The colloidal gold nanoparticle-based lateral flow immunoassay for fast and simple detection of plant-derived doping agent,higenamine

Higenamine (HM), an alkaloid found in various plant species, is obtained when norcoclaurine synthase selectively condenses dopamine and 4-hydroxyphenylacetaldehyde to give (S)-higenamine ((S)-HM). The World Anti-doping Agency has listed HM as a prohibited agent in athletics. As a result, many commercial, academic, and regulatory bodies across the globe are invested in finding a rapid method for (S)-HM detection. In the current study, a lateral flow immunoassay (LFA) was developed in which the relevant biosensor was generated as a conjugate of the monoclonal antibody against (S)-HM (namely, MAb E8) and colloidal gold nanoparticles. The HM–γ-globulin conjugates and rabbit anti-mouse IgG antibodies were placed in the test and control zones, respectively. The free (S)-HM molecules in the samples and the immobilized HM–γ-globulin conjugates competitively reacted with the developed biosensor in the LFA. An inverse relationship existed between the biosensors' visible response, which was noted by the variation in the intensity of a pinkish spot in the test zone, and the content of the free (S)-HM. The limit of detection of the developed LFA was 156 ng/mL. Various validation methods confirmed that the LFA exhibited sufficient sensitivity, selectivity, repeatability, and reliability, making it ideal for (S)-HM detection in plant samples and plant-containing products. The developed system required only a small sample volume (20 μL) and a concise sample preparation time compared with conventional LFAs. Thus, the LFA reported in this study could serve as a rapid response kit for the detection of (S)-HM in plant samples.     

Development,validation, and application of an LC–MS/MS method for mitragynine and 7‐hydroxymitragynine analysis in hair

The entire scalp hair of a self‐declared Kratom consumer of 3 grams per day was acquired during an ethical committee approved study. As no values of the concentration in hair of the two Kratom alkaloids mitragynine or 7‐hydroxymitragynine were found in the literature, an already established method for the analysis of benzodiazepines/z‐substances was extended for the detection of mitragynine and 7‐hydroxymitragynine with LC–MS/MS, and successfully validated. The limits of detection and quantification for mitragynine were 2 pg/mg and 4 pg/mg, respectively. Those of 7‐hydroxymitragynine were 20 pg/mg and 30 pg/mg, respectively. The method was applied to the entire scalp hair, divided in 91 regions, of the study participant. A narrow mitragynine concentration distribution with values between 1054 pg/mg and 2244 ng/mg (mean 1517 ng/mg) and no clear scalp region associated distribution pattern was obtained. 7‐Hydroxymitragynine was not detected in any hair sample. After validation, the method was established as routine and subsequently 300 samples (mainly abstinence controls for drugs of abuse) were analyzed, allowing the investigation of the prevalence of Kratom consumption in our population. None of the analyzed routine hair samples were positive for mitragynine or 7‐hydroxymitragynine, providing no evidence that Kratom consumption is prevalent in the investigated population.     

Development of an immunochromatographic assay as a screen for detection of total phthalate acid esters in cooking oil

Phthalate acid esters (PAEs) contamination raised concerns as a result of migration from food packaging and environmental exposure. Because of the adverse effects of PAE reported in humans, the aim of this study was to examine the ability to screen for the detection these chemicals as an indicator of potential exposure. Too develop a sensitive screening test to determine PAE, a specific polyclonal antibody against phthalic acid (PA), the hydrolysate of PAEs, was used as a marker of total PAEs. This method involved the use of 4-aminophthalic acid (APA) as an immunizing hapten to generate antibody. Subsequently, this antibody conjugated with labeled gold nanoparticles (GNPs) was then used to develop an immunochromatographic assay (ICA) for visually detecting PA. After establishing optimal assay conditions, the ICA strip detected visually PA at 3 μg/ml rapidly in less than 5 min. Further, this assay exhibited reliable specificity for PA with no apparent cross-reactivity with structurally related PAEs. A significant correlation between data obtained with the ICA strip and high-performance liquid chromatography (HPLC) analysis was achieved using cooking oils as model spiked samples. The proposed use of ICA offers an effective tool for rapid on-site screening for total PAEs in cooking oils.     

Solubilized candida cell Wall β‐glucan,CSBG, is an epitope of natural human antibody

We have recently developed a protocol to obtain a soluble Candida spp. β1, 3‐glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (DMSO) extraction. CSBG is composed mainly of β‐1,3 and β‐1,6‐glucosidic linkages with a small amount of branching. Antibody to β‐glucan is generally difficult to produce. In the present study, the specificity of the human sera to CSBG was examined by ELISA. Using CSBG‐coated plates, sera from normal human volunteers showed high reactivity that was neutralized by adding soluble CSBG as a competitor. However, the reactivity could not be neutralized by a β‐1,6 branched β‐1,3‐glucan, grifolan. Similar specificity of the immunoglobulin (Ig) was detected in commercially available γ‐globulin prepared from pooled human sera. Comparing various standard glucans, a major epitope was present on β‐1,6 glucan segment. Drug Dev. Res. 58:179–189, 2003. © 2003 Wiley‐Liss, Inc.     

Development of an in vivo anti‐androgenic activity detection assay using fenitrothion in Japanese medaka (Oryzias latipes)

The effects of endocrine disruptors, including anti‐androgenic chemicals, on aquatic environments have received increased attention in recent years. Currently, the method used to screen chemicals for anti‐androgenic activity is called the androgenized female stickleback screen, and it was established by the Organization of Economic Cooperation and Development in 2011 using the three‐spined stickleback. However, screening chemicals for anti‐androgenic activity has yet to be established using Japanese medaka. Thus, the purpose of this study was to establish a screening method for anti‐androgenic activity utilizing the number of papillary processes in Japanese medaka (Oryzias latipes ) as an indicator of the chemical's anti‐androgenic activity. Thus, at 35 days post‐fertilization, medaka were exposed to fenitrothion, an anti‐androgenic compound, for 28 days. In the control group, the formation of papillary processes was observed in XY medaka, but not in XX medaka. However, after fenitrothion exposure, the number of papillary processes was significantly decreased in a dose‐dependent manner in XY medaka; in the 300 μg l⁻¹ concentration group, four of 11 XY medaka showed no papillary processes even if there were no significant effects on total length and wet body weight compared with the control group. Our results indicate that the number of papillary processes in Japanese medaka can be used as an indicator of anti‐androgenic activity and that this model may prove useful as a chemical screening method. Copyright © 2016 John Wiley & Sons, Ltd.     

Feasibility of the radioastatination of a monoclonal antibody with astatine‐211 purified by wet extraction

Astatine‐211, a most promising α‐particle emitter for targeted radiotherapy, is generally obtained by high‐temperature distillation. However, a liquid–liquid extraction procedure (wet extraction) has also been described. The purpose of this study was to develop and optimize the labelling of the stannylated‐activated ester N‐hydroxysuccinimidyl‐meta‐trimethylstannylbenzoate ester (MeSTB) with astatine‐211 extracted in di‐isopropylether (DIPE) in the presence of the oxidant N‐chlorosuccinimide (NCS). The effect of final volume, incubation duration and NCS amounts on radiolabelling yield was studied. The best yields (85–90%) of N‐hydroxysuccinimidyl‐meta‐[²¹¹At]astatobenzoate ester (SAB) were obtained with 20 nmol of MeSTB, 100 nmol of NCS in 120 µL of DIPE after 15 min. The astatine‐211‐labelled‐activated ester was then used to radiolabel a monoclonal antibody (mAb). The labelling yield was 20–25% and the radiochemical purity was 97–99%. These results show that mAbs may be efficiently labelled with astatine‐211 obtained by wet extraction, a fully automatable technique that may prove to be a useful alternative to dry distillation for high activity labelling of radiopharmaceuticals. Copyright © 2008 John Wiley & Sons, Ltd.     

Development of antibody‐based therapeutics for oncology indications

Monoclonal antibodies have emerged as novel oncology therapeutics. These biologics exert anticancer effects via a variety of mechanisms of action including modulating the function of key regulatory molecules and signaling pathways of tumor cells such as blocking growth factor/receptor interaction and/or down‐regulating expression of oncogenic proteins (e.g., growth factor receptors) on the cell surface; recruiting effector mechanisms of the immune system, such as antibody‐dependent cellular cytotoxicity and complement‐mediated cytotoxicity; as a targeting device in immnuoconjugates; and other mechanisms such as anti‐idiotype, catalytic antibodies, or antibodies that modulate patient's immune responses to tumors. In this review, we focus on the research and development experience of four such representative antibody therapeutics, rituximab (Rituxan^®), trastuzumab (Herceptin^®), cetuximab (Erbitux^®), and bevacizumab (Avastin^®), approved for treating non‐Hodgkin's lymphoma, metastatic breast cancer, and colorectal cancer, respectively. The biology behind each antibody target, their proposed mechanisms of action, as well as preclinical and clinical development of these antibodies are the topics of this article. Experience drawn from the development process of these four antibodies is instrumental for ongoing and future antibody development activities. In addition, perspective views on challenges and opportunities of oncology antibody therapeutics are presented. Drug Dev. Res. 67:699–728, 2006. © 2006 Wiley‐Liss, Inc.     

Radioimmunoimaging and targeting treatment in an immunocompetent murine model of triple‐negative breast cancer using radiolabeled anti–programmed death‐ligand 1 monoclonal antibody

The overall aim of this study was to evaluate whether iodine‐131 radiolabeled monoclonal antibody (mAb) targeting programmed death‐ligand 1 (PD‐L1) can be used for imaging of PD‐L1 expression noninvasively in vivo and playing synergistic effect combined with immunotherapy. Anti‐PD‐L1 mAb was radiolabeled with iodine‐131 (¹³¹I‐PD‐L1 mAb) and was characterized in vitro. Biodistribution and imaging in vivo were performed periodically. Therapy study was conducted in triple‐negative breast cancer–bearing BALB/c mice. As results, the labeling efficiencies of ¹³¹I‐PD‐L1 mAb reached 80% ± 3%, with radiochemical purity of 97% ± 1%. ¹³¹I‐PD‐L1 mAb preserved the capacity to bind living PD‐L1‐expressing cells specifically in vitro. Tumor radioactivity uptake of ¹³¹I‐PD‐L1 mAb was significantly higher than that of control groups. The xenografts were clearly imaged from 48 to 72 hours noninvasively after injection of ¹³¹I‐PD‐L1 mAb, while the xenografts were not imaged in control groups. Tumor growth was significantly inhibited, and median survival time was remarkably prolonged in combination therapy group compared with control groups. It was concluded that ¹³¹I‐PD‐L1 mAb can be a potential theranostic candidate for visualizing of PD‐L1 expression noninvasively and performing synergistic therapy in carcinomas.     

Preparation and Evaluation of 99mTc‐labeled anti‐CD11b Antibody Targeting Inflammatory Microenvironment for Colon Cancer Imaging

CD11b, an active constituent of innate immune response highly expressed in myeloid‐derived suppressor cells (MDSCs), can be used as a marker of inflammatory microenvironment, particularly in tumor tissues. In this research, we aimed to fabricate a ^99mTc‐labeled anti‐CD11b antibody as a probe for CD11b⁺ myeloid cells in colon cancer imaging with single‐photon emission computed tomography (SPECT). In situ murine colon tumor model was established in histidine decarboxylase knockout (Hdc^?/?) mice by chemicals induction. ^99mTc‐labeled anti‐CD11b was obtained with labeling yields of over 30% and radiochemical purity of over 95%. Micro‐SPECT/CT scans were performed at 6 h post injection to investigate biodistributions and targeting of the probe. In situ colonic neoplasma as small as 3 mm diameters was clearly identified by imaging; after dissection of the animal, anti‐CD11b immunofluorescence staining was performed to identify infiltration of CD11b+ MDSCs in microenvironment of colonic neoplasms. In addition, the images displayed intense signal from bone marrow and spleen, which indicated the origin and migration of CD11b⁺ MDSCs in vivo, and these results were further proved by flow cytometry analysis. Therefore, ^99mTc‐labeled anti‐CD11b SPECT displayed the potential to facilitate the diagnosis of colon tumor in very early stage via detection of inflammatory microenvironment.     

Advantages of a Synthetic Peptide Immunogen Over a Protein Immunogen in the Development of an Anti‐Pilus Vaccine for Pseudomonas aeruginosa

The type IV pilus is an important adhesin in the establishment of infection by Pseudomonas aeruginosa. We have previously reported on a synthetic peptide vaccine targeting the receptor‐binding domain of the main structural subunit of the pilus, PilA. The receptor‐binding domain is a 14‐residue disulfide loop at the C‐terminal end of the pilin protein. The objective of this study was to compare the immunogenicity of a peptide‐conjugate to a protein subunit immunogen to determine which was superior for use in an anti‐pilus vaccine. BALB/c mice were immunized with the native PAK strain pilin protein and a synthetic peptide of the receptor‐binding domain conjugated to keyhole limpet haemocyanin. A novel pilin protein with a scrambled receptor‐binding domain was used to characterize receptor‐binding domain‐specific antibodies. The titres against the native pilin of the animals immunized with the synthetic peptide‐conjugate were higher than the titres of animals immunized with the pilin protein. In addition, the affinities of anti‐peptide sera for the intact pilin receptor‐binding domain were significantly higher than affinities of anti‐pilin protein sera. These results have significant implications for vaccine design and show that there are significant advantages in using a synthetic peptide‐conjugate over a subunit pilin protein for an anti‐pilus vaccine.     

被动凝集法、间接免疫荧光法和胶体金法联合检测肺炎 支原体抗体对儿童肺炎支原体感染的诊断价值

目的 分析被动凝集法（PA）、间接免疫荧光法（IFA）和胶体金法（GICT）联合检测对儿童肺炎支原体（MP） 感染的诊断价值。方法 选取进行MP抗体检测的患儿617例,以临床诊断为判断标准,分为MP感染组（345例）和 非MP感染组（272例）。所有患儿均经PA检测MP总抗体,经IFA和GICT检测MP-IgM抗体。分析PA、IFA和GICT 这3种方法单独检测及两两联合检测与临床诊断的一致性,受试者工作特征（ROC）曲线评价其对MP感染的诊断价 值,分析PA联合IFA检测2组患儿抗体情况。结果 MP感染组PA检测MP总抗体、IFA和GICT检测MP-IgM抗体的 阳性率较非MP感染组高（P＜0.01）。PA检测MP总抗体的阳性检出率高于IFA和GICT检测MP-IgM抗体的阳性检 出率（P＜0.01）。PA联合IFA与临床诊断为中度一致（Kappa值=0.41,P＜0.05）。3种方法单独检测和两两组合检测 中PA联合IFA的曲线下面积、敏感度、总符合率、阴性预测值最高,阴性似然比最低。GICT单独检测特异度最高。 IFA 单独检测阳性预测值和阳性似然比最高。当 MP-IgM 抗体阳性时,MP 感染组 23.44% 的患儿总抗体滴度＜1︰ 160,非MP感染组47.22%的患儿总抗体滴度≥1︰160。当MP-IgM抗体阴性时,MP感染组91.91%的患儿MP总抗体 滴度≥1︰160,非MP感染组有73.50%的患儿总抗体滴度＜1︰160。结论 PA和IFA联合检测可为临床诊断儿童MP 感染提供更客观、准确的检测结果。     

In vitro evaluation of concentration,labeling effectiveness and stability for 131I‐labeled radioimmunoassay ligand using real‐time detection technology

Radioimmunoassay belongs to the analytical method enabling highly specific and sensitive quantification of molecules. The verification of the real‐time radioimmunoassay technology usefulness for ligand‐quality characteristics evaluation such as concentration, influence of radiolabeling on binding affinity and stability was estimated. The anti‐epidermal growth factor receptor antibody ¹³¹I‐cetuximab was employed as the ligand antibody. The concentration of ¹³¹I‐cetuximab was derived from the shape of binding curves coming from the ligand‐receptor interaction. The binding curves also allowed the estimation of ¹³¹I‐cetuximab binding affinity for different radiolabeling procedures (incubation times 1, 5, and 10 minutes) in stability testing up to 96 hours at 4°C. The stability testing also included comparative analysis by size exclusion high‐performance liquid chromatography. The assessment of cetuximab concentrations using real‐time method showed acceptable accordance between real and calculated values. The real‐time method revealed that 1‐minute radiolabeling proved to be the optimal incubation time for direct radioiodination of cetuximab. Stability testing showed the significant change in radioligand affinity by one order at the longest incubation times (72 and 96 hours). Characterization of stability and binding behavior of radiolabeled monoclonal antibodies by the verified real‐time method before use in other assays may be employed to eliminate variability and suboptimal antibody performance.     

Development and clinical applications of the dried blood spot method for therapeutic drug monitoring of anti‐epileptic drugs

Anti‐epileptic drugs (AEDs) have various pharmacokinetic profiles, inter‐individual variabilities, high possibilities of drug‐drug interactions and narrow therapeutic indices. To provide optimal treatment for patients, therapeutic drug monitoring (TDM) is necessary. However, TDM requires sufficient quantities of blood samples to measure drug concentrations. Therefore, TDM could be a burden, particularly in paediatric cases. A good alternative that overcomes these disadvantages is the dried blood spot (DBS) method, which is simple, convenient to use and less invasive, requiring a lower quantity of blood than traditional blood sampling methods. However, the DBS method is affected by haematocrit (Hct) levels to varying extents depending on the drug properties. In addition, different papers with varying characteristics are available for use when applying the DBS method. Therefore, it has not yet been applied to TDM in clinical practice. To achieve this, several steps are required, including method development, method validation and clinical validation. Currently, the development status of the DBS method is different for each AED and unclear. Therefore, we assessed the development status of the following 19 AEDs in 26 studies: lamotrigine, valproic acid, levetiracetam, phenytoin, topiramate, carbamazepine, carbamazepine epoxide, gabapentin, phenobarbital, pregabalin, clobazam, clonazepam, ethosuximide, felbamate, monohydroxycarbamazepine, nitrazepam, rufinamide, vigabatrin and zonisamide. Among them, carbamazepine, lamotrigine, topiramate and valproic acid have been developed such that they are nearly available for TDM. In addition, Whatman 903 Protein Saver Cards and concentration analysis by liquid chromatography with triple quadrupole mass spectrometer were used most often.     

Development of a lateral flow dipstick for simultaneous and semi‐quantitative analysis of dihydroartemisinin and piperaquine in an artemisinin combination therapy

Dihydroartemisinin (DHA) and piperaquine (PPQ) are two drugs used in an artemisinin‐based combination therapy (ACT). The circulation of counterfeit antimalarial drugs demands the development of simple, point‐of‐care (POC) tests for monitoring drug quality. Here we aimed to design an antibody‐based lateral flow dipstick assay for simultaneous quality control of DHA and PPQ. To obtain a monoclonal antibody (mAb) for PPQ, one structural unit of the symmetric PPQ molecule was used to derive a carboxylic acid for linkage to a carrier protein as immunogen. Screening of hybridoma cells identified an mAb 4D112B2 that reacted with the PPQ‐based immunogen. A highly‐sensitive icELISA was designed based on this mAb, which showed 50% inhibition concentration of PPQ at 1.66 ng/mL and a working range of 0.35 – 7.40 ng/mL. The mAb showed 10.2, 15.9 and 30.4% cross reactivity to hydroxychloroquine sulfate, chloroquine and amodiaquine, respectively. No cross reactivity was observed to lumefantrine, mefloquine artemisinin and its derivatives. Using our previous DHA dipstick design, a lateral flow dipstick for simultaneous analysis of PPQ and DHA was developed. The indicator ranges for PPQ and DHA were 2 – 5 μg/mL and 250 – 500 ng/mL, respectively. The dipstick was used to semi‐quantitatively analyze PPQ and DHA content in commercial ACT drugs, which produced agreeable results to those determined by high‐performance liquid chromatography. This combination dipstick makes it a potential POC device for quality control of the two active ingredients in a commonly used ACT.     

Synthesis and biodistribution studies of iodine‐131 D‐amino acid YYK peptide as a potential therapeutic agent for labeling an anti‐CD20 antibody

A major drawback of conventionally radioiodinated monoclonal antibodies for radioimmunotherapy is in vivo dehalogenation of iodine as a result of deiodinase recognition. To solve this problem we have synthesized a YYK tri‐peptide consisting of non‐metabolizable D ‐amino acids modified with the N‐succinimidyl (N‐Succ) function. The chemical purity of the synthesized peptide as assessed by analytical high performance liquid chromatography was 95%. Labeling of the Fmoc‐D ‐Tyr(_tBu)‐D ‐Tyr(_tBu)‐D ‐Lys(Boc)‐N‐Succ was performed using the chloramine‐T method and the conventional extraction, resulting in a radiochemical yield of 50–71% and a radiochemical purity of >95%. Radioiodination of the peptide was followed by conjugation to anti‐CD20 antibody with 65–75% labeling efficiency and 90% radiochemical purity. The effect of radioiodinated peptide on the biological behavior of the conjugate was evaluated through biodistribution studies in normal Lewis rats. Thyroid and stomach levels from Rituximab labeled with [¹³¹I]‐YYK‐peptide were two‐ to four‐fold less than those with directly labeled [¹³¹I]‐Rituximab, suggesting low recognition of its D ‐iodotyrosine residue by endogenous deiodinases. The favorable in vitro/in vivo stability and biodistribution profiles suggest that this radioiodine‐labeled YYK peptide is a good candidate for further exploration of its potential clinical application. Copyright © 2009 John Wiley & Sons, Ltd.     

Differentiation between microcystin contaminated and uncontaminated fish by determination of unconjugated MCs using an ELISA anti‐adda test based on receiver‐operating characteristic curves threshold values: Application to Tinca tinca from natural ponds

The aim of this study was to evaluate whether the enzyme‐linked immunosorbent assay (ELISA) anti‐Adda technique could be used to monitor free microcystins (MCs) in biological samples from fish naturally exposed to toxic cyanobacteria by using receiver operating characteristic (ROC) curve software to establish an optimal cut‐off value for MCs. The cut‐off value determined by ROC curve analysis in tench (Tinca tinca) exposed to MCs under laboratory conditions by ROC curve analysis was 5.90‐μg MCs/kg tissue dry weight (d.w.) with a sensitivity of 93.3%. This value was applied in fish samples from natural ponds (Extremadura, Spain) in order to asses its potential MCs bioaccumulation by classifying samples as either true positive (TP), false positive (FP), true negative (TN), or false negative (FN). In this work, it has been demonstrated that toxic cyanobacteria, mainly Microcystis aeruginosa, Aphanizomenon issatchenkoi, and Anabaena spiroides, were present in two of these ponds, Barruecos de Abajo (BDown) and Barruecos de Arriba (BUp). The MCs levels were detected in waters from both ponds with an anti‐MC‐LR ELISA immunoassay and were of similar values (between 3.8–6.5‐μg MC‐LR equivalent/L in BDown pond and 4.8–6.0‐μg MC‐LR equivalent/L in BUp). The MCs cut‐off values were applied in livers from fish collected from these two ponds using the ELISA anti‐Adda technique. A total of 83% of samples from BDown pond and only 42% from BUp were TP with values of free MCs higher than 8.8‐μg MCs/kg tissue (d.w.). © 2009 Wiley Periodicals, Inc. Environ Toxicol 26: 45–56, 2011.     

Synthesis of 13C215N2‐labeled anti‐inflammatory and cytoprotective tricyclic bis(cyanoenone) ([13C215N2]‐TBE‐31) as an internal standard for quantification by stable isotope dilution LC‐MS method

Tricyclic bis(cyanoenone), TBE‐31, one of the most potent activators of the Keap1/Nrf2/antioxidant response element pathway, has been developed as a new anti‐inflammatory and cytoprotective agent. ¹³C₂¹⁵N₂‐labeled TBE‐31 ([¹³C₂¹⁵N₂]‐TBE‐31), which has two ¹³C and two ¹⁵N atoms in two cyano groups, was designed to develop a method for quantification of cell, tissue, and plasma levels of TBE‐31 that involves chromatography/mass spectrometry coupled with the use of a stable isotope‐labeled internal standard. [¹³C₂¹⁵N₂]‐TBE‐31 was successfully synthesized in four steps from a previously reported intermediate, which is prepared in 11 steps from cyclohexanone, by introduction of two ¹³C atoms with ethyl [¹³C]formate and two ¹⁵N atoms with hydroxyl[¹⁵N]amine. The stable isotope dilution liquid chromatography–mass spectrometry method for quantification of TBE‐31 was successfully developed using [¹³C₂¹⁵N₂]‐TBE‐31 to compensate for any variables encountered during sample processing and analysis.     

LC−MS/MS assay for the determination of a novel anti‐fibrotic candidate mefunidone in monkey plasma and its application to a pharmacokinetics study

Mefunidone (MFD) is a promising anti‐fibrotic candidate molecule with greater anti‐fibrotic activity than pirfenidone (PFD). However, there has been no report on the methodology used for the quantification of MFD or on any investigation of its pharmacokinetics. In this study, an efficient and reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to assay MFD in monkey plasma. This assay method was validated and applied to a pharmacokinetics study in monkeys. The lower limit of quantification of this assay was 0.1 μg·mL^?1, and the linear calibration curve was acquired with R² > 0.99 between 0.1 and 60 μg·mL^?1. The intra‐day and inter‐day precision were evaluated with coefficient of variations of 1.5%–5.8%, whereas the mean accuracy ranged from 91.7% to 106.9%. A negligible matrix effect and good recovery were obtained using this assay, with average extraction recoveries of MFD and the internal standard (IS) in the range of 85.5%–124.8% and 84.1%–94.0%, respectively. The precision of the absolute matrix effect of MFD and the IS was 1.2–3.0% and 1.2–7.3%, respectively. The samples were stable under all experimental conditions. Linear pharmacokinetics were observed for MFD in monkeys, where the exposures of MFD increased proportionally with increasing MFD doses at the range of 10–90 mg·kg^?1. Moderate elimination of MFD from the body was observed, with t_1/2 of 5–7 h, and the elimination rate of MFD was stable during multiple dosing. In conclusion, this method provides an reliable analytical approach for quantification of MFD in plasma and was successfully applied to a pharmacokinetics study in monkeys.