Lipid metabolism imbalance and inflammation are important mechanisms driving osteoporosis. Gushudan (GSD) has the effect of tonifying the kidney and strengthening bones. However, the preventive mechanism of GSD against postmenopausal osteoporosis (PMOP) by modulating lipid metabolism remains to be further elucidated. A multi-omics strategy integrating lipidomics, transcriptomics, network pharmacology, and molecular docking was developed to investigate the potential mechanistic targets and pharmacodynamic material basis of GSD in preventing PMOP. Firstly, lipidomics analysis employing the dual-MS platform of UHPLC-Q-Orbitrap HRMS and UHPLC-MS/MS demonstrated that GSD improved the disorder of the lipid-inflammatory crosstalk mechanism involving glycerophospholipids and arachidonic acid in PMOP rats. Secondly, the combined analysis of lipidomics and network pharmacology predicted that Pla2g2a might be a potential mechanistic target linking lipid metabolism disorder to PMOP. Furthermore, high-throughput RNA sequencing transcriptomics was conducted to reveal that GSD significantly regulated 49 differentially expressed genes in PMOP rats, with the reversed genes (represented by Pla2g2a) markedly enriched in the arachidonic acid metabolism pathway. Finally, molecular docking was used to screen four active ingredients: przewaquinone A, naringenin, salvianolic acid D, and tricin, which were found to target Pla2g2a. In conclusion, this work achieved multi-dimensional cross-analysis by integrating multiple approaches, suggesting that GSD might ameliorate the lipid-inflammatory crosstalk disorder in PMOP rats by downregulating Pla2g2a expression, thereby restoring bone homeostasis. These findings provide new research clues and a potential target basis for the mechanistic investigation of PMOP.

Serum neurotransmitters (NTs) metabolic network plays a crucial role in bone metabolism. However, due to low concentration, poor chromatographic retention, and limited sensitivity of conventional detection methods, the metabolic characteristics and regulatory mechanisms of the NT metabolic network remain unclear. In this study, a highly specific, sensitive, and accurate strategy integrating d0/d6 DnsCl-based isotope-coded derivatization (ICD) with fragment-imprinted magnetic molecularly imprinted polymers (MMIPs) was established, which enables reliable quantitative determination of 22 NTs (such as HVA, 3-MT, Trpm, Tyr, NAS) in serum. The ICD strategy enhanced chromatographic separation, ionization efficiency, and enabled accurate quantification. The detection sensitivity was enhanced 5.50-346-fold compared with nonderivatized methods. In addition, 4-VP was selected as the optimal functional monomer via molecular docking, and DFT calculations further indicated that the DnsA/4-VP complex is stabilized through the synergistic effects of hydrogen bonding, π-π stacking, and van der Waals forces. The synthesized MMIPs efficiently enriched labeled NTs, with good selectivity (imprinting factors 1.46-2.28), remarkable adsorption capacity (85.0 mg·g-1), and excellent regeneration ability (8 times). This method was further applied to serum samples of ovariectomized postmenopausal osteoporosis rats, and 2 dysregulated metabolic pathways (the Trp metabolic disorder and the Phe/Tyr metabolic disorder) were identified. Longitudinal analysis further identified 7 potential time-dependent biomarkers (such as NAS, Tyr, GABA) associated with bone metabolism. In conclusion, this study developed an integrated approach combining the d0/d6 DnsCl ICD strategy with fragment-imprinted MMIPs to profile the serum NT metabolic network, demonstrating promising application prospects for submetabolome analysis in complex biological matrices.

The comprehensive characterization of natural product constituents by mass spectrometry remains challenging due to their structural complexity and chemical diversity, emphasizing the need for effective post-processing of mass spectrometry data. To address this need, MDFocus+ was developed as a novel all-in-one software platform with a user-friendly graphical user interface for post-processing LC-MS data in natural product analysis. Distinct from conventional post-processing tools, MDFocus+ integrates user-defined polygonal mass defect filtering (MDF) with visualized outputs, combines MDF, diagnostic ion filtering (DIF), and neutral loss filtering (NLF) within a single visual interface, and automates the association of precursor ions with MS/MS data for structural annotation. The effectiveness of MDFocus+ was demonstrated through successful application to the analysis of phenolic acids (PAs) from Salvia miltiorrhiza Bunge. As a result, polygonal MDF windows were constructed for monomers, dimers, trimers, and tetramers, respectively, enabling preliminary targeted screening. Notably, polygonal MDF alone eliminated 80.46%, 95.41%, 93.06%, and 96.59% of non-target precursor ions for each polymerization degree, significantly reducing false-positive interference. Moreover, refined filtering by DIF and NLF, combined with a knowledge-guided identification strategy based on characteristic fragmentation behaviors and fragment ion abundance patterns, enabled confident discrimination of PA isomers. Ultimately, a total of 120 PAs were characterized, including 5 previously unreported compounds. In conclusion, MDFocus+ provides a robust and scalable open-source solution for the efficient screening and characterization of natural products in LC-MS data analysis.

AIM:A rapid and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for epalrestat detection in human plasma. MATERIALS AND METHODS:A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source was used to quantify epalrestat and the internal standard epalrestat-d5 in the negative ion mode using multiple reaction monitoring (MRM). After acetonitrile-mediated protein precipitation, chromatographic separation was achieved using a reversed-phase C18 column (ACQUITY UPLC BEH, 2.1 × 50 mm, 1.7 μm; Waters Corp) with acetonitrile and 2 mM ammonium acetate in water as the mobile phase through gradient elution. RESULTS AND CONCLUSION:The retention time of epalrestat was 0.92 min and the entire run time was only 2.00 min. The calibration curve was linear in the range 10.0-8.00 × 103 ng/mL (r ≥ 0.99). The within-run and between-run relative standard deviations (RSDs) were < 9.3%. The within-run and between-run relative errors (REs) ranged -8.4-4.2%. No significant matrix effects were observed and the recovery rate was high. The method was fully validated, including reinjection reproducibility in human plasma, and was successfully applied in a pharmacokinetic study, in which 100% incurred sample reanalysis met the criteria.

Aflatoxins are highly toxic environmental contaminants that pose serious health risks. Traditionally, naturally occurring aflatoxins were believed to exclusively possess the (-)-configuration. This study reports the isolation and identification of two novel natural enantiomers, (+)-aflatoxin B2 and (+)-aflatoxin G2, from Aspergillus flavus. The previous lack of detection of these enantiomers was likely due to immunoaffinity-based detection methods designed to recognize only the (-)-forms, combined with the relatively low natural abundance of AFB2 and AFG2. Targeted screening of the collected reference materials and herbal medicines, revealed the occurrence of the two (+)-enantiomers in multiple samples. This finding expands current understanding of the natural occurrence and stereochemical diversity of aflatoxins, and highlights the importance of incorporating enantiomer detection into future analytical assays to improve both accuracy and comprehensiveness.

The photocatalytic system was developed through the self-assembly of lignocellulosic-based waste Panax notoginseng residue converted into hydrochar (PHC) with the organic semiconductor perylene diimide (PDI) to facilitate the visible-light photocatalytic degradation of minocycline hydrochloride (Mino-HCl) with the assistance of peroxodisulfate (PDS). The optimal PHC/PDI ratio of 1:4 was selected for further experiments, achieving 90.8 % minocycline removal under visible light after 2 h. At the optimal ratio, the visible light photocatalytic performance of PHC/PDI composites under PDS activation was 5.18 and 8.61 times higher than that of PDI and PHC, respectively. The system also exhibited excellent mineralization of minocycline, with a mineralization rate of 73.7 %. The PHC/PDI/PDS (P-P-P) system demonstrated good stability under visible light and showed general adaptability to different environmental conditions, including pH, coexisting anions and cations, and real water matrix. Theoretical calculations identified the O5, O7, N9, C20 and C29 sites of Mino-HCl as key targets for free radical attack. Toxicity assessments indicated significant reductions in both acute and chronic toxicity, particularly for fish and algae, indicating the transformation of Mino-HCl into less harmful products. This work provided a theoretical basis and reference for the research on waste management and wastewater treatment by employing lignocellulosic-based waste residues converted into biochar and an organic semiconductor PDI composite under PDS activation for visible light photocatalysis.

A rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination of bisoprolol in human plasma was established and validated. The sample was pretreated by methanol precipitation protein, and the isotope bisoprolol-d5 was used as the internal standard. The chromatographic column was ACQUITY UPLC BEH-C18 column (2.1 × 50 mm, 1.7 μm), with methanol and 0.2% formic acid aqueous solution as mobile phase for gradient elution. The electrospray ionization (ESI) source was used in the positive ion mode, and the multiple reaction monitoring (MRM) mode was used. The total running time was only 2.00 min. The correlation coefficient was good (r > 0.99) in the linear range of 0.0200-40.0 ng/mL. The lower limit of quantitation (LLOQ) was 20.0 pg/mL. The intrabatch and interbatch precisions were not more than 8.9% and 9.2%. The intrabatch and interbatch accuracies were -7.9% ~ 6.3% and -6.9% ~ 5.0%. The method was fully validated including whole blood stability and reinjection reproducibility and successfully applied to the pharmacokinetic study of 5-mg bisoprolol in healthy volunteers, which 93.1% incurred samples reanalysis (ISR) met the criteria. Compared with the reported methods, this method had the highest sensitivity and fast analysis speed.

Phthalates （PAEs） are widely employed as plasticizers in plastic products that are used in industrial， agricultural， food， medical， and other fields. PAEs are relatively weakly bonded to plastic products through non-covalent interactions. Consequently， PAEs can easily leak from the product into the environment， which exposes the public to PAEs through food intake， skin absorption from personal care products， and by inhaling air. Related studies have shown that PAEs are endocrine-disrupting substances and that long-term exposure to PAEs may result in diseases of the nervous， reproductive， cardiovascular and immune systems. In addition， excessive exposure to PAEs may trigger inflammatory responses and induce tumors. Therefore， establishing a highly sensitive assay for determining PAE levels in the human body following exposure is an important objective. PAEs generally have half-lives of less than 24 h； they are rapidly metabolized through enzymatic hydrolysis after entering the human body and excreted through urine. Therefore， most studies have focused on PAE metabolites as target compounds； hence， human body exposure to PAEs can be assessed by analyzing the types and levels of these metabolites. Herein， we established a method for simultaneously determining ten phthalate （PAE） metabolites in human urine using ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）. The ten PAE metabolites in urine were separated using an ACQUITY UPLC BEH Phenyl column （50 mm×2.1 mm， 1.7 μm）. Gradient elution was performed using 0.1% formic acid aqueous solution and 0.1% formic acid in acetonitrile as the mobile phases， at a flow rate of 0.5 mL/min， a column temperature of 40 ℃， and a sample size of 20 μL. Data were acquired in negative-ion electrospray ionization （ESI） and multiple reaction monitoring （MRM） modes， and quantified using the isotope internal standard method. The method was found to be highly specific， with the ten PAE metabolites exhibiting good linearities in their linear ranges， with limits of detection （LODs） and quantification （LOQs） of 0.03-0.3 and 0.1-1 ng/mL， respectively. Under the four quality control （QC） levels， the intra-day and inter-day precisions of the ten PAE metabolites were all ≤8.3%， and the accuracy ranged from ‒10.5% to 7.3%. The method was used to assess the exposure levels of PAE metabolites in the urine samples of 60 volunteers， with 1‒6 kinds of PAE metabolites detected in the urine of each volunteer. This method is sensitive， accurate， simple， efficient， and suitable for the large-scale biological monitoring of PAE metabolites.

In this study a bile acid (BA)-imprinted covalent organic framework (COF) was constructed via Schiff base reactions, which integrated the advantages of both inherent structural stability of COF and exceptional selectivity of molecular imprinting technology. Besides, it was found that the addition of graphene oxide (GO) effectively increased the dispersibility of nanoparticles, ultimately resulting in a GO-based molecularly imprinted COF (GO@MICOF). The GO@MICOF was identified with the characteristics of excellent mass transfer performance (20.09 mg g–1), specific surface area (152.35 m2 g–1), selectivity (IFs = 2.4), and regeneration ability (n ≥ 10). By coupling the GO@MICOF-based pretreatment method with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis, sensitive and accurate validation results (LOQs, 0.01–2.5 µmol L–1; extraction efficiency, 81.1–118.9

Epimedium-Rhizoma drynariae (EP-RD) was a well-known herb commonly used to treat bone diseases in traditional Chinese medicine. Nevertheless, there was incomplete pharmacokinetic behavior, metabolic conversion and chemical characterization of EP-RD in vivo. Therefore, this study aimed to establish metabolic profiles combined with multicomponent pharmacokinetics to reveal the in vivo behavior of EP-RD. Firstly, the diagnostic product ions (DPIs) and neutral losses (NLs) filtering strategy combined with UHPLC-Q-Orbitrap HRMS for the in vitro chemical composition of EP-RD and metabolic profiles of plasma, urine, and feces after oral administration of EP-RD to rats were proposed to comprehensively characterize the 47 chemical compounds and the 97 exogenous in vivo (35 prototypes and 62 metabolites), and possible biotransformation pathways of EP-RD were proposed, which included phase I reactions such as hydrolysis, hydrogenation, dehydrogenation, hydroxylation, dehydroxylation, isomerization, and demethylation and phase II reactions such as glucuronidation, acetylation, methylation, and sulfation. Moreover, a UHPLC-MS/MS quantitative approach was established for the pharmacokinetic analysis of seven active components: magnoflorine, epimedin A, epimedin B, epimedin C, icariin, baohuoside II, and icariin II. Results indicated that the established method was reliably used for the quantitative study of plasma active ingredients after oral administration of EP-RD in rats. Compared to oral EP alone, the increase in area under curves and maximum plasma drug concentration (P < 0.05). This study increased the understanding of the material basis and biotransformation profiles of EP-RD in vivo, which was of great significance in exploring the pharmacological effects of EP-RD.

Small-molecule prodrug nanoassembly technology with a unique advantage in off-target toxicity reduction has been widely used for antitumor drug delivery. However, prodrug activation remains a rate-limiting step for exerting therapeutic actions, which requires to quickly reach the minimum valid concentrations of free drugs. Fortunately, we find that a natural compound (BL-193) selectively improves the chemotherapy sensitivity of breast cancer cells to podophyllotoxin (PPT) at ineffective dose concentrations. Based on this, we propose to combine prodrug nanoassembly with chemotherapy sensitization to fully unleash the chemotherapeutic potential of PPT. Specifically, a redox-sensitive prodrug (PSSF) of PPT is synthesized by coupling 9-fluorenyl-methanol (Fmoc-OH) with PPT linked via disulfide bond. Intriguingly, PSSF with a π-conjugated structure readily co-assembles with BL-193 into stable nanoassembly. Significantly, BL-193 serves as an excellent chemosensitizer that creates an ultra-low-dose chemotherapeutic window for PPT. Moreover, prodrug design and precise hybrid nanoassembly well manage off-target toxicity. As expected, such a BL-193-empowered prodrug nanoassembly elicits potent antitumor responses. This study offers a novel paradigm to magnify chemotherapy efficacy-toxicity benefits.

A novel pH-responsive magnetic graphene oxide composite (MGO@PEI-BA) is proposed for the first time as an adsorbent for the rapid capture and detection of nucleosides (cytidine, uridine, guanosine, and adenosine). The morphology, structure, and magnetic properties of the composite were evaluated using various characterization techniques. The results indicated that the composite was successfully fabricated. A series of parameters that affect extraction and elution were optimized through one-factor-at-a-time and Box-Behnken design of response surface methodology (BBD-RSM). The unique layered structures and easily accessible active sites of the composite facilitated molecular transport, resulting in instantaneous equilibrium of nucleosides adsorption within 5 min. Based on this study, a magnetic dispersive micro-solid-phase extraction (MD-μ-SPE) method assisted by the MGO@PEI-BA was developed in combination with UHPLC-UV analysis for the determination of nucleosides in rat urine. Under the optimum conditions, a wide linear range (10–2000 ng mL−1), good linearity (r > 0.99), low detection limits (1–3 ng mL−1), low relative standard deviations (RSDs ≤ 3.9

Kidney-yang-deficiency-syndrome is a neuroendocrine disease caused by the dysfunction of adrenal-pituitary-target gland axis. Gushudan is a traditional Chinese medicine prescription with functions of tonifying kidney and strengthening bone, and its bone-strengthening effect has been confirmed by previous anti-osteoporosis research. However, its kidney-tonifying mechanism has not been clear so far. In this study, the renal metabolomics and lipidomics based on gas chromatography-mass and ultra-high performance liquid chromatography-high resolution mass were integrated to find the metabolic disorders in kidney-yang-deficiency-syndrome rats. Protein precipitation and liquid-liquid extraction were used to extract metabolome and lipidome from kidney. Gushudan regulated abnormal levels of amino acids, lipids, purines and carbohydrates, such as L-arginine, hypoxanine, stearic acid and phosphatidylethanolamine (P-18:1/20:4), which had effects on many metabolic pathways, such as glycerophospholipid metabolism, sphingolipid metabolism, glycine, serine and threonine metabolism and purine metabolism, etc. By integrating metabolomics and lipidomics, this study comprehensively revealed the abnormal metabolic activities of amino acids, lipids and nucleotides in kidney-yang-deficiency-syndrome, and the metabolic regulation mechanism of Gushudan in preventing kidney-yang-deficiency-syndrome, as well as the improvement of Gushudan in maintaining renal cell structure, mitochondrial function and energy supply, which also provided some new evidence and connotation for "kidney-bone" axis. This article is protected by copyright. All rights reserved.

Flurbiprofen axetil (FA) is a prodrug of flurbiprofen (FP), and it is hydrolyzed to the active FP by carboxylesterase in plasma after intravenous injection. The pharmacological action of FP is closely related to its chirality, and S-FP shows better analgesic effects than R-FP. Therefore, it is of great significance to compare the in vivo pharmacokinetic behaviors of R-FP and S-FP. In this study, we designed a sensitive high performance liquid chromatography-tandem mass spectrometry method and used CHIRALPAK-IG3 column for chiral separation to quantify the concentrations of R-FP and S-FP in rat plasma. The results show that this method can accurately and effectively analyze the contents of R-FP and S-FP in plasma. In addition, the systemic exposure was approximately 3.09-folds for the S-FP compared with the R-FP following intravenous administration of the FA to rats at a single dose of 4.5 mg/kg. More importantly, the clearance rate of S-FP is significantly smaller than that of R-FP. Therefore, the development of S-FA injectable emulsion for clinical treatment of postoperative pain is very necessary.

Gushudan (GSD), a compound prescription on the basis of traditional Chinese medicine (TCM) theory and clinical practice, has been used in the treatment of osteoporosis (OP) for many years. Although studies have shown that GSD can treat OP, there is a lack of systematic screening method to explore the bioactive components, which are still unclear. Therefore, this study was aimed to establish an integrated method to screen and determine bioactive ingredients of GSD in the treatment of OP by serum pharmacochemistry, network pharmacology and pharmacokinetics. Firstly, 112 components of the GSD extract and 90 serum migrating constituents were identified by the ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap highresolution mass spectrometry (UHPLC-Q-Orbitrap HRMS), most of which were derived from flavonoids, tanshinones, coumarins and organic acids. Secondly, based on the network pharmacological analysis of the serum migrating constituents, 37 core targets and 20 main pathways related to both GSD and OP were obtained. More importantly, 7 bioactive ingredients were further screened as the PK markers by the network topology parameters including icariin, icariside II, isopimpinellin, bergapten, imperatorin, osthole and tanshinone IIA. Finally, a sensitive and accurate quantitative method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was established and validated for simultaneous determination of the 7 bioactive ingredients in the rat plasma after oral administration of GSD extract, which was then applied to pharmacokinetic study. Besides, the overall pharmacokinetic characteristics were further calculated: Cmax was 180.52 +/- 31.18 ng/mL, T-max was 0.46 +/- 0.20 h, t(1/2) was 4.09 +/- 0.39 h, AUC0-8 was 567.24 +/- 65.29 ng.h/mL, which displayed quick absorption and medium elimination in rats after oral administration of GSD extract. This study provided a new and holistic insight for exploring bioactive constituents and main targets to decode the therapeutic material basis of GSD against OP.

In this study, molecularly imprinted microfiltration membranes (MIMFMs) based on polydopamine and metal-organic framework were innovatively proposed, and successfully prepared for capturing thiamphenicol, florfenicol and chloramphenicol from milk samples through syringe filter device. The provision of PDA and coupling of MOF particles enabled the filter membranes to have great permeability and binding capacity. Cleverly, hydrophilicity and heightened rebinding selectivity (IF = 2.42) of filter membranes was provided using two functional monomers (methacrylate and acrylamide), and microfiltration-grade composite membranes were generated by adjusting the imprinting polymerization time. The combination of the selective filter membranes and the syringe filter devices can effectively simplify the operation process, minimize the extraction time and reduce the tested sample volume. The method was validated by coupling with ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) and obtained a good correlation (r2 > 0.99). The limit of detection (LOD) of three analytes was 0.0800-0.6583 & mu;g kg-1 and the mean recovery ranged from 85.5% to 91.2% within relative standard deviations (RSDs) less than 8.2%. Finally, an effective MIMFMs-UHPLC-MS/ MS method was established for the recognition and detection of three amphenicol antibiotics in milk samples. The MIMFMs filtration technique offered in this work provides valuable science references and potential application prospects for complicated sample pretreatment.

The efficiency of reactive oxygen species (ROS)-based photodynamic therapy (PDT) is far from satisfac-tory, because cancer cells can adapt to PDT by upregulating glutathione (GSH) levels. The GSH levels in tumor cells are determined based on glutamine availability via alanine-serine-cysteine transporter 2 (ASCT2)-mediated entry into cells. Herein, we develop co-assembled nanoparticles (PPa/V-9302 NPs) of the photosensitizer pyropheophorbide a (PPa) and V-9302 (a known inhibitor of ASCT2) in a 1:1 M ratio using a one-step precipitation method to auto-enhance photodynamic therapy. The computational simulations revealed that PPa and V-9302 could self-assemble through different driving forces, such as 7C-7C stacking, hydrophobic interactions, and ionic bonds. Such PPa/V-9302 NPs could disrupt the intracel-lular redox homeostasis due to enhanced ROS production via PPa-induced PDT and reduced GSH synthe-sis via inhibition of the ASCT2-mediated glutamine flux by V-9302. The in vivo assays reveal that PPa/V-9302 NPs could increase the drug accumulation in tumor sites and suppress tumor growth in BALB/c mice bearing mouse breast carcinoma (4 T1) tumor. Our findings provide a new paradigm for the rational design of the PDT-based combinational cancer therapy.(c) 2022 Elsevier Inc. All rights reserved.

Photodynamic therapy (PDT), extensively explored as a non-invasive and spatio-temporal therapeutic modality for cancer treatment, encounters challenges related to the brief half-life and limited diffusion range of singlet oxygen. Lipid peroxides, formed through the oxidation of polyunsaturated fatty acids by singlet oxygen, exhibit prolonged half-life and potent cytotoxicity. Herein, we employed small molecule co-assembly technology to create nanoassemblies of pyropheophorbide a (PPa) and docosahexaenoic acid (DHA) to bolster PDT. DHA, an essential polyunsaturated fatty acid, co-assembled with PPa to generate nanoparticles (PPa@DHA NPs) without the need for additional excipients. To enhance the stability of these nanoassemblies, we introduced 20% DSPE-PEG2k as a stabilizing agent, leading to the formation of PPa@DHA PEG2k NPs. Upon laser irradiation, PPa-produced singlet oxygen swiftly oxidized DHA, resulting in the generation of cytotoxic lipid peroxides. This process significantly augmented the therapeutic efficiency of PDT. Consequently, tumor growth was markedly suppressed, attributed to the sensitizing and amplifying impact of DHA on PDT in a 4T1 tumor-bearing mouse model. In summary, this molecule-engineered nanoassembly introduces an innovative co-delivery approach to enhance PDT with polyunsaturated fatty acids.

A rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method was developed to determine flurbiprofen in rat plasma. A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source was used in negative ion mode. Acetonitrile precipitation was selected to prepare samples. Flurbiprofen and internal standard flurbiprofen-d5 were analyzed on an Acquity UPLC BEH C18 column with the mobile phase consisting of acetonitrile and water, and a gradient procedure was used for separation. The retention time of flurbiprofen was 0.67 min, and the whole running time was only 1.2 min. The detection was performed on a triple quadrupole tandem mass spectrometer using multiple reaction monitoring mode via an ESI source with optimized mass spectrometry parameters. The calibration curve was linear in the range of 25.0-1.00 × 104  ng/mL (r ≥ 0.99). The within-run and between-run relative standard deviations were not more than 13.9%. The within-run and between-run relative errors were from -9.0% to 3.4%. There was no significant matrix effect, and recovery was high. This method was fully validated, including whole blood stability in rat plasma, and successfully applied to the pharmacokinetic study in which 100% incurred sample reanalysis met the criteria.