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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]

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]