toxicological effects of kansui were closely related to the gastrointestinal tract. The ingenane-type and jastrophane-type diterpenoids are both toxic and active components of kansui. The contents of kansuiphorin C (KPC) and kansuinin A (KA) take highest accounts in each type of diterpene. Hence, in this study, the efficacy and toxicity of KPC and KA on normal rats and MA rats were firstly evaluated by serum liver enzymes (ALT and AST), oxidative damage indicators (GSH, SOD, MDA and LDH), inflammatory indexes (TNF-α, IFN-γ and IL-2) and the volume of ascites. Changes in the levels of these indices showed that although the toxicity of KPC on normal rats was stronger than KA, KPC exhibited better efficacy to the malignant ascites with no obvious side effects at the dose of 10 mg·kg−1. Then, accurate and reliable methods for the determination of KPC and KA in the rat feces by ultra-fast liquid chromatography coupled with MS/MS detector (UFLC-MS/MS) were established, detected by the multiple reaction monitoring mode. The chromatographic separation was conducted on an XBbridge C18 column (50 mm × 2.1 mm, 2.5 μm) using gradient elution composed of 0.1% formic acid in water and acetonitrile. The flow rate was 0.5 mL·min−1 and column temperature was 30 °C. The method was finally applied to the comparative study on normal and malignant ascites rats given KPC and KA, respectively. Interestingly, the results showed that KPC's accumulative fecal excretion rate (normal, 19.22%±5.36%; model, 15.96%±3.47%) were much higher than that of KA (normal, 2.928%±0.741%; model, 2.835%±0.873%) at the same dose within 48 h. This suggested KPC had higher in-vivo transformations in comparison with KA, providing guidance for the further preclinical research of KPC and KA as promising compounds treating MA. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Supercritical fluid chromatography resolves difficult isomers with high molecular mass. • Method optimization with central composite design is fast (4 h experiments). • Scale-up to gram-scale provides a facile route to metabolites for activity studies. Abstract A stressed degradation (oxidation) was employed to produce metabolites from an active pharmaceutical ingredient (API) with large molecular weight (about 900 g/mol). An analytical chromatographic method was desired to compare the products generated by different degradation methods while a multi-gram-scale preparative chromatographic method was necessary to purify the produced metabolites. Supercritical fluid chromatography (SFC) was selected for both tasks as no other chromatographic method had achieved the resolution of the API and metabolites (two isomeric mono-oxide species and one di-oxide). First, an analytical-scale method was developed with ultra-high performance supercritical fluid chromatography (UHPSFC). Achiral stationary phases containing sub-2 μm fully porous particles or sub-3 μm superficially porous particles, and chiral phases containing 3 and 5 μm fully porous particles were selected for a first screening with gradient elution (carbon dioxide – methanol containing additives). The stationary phase providing the most promising results was ACQUITY Torus 2-PIC (100 x 3 mm, 1.7 μm, Waters). A central composite design (CCD) was conducted to optimize the gradient program and oven temperature. Final gradient conditions were as follows: 50–70% methanol in 3.8 min with oven temperature set at 36 °C, back-pressure set at 11 MPa and flow-rate at 0.8 mL/min. The optimized method was employed to analyze samples obtained with different degradation conditions. Then the method was adapted and transferred to preparative-scale SFC on a 5 μm-particles Torus 2-PIC stationary phase (150 x 30 mm). The method was modified to comprise an isocratic step followed by a gradient, favoring peak shape of the last eluting compound and minimal volume of collected fractions. Batch injections in gradient mode were carried out to purify six grams of crude product. [ABSTRACT FROM AUTHOR]

Highlights • A method for simultaneously measuring epigallocatechin-3-gallate, silibinin, curcumin. • The three components are mainly distributed in the small intestine. • The three components weren't accumulated after multiple administrations of Protandim. Abstract Protandim is an over-the-counter herbal dietary supplement. The key components of Protandim, i.e., epigallocatechin-3-gallate (EGCG), silibinin (SIL), and curcumin (CUR) were simultaneously analyzed through a liquid chromatography-tandem mass spectrometric (LC–MS/MS) method in plasma and different tissues after administration of Protandim in rats. The developed and validated method was employed to assess the pharmacokinetic profiles and the accumulation of EGCG, SIL, CUR in rat plasma and tissue homogenates. The plasma and tissue homogenates were subjected to liquid-liquid extraction and separated on a Hypurity C18 column (50 × 4.6 mm) with a gradient elution of water and acetonitrile. Mass spectrometric detection was performed in the multiple reaction monitoring mode (MRM) following the transitions: m / z 457.3/169.3, m / z 481.3/125.0, m / z 367.3/149.3 and m / z 609.4 /300.2 for EGCG, SIL, CUR, and RU (rutin), respectively. The concentrations of all the analytes in the range from 2 to 1000 ng/mL showed linear relationships with respective peak areas in different matrices. For all matrices , the values of inter-day and intra-day precisions and accuracies were less than 10.3% of the nominal concentration. The matrix effect, extraction recovery, dilution integrity, and stability values were all within acceptable levels. This method was successfully applied for determining the pharmacokinetics and tissue distribution of the components in rats after the intragastrical administration of a single-dose (364.5 mg/kg) or multiple-doses (1458 mg/kg) of Protandim. The data showed that EGCG, SIL, and CUR did not accumulate in rats after multiple doses of Protandim, and the three main components were distributed mainly in the small intestine. [ABSTRACT FROM AUTHOR]

Highlights • Fully validated HPLC-MS/MS method for measuring minocycline in human plasma and urine. • Novel but simple and robust method that is selective with respect to the drug's 4-epimer. • Plasma PK and urine data successfully generated from a Phase I study. • Acidic deproteinising agent, trichloroacetic acid, does not catalyse epimerization. • Ion pairing agent not required for chromatographic separation. Abstract New treatments are urgently required to treat infections caused by multi-drug resistant Acinetobacter baumanni,. To address this need, a new formulation of Minocin®, (minocycline for injection) has been developed that allows for higher doses of minocycline to be administered. Phase 1 clinical trials were conducted in healthy volunteers to assess the safety and pharmacokinetics (PK) of this new formulation at higher doses. In order to generate PK data, novel, selective and simple HPLC-MS/MS based assays were developed and validated for the determination of minocycline (MC) in human plasma and urine. The respective working ranges were 0.05 to 30 mg/L and 0.1 to 30 mg/L. Removal of endogenous proteins with trichloroacetic acid was used as a simple means of extracting MC from the samples. An analogue, tetracycline was used as the internal standard (IS). Chromatographic separation, including that of MC from its 4-epimer (4-EMC), was achieved on a Waters XBridge BEH C18 column (50 x 4.6 mm ID, 5 μm) with gradient elution. The mobile phases comprised water containing 5 mM ammonium formate at a pH of 2.5, and methanol containing 5 mM ammonium formate. The internal standard (IS) was tetracycline, a structural analogue of minocycline. The methods were fully validated and met regulatory acceptance criteria for intra-run and inter-run accuracy and precision, carryover, dilution integrity and matrix effects. Mean extraction recoveries ranged between 64.3% and 84.6% for MC and 64.3% for the IS. There was no significant ion suppression or enhancement for MC or the IS. The validated assays were successfully applied to 1423 plasma and 689 urine samples from a Phase 1 clinical study. There was no evidence of instability, or significant interconversion between MC and 4-EMC, in stored clinical samples, spiked plasma and urine samples, or their extracts, under various test conditions. [ABSTRACT FROM AUTHOR]

Highlights • 3D-UPLC-DAD fingerprint was firstly applied to provide the quality control of NLXTD. • Combined with "new method" was used to provide the semi-quantitative and qualitative analysis of chemical components in NLXTD. • This study has conducted a preliminary discussion the relationship between the bio-active components and traditional Chinese medicine theory. Abstract Naoluoxintong decoction (NLXTD) is a traditional Chinese formula which has been used for the management of ischemic stroke in China for two hundred years. In this study, we developed a comprehensive and reliable analytical method to qualitatively analyze the components in NLXTD. This novel method was based on three-dimensional ultra-fast high performance liquid chromatography coupled with diode array detector (3D-UPLC-DAD) with an additional component validation method via incorporation of the mixture standard compounds during the verification step. In addition, the relationship between active components and "Monarch drug, Minster drug, Assistant drug, Guide drug" were determined. Our results showed that gradient elution with the mobile phase of 0.02% formic acid and methanol was the optimum condition to separate peaks. A total of 35 common peaks were established by comparing ten batches of NLXTD, and eight components were identified, including Calycosin, Calycosin-7-O-β- d -glucoside and Ononin in Astragali radix (Monarch drug); Ligustrazine in Chuanxiong Rhizoma (Minster drug); 4-Hydroxbenzyl alcohol and Parishin A in Gastrodiae rhizome (Assistant drug); Ferulic acid in Angelicae sinensis radix (Guide drug). The validation method of verification by adding mixture standard compounds combined with 3D-UPLC-DAD method, with the merits of greater resolution, higher speed of analysis and higher sensitivity, provided a semi-quantitative and qualitative analysis method to assess traditional Chinese medicinal prescription consisting of many bio-active components. Finally, our study has provided systemic and scientific evidence to explain the relationship between the bio-active components in the NLXTD and traditional Chinese medicine theory. [ABSTRACT FROM AUTHOR]