Simultaneous Analysis for Quality Control of Traditional Herbal Medicine,Gungha-Tang,Using Liquid Chromatography–Tandem Mass Spectrometry

Gungha-tang (GHT), a traditional herbal medicine, consists of nine medicinal herbs (Cnidii Rhizoma, Pinelliae Tuber, Poria Sclerotium, Citri Unshius Pericarpium, Citri Unshius Pericarpium Immaturus, Aurantii Fructus Immaturus, Atracylodis Rhizoma Alba, Glycyrrhizae Radix et Rhizoma, and Zingiberis Rhizoma Recens). It has been used for various diseases caused by phlegm. This study aimed to develop and verify the simultaneous liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis method, using nine marker components (liquiritin apioside, neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, liquiritigenin, glycyrrhizin, and 6-shogaol) for quality control of GHT. LC–MS/MS analysis was conducted using a Waters TQ-XS system. All marker analytes were separated on a Waters Acquity UPLC BEH C₁₈ column (2.1 × 100 mm, 1.7 μm) using gradient elution with a distilled water solution (containing 5 mM ammonium formate and 0.1% [v/v] formic acid)–acetonitrile mobile phase. LC–MS/MS multiple reaction monitoring (MRM) analysis was carried out in negative and positive ion modes of an electrospray ionization source. The developed LC–MS/MS MRM method was validated by examining the linearity, limits of detection and quantification, recovery, and precision. LOD and LOQ values of nine markers were calculated as 0.02–8.33 ng/mL and 0.05–25.00 ng/mL. The recovery was determined to be 89.00–118.08% and precision was assessed with a coefficient of variation value of 1.74–8.64%. In the established LC–MS/MS MRM method, all markers in GHT samples were detected at 0.003–16.157 mg/g. Information gathered during the development and verification of the LC–MS/MS method will be useful for the quality assessment of GHT and other herbal medicines.     

The Development of an Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry Method for Biogenic Amines in Fish Samples

Biogenic amines (BAs) are a group of substances that are formed from amino acids by decarboxylation or amination and transamination of aldehydes and ketones. They may have either an aliphatic, aromatic, or heterocyclic structure. Their quantity determines their effects and optimum amounts are essential for physiological functions, but excess BAs causes various toxic effects throughout the human body. In our study, to rapidly determine 14 BAs (histamine, tyramine, dopamine, tryptamine, serotonin, putrescine, spermine, spermidine, octopamine, benzylamine, 1-Phenylethanamine, cadaverine, 2-Phenethylamine, and agmatine) in real fish samples, an ultra-performance liquid chromatography–tandem mass spectrometry method was established. The fish sample was extracted by acetonitrile with 0.1% formic acid and stable biogenic amine derivatives could be obtained by benzoyl chloride derivatization with a shorter reaction time. The method showed good linearity with a linear range of 3–4 orders of magnitude and regression coefficients ranging from 0.9961 to 0.9999. The calculated LODs ranged from 0.1 to 20 nM and the LOQs ranged from 0.3 to 60 nM. Satisfactory recovery was obtained from 84.6% to 119.3%. The proposed method was employed to determine the concentration levels of biogenic amine derivatives in different fish. The results indicated that this method was suitable for the analysis of biogenic amines.     

Multi-Class Procedure for Analysis of 50 Antibacterial Compounds in Eggshells Using Ultra-High-Performance Liquid Chromatography–Tandem Mass Spectrometry

In this work, for the first time, Ultra-High-Performance Liquid Chromatography–Tandem Mass Spectrometry (UHPLC–MS/MS) method was developed for qualitative and quantitative analysis of veterinary antibiotics (cephalosporins, diaminopyrimidines, fluoro(quinolones), lincosamides, macrolides, penicillins, pleuromutilins, sulfonamides, tetracyclines, and sulfones) in hen eggshells. The sample preparation method is based on a liquid–liquid extraction with a mixture of metaphosphoric acid, ascorbic acid, EDTA disodium salt dihydrate, and acetonitrile. The chromatographic separation was performed on Luna^® Omega Polar C18 10 column in gradient elution mode and quantitated in an 8 min run. Validation such as linearity, selectivity, precision, recovery, matrix effect, limit of quantification (LOQ), and limit of detection (LOD) was found to be within the acceptance criteria of the validation guidelines of the Commission Decision 2002/657/EC and EUR 28099 EN. Average recoveries ranged from 81–120%. The calculated LOQ values ranged from 1 to 10 µg/kg, the LOD values ranged from 0.3 to 4.0 µg/kg, depending on analyte. The developed method has been successfully applied to the determination of antibacterial compounds in hen eggshell samples obtained from different sources. The results revealed that enrofloxacin, lincomycin, doxycycline, and oxytetracycline were detected in hen eggshell samples.     

Development of an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Hydroxylated Polychlorinated Biphenyls in Animal-Derived Food

Hydroxylated polychlorinated biphenyls (OH-PCBs) are a group of metabolites biotransformed from polychlorinated biphenyls by animals with higher toxicities than their parent compounds. The present work developed and validated an analytical method for determinating penta-, hexa-, and hepta-chlorine substituted OH-PCBs in animal-derived food based on ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) with isotope-dilution. The target analytes were extracted with a 50% n-hexane/dichloromethane (v/v), purified by sulfuric acid-silica gel, and separated by 5% hydrated silica gel, achieving a final concentration of 100 times before injection to LC–MS/MS. The limits of detection (LOD) and quantification (LOQ) for target OH-PCBs were within the ranges of 0.003–0.010 μg/kg and 0.009–0.030 μg/kg, respectively. Average recoveries ranged between 76.7% and 116.5%, with relative standard deviations of less than 18.4%. The proposed method is simple, time-saving, sensitive, and accurate, making it a powerful tool for risk monitoring of OH-PCBs in animal-derived food.     

Development and Validation of Multi-Residue Method for Drugs Analysis in Human Feces by Liquid Chromatography–Tandem Mass Spectrometry

The use of veterinary drugs in animal production is a common practice to secure animal and human health. However, residues of administrated drugs could be present in animal food products. Levels of drugs in food of animal origin are regulated within the European Union. In recent years, residues have been detected not only in food, but also in the environmental elements such as water or soil, meaning that humans are involuntarily exposed to these substances. This article presents a multiclass method for the analysis of various therapeutic groups of pharmaceuticals in human feces. Pharmaceuticals are extracted from feces with an acid extraction solvent, and after filtration the extract was analyzed by HPLC–MS/MS. A limit of detection of 10 ng/g was achieved for 9 pharmaceuticals, with linearity over 0.99 and repeatability and reproducibility lower than 20%. The method was satisfactorily applied in 25 feces samples of individuals that had declared not to be under medical treatment for the last two months. Results indicate the presence of six different compounds at concentration between 10 and 456 ng/g. This preliminary study showed the involuntary exposure of human gut microbiota to active substances such as pharmaceuticals     

Online In-Tube Solid-Phase Microextraction Coupled to Liquid Chromatography–Tandem Mass Spectrometry for the Determination of Tobacco-Specific Nitrosamines in Hair Samples

Active and passive smoking are serious public health concerns Assessment of tobacco smoke exposure using effective biomarkers is needed. In this study, we developed a simultaneous determination method of five tobacco-specific nitrosamines (TSNAs) in hair by online in-tube solid-phase microextraction (SPME) coupled to liquid chromatography-tandem mass spectrometry (LC–MS/MS). TSNAs were extracted and concentrated on Supel-Q PLOT capillary by in-tube SPME and separated and detected within 5 min by LC–MS/MS using Capcell Pak C18 MGIII column and positive ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. The calibration curves of TSNAs showed good linearity in the range of 0.5–1000 pg mL^–1 using their stable isotope-labeled internal standards. Moreover, the limits of detection (S/N = 3) of TSNAs were in the range of 0.02–1.14 pg mL^–1, and intra-day and inter-day precisions were below 7.3% and 9.2% (n = 5), respectively. The developed method is highly sensitive and specific and can easily measure TSNA levels using 5 mg hair samples. This method was used to assess long-term exposure levels to tobacco smoke in smokers and non-smokers.     

Detection of Methylphenidate in Equine Hair Using Liquid Chromatography–High-Resolution Mass Spectrometry

Methylphenidate is a powerful central nervous system stimulant with a high potential for abuse in horse racing. The detection of methylphenidate use is of interest to horse racing authorities for both prior to and during competition. The use of hair as an alternative sampling matrix for equine anti-doping has increased as the number of detectable compounds has expanded. Our laboratory developed a liquid chromatography–high-resolution mass spectrometry method to detect the presence of methylphenidate in submitted samples. Briefly, hair was decontaminated, cut, and pulverized prior to liquid–liquid extraction in basic conditions before introduction to the LC-MS system. Instrumental analysis was conducted using a Thermo Q Exactive mass spectrometer using parallel reaction monitoring using a stepped collision energy to obtain sufficient product ions for qualitative identification. The method was validated and limits of quantitation, linearity, matrix effects, recovery, accuracy, and precision were determined. The method has been applied to confirm the presence of methylphenidate in official samples submitted by racing authorities.     

高效液相色谱-串联质谱对蜂王浆中27种药物残留的同时测定

建立了蜂王浆中氯霉素、磺胺类、磺胺增效剂、氟喹诺酮类、林可胺类、硝基咪唑类、大环内酯类等7大类27种药物残留的高效液相色谱-串联质谱同时测定的分析方法。对提取溶剂、提取pH值、色谱柱、流动相、质谱条件进行了优化。结果表明,氯霉素的线性范围为0.3~10μg/kg,其余26种药物的线性范围为2~100μg/kg;氯霉素的检出限(S/N=3)为0.1μg/kg,其余26种药物的检出限均为1μg/kg;回收率达70%~119%,相对标准偏差为4.3%~14.9%。该方法满足蜂王浆中27种药物残留的同时测定要求,具有样品预处理简单、灵敏度高、分析时间短的优点。     

Study on Quality Control of Compound Anoectochilus roxburghii (Wall.) Lindl. by Liquid Chromatography–Tandem Mass Spectrometry

Compound Anoectochilus roxburghii (Wall.) Lindl. (A. roxburghii) oral liquid (CAROL) is a hospital preparation of A. roxburghii and Ganoderma lucidum (G. lucidum), which have hepatoprotective effects. Eight active components (five nucleosides/nucleobases and three triterpenoid acids) in CAROL, A. roxburghii, and G. lucidum were simultaneously detected by high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS). The multiple reaction monitoring (MRM) mode was applied for the detection of analytes. These eight compounds were separated well within 12 min and quantified using the internal standard working curve method. The method showed good linearity (R² > 0.9935) and high sensitivity (limit of detection = 0.29 ng/mL). The analyte recovery ranged from 85.07% to 97.50% (relative standard deviation < 3.31%). The content of the target analytes in four batches of CAROL, and the raw materials of G. lucidum and A. roxburghii from the five regions was determined using this method. The contents of guanosine and ganoderic acid A in four batches of oral liquid were high and stabilized and could be recommended as quality markers (Q-marker) for CAROL. Simultaneous qualitative and quantitative analysis of nucleosides and triterpenoid acids in CAROL, A. roxburghii, and G. lucidum by LC–MS/MS based on the MRM model was reported for the first time. The proposed method provides a sensitive, rapid, and reliable approach for the quality control of Chinese medicinal products.     

Determination of Methylene Blue and Its Metabolite Residues in Aquatic Products by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

A sensitive and reliable method was developed to determine methylene blue (MB) and its metabolite residues, including azure A (AZA), azure B (AZB), and azure C (AZC) in aquatic products by HPLC–MS/MS. The samples were extracted by acetonitrile and cleaned up by alumina-neutral (ALN) cartridges. The analytes were separated on a Sunfire C18 column (150 mm × 2.1 mm, 5 µm). The method was validated according to the European criteria of Commission Decision 2002/657/CE. Good linearity between 1–500 µg/L was obtained with correlation coefficients (R²) greater than 0.99. The limit of quantification (LOQ) was 1.0 µg/kg. The average recoveries at three levels of each compound (1, 5, and 10 µg/kg) were demonstrated to be in the range of 71.8–97.5%, with relative standard deviations (RSDs) from 1.05% to 8.63%. This method was suitable for the detection of methylene blue and its metabolite residues in aquatic products.     

Determination of Genotoxic Impurity N-Nitroso-N-methyl-4-aminobutyric Acid in Four Sartan Substances through Using Liquid Chromatography–Tandem Mass Spectrometry

N-nitroso-N-methyl-4-aminobutyric acid (NMBA) is the third N-nitrosamine impurity found in sartans. Herein, a sensitive and stable LC-MS/MS method with multiple reactions monitoring mode has been developed for the quantitative determination of NMBA in four sartan substances. The effective separation of NMBA and sartan substances was achieved on a C18 column under gradient elution conditions. The mass spectrometry method of the atmospheric pressure chemical ionization source and internal standard method was selected as the quantitative analysis method of NMBA. Then, this proposed LC-MS/MS analysis method was validated in terms of specificity, sensitivity, linearity, accuracy, precision and stability. Good linearity with correlation coefficient over 0.99 was obtained at the NMBA concentration of 3–45 ng/mL, and the limit of quantification was 3 ng/mL. Additionally, the recoveries of NMBA in four sartan substances ranged from 89.9% to 115.7%. The intra-day and inter-day relative standard deviation values were less than 5.0%. In conclusion, this developed determination method for NMBA through liquid chromatography–tandem mass spectrometry showed the characteristics of good sensitivity, high accuracy and precision, which will be of great help for the quantitative analysis of NMBA in sartan products.     

Online In-Tube Solid-Phase Microextraction Coupled with Liquid Chromatography–Tandem Mass Spectrometry for Automated Analysis of Four Sulfated Steroid Metabolites in Saliva Samples

Accurate measurement of sulfated steroid metabolite concentrations can not only enable the elucidation of the mechanisms regulating steroid metabolism, but also lead to the diagnosis of various related diseases. The present study describes a simple and sensitive method for the simultaneous determination of four sulfated steroid metabolites in saliva, pregnenolone sulfate (PREGS), dehydroepiandrosterone sulfate (DHEAS), cortisol sulfate (CRTS), and 17β-estradiol-3-sulfate (E2S), by online coupling of in-tube solid-phase microextraction (IT-SPME) and stable isotope dilution liquid chromatography–tandem mass spectrometry (LC–MS/MS). These compounds were extracted and concentrated on Supel-Q PLOT capillary tubes by IT-SPME and separated and detected within 6 min by LC–MS/MS using an InertSustain swift C18 column and negative ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. Calibration curves using their stable isotope-labeled internal standards showed good linearity in the range of 0.01–2 ng mL⁻¹ for PREGS, DHEAS, and CRTS and of 0.05–10 ng mL⁻¹ for E2S. The limits of detection (S/N = 3) of PREGS, DHEAS, CRTS, and E2S were 0.59, 0.30, 0.80, and 3.20 pg mL⁻¹, respectively. Moreover, intraday and interday variations were lower than 11.1% (n = 5). The recoveries of these compounds from saliva samples were in the range of 86.6–112.9%. The developed method is highly sensitive and specific and can easily measure sulfated steroid metabolite concentrations in 50 μL saliva samples.     

High Performance Liquid Chromatography–Tandem Mass Spectrometry Method for Correlating the Metabolic Changes of Lactate,Pyruvate and L-Glutamine with Induced Tamoxifen Resistant MCF-7 Cell Line Potential Molecular Changes

Breast cancer is one of the most prevalent cancers worldwide usually treated with Tamoxifen. Tamoxifen resistance development is the most challenging issue in an initially responsive breast tumor, and mechanisms of resistance are still under investigation. The objective of this study is to develop and validate a selective, sensitive, and simultaneous high performance liquid chromatography–tandem mass spectrometry method to explore the changes in substrates and metabolites in supernatant media of developed Tamoxifen resistance MCF-7 cells. We focus on the determination of lactate, pyruvate, and L-glutamine which enables the tracking of changes in metabolic pathways as a result of the resistance process. Chromatographic separation was achieved within 3.5 min. using a HILIC column (4.6 × 100 mm, 3.5 µm particle size) and mobile phase of 0.05 M acetic acid–ammonium acetate buffer solution pH 3.0: Acetonitrile (40:60 v/v). The linear range was 0.11–2.25, 0.012–0.227, and 0.02–0.20 mM for lactate, pyruvate, and L-glutamine, respectively. Within- and between-run accuracy was in the range 98.94-105.50% with precision (CV, %) of ≤0.86%. The results revealed a significant increase in both lactate and pyruvate production after acquiring the resistant. An increase in L-glutamine levels was also observed and could be attributed to its over production or decline in its consumption. Therefore, further tracking of genes responsible of lactate, pyruvate, and glutamine metabolic pathways should be performed in parallel to provide in-depth explanation of resistance mechanism.     

Nontargeted Screening Using Gas Chromatography–Atmospheric Pressure Ionization Mass Spectrometry: Recent Trends and Emerging Potential

Gas chromatography–high-resolution mass spectrometry (GC–HRMS) is a powerful nontargeted screening technique that promises to accelerate the identification of environmental pollutants. Currently, most GC–HRMS instruments are equipped with electron ionization (EI), but atmospheric pressure ionization (API) ion sources have attracted renewed interest because: (i) collisional cooling at atmospheric pressure minimizes fragmentation, resulting in an increased yield of molecular ions for elemental composition determination and improved detection limits; (ii) a wide range of sophisticated tandem (ion mobility) mass spectrometers can be easily adapted for operation with GC–API; and (iii) the conditions of an atmospheric pressure ion source can promote structure diagnostic ion–molecule reactions that are otherwise difficult to perform using conventional GC–MS instrumentation. This literature review addresses the merits of GC–API for nontargeted screening while summarizing recent applications using various GC–API techniques. One perceived drawback of GC–API is the paucity of spectral libraries that can be used to guide structure elucidation. Herein, novel data acquisition, deconvolution and spectral prediction tools will be reviewed. With continued development, it is anticipated that API may eventually supplant EI as the de facto GC–MS ion source used to identify unknowns.     

Simultaneous Determination of 21 Sulfonamides in Poultry Eggs Using Ionic Liquid-Modified Molecularly Imprinted Polymer SPE and UPLC–MS/MS

An ionic liquid-modified molecularly imprinted polymer (IL-MIP) composite with sulfamethazine as a template molecule and methyl acrylic acid and 1-aminopropyl-3-methylimidazolium bromide as functional monomers was successfully synthesized. The achieved IL-MIP was characterized and evaluated in detail and utilized in the extraction and cleanup of sulfonamides (SAs) in poultry egg samples. The results demonstrated that the IL-MIP possessed a broad reorganization toward SAs and could selectively adsorb 21 kinds of SA compounds. Furthermore, the solid-phase extraction column based on the IL-MIP was used in the extraction and cleanup of 21 SAs in eggs, and the confirmatory detection of SAs was performed using ultraperformance liquid chromatography–tandem mass spectrometry. Under optimum conditions, the limits of detection (LODs) for all SAs ranged from 0.1 ng·g⁻¹ to 1.5 ng·g⁻¹, and the LOD of this method was better than those of the existing methods. The recoveries of SA compounds spiked in egg samples ranged from 84.3% to 105.8%, with low relative standard deviations (<15%). The developed method based on the IL-MIP extraction and cleanup was successfully used in the detection of 21 SAs in more than 100 real poultry egg samples. The results indicated that the proposed method was suitable for detecting 21 SAs in poultry eggs.     

In Vivo Detection of Tetrodotoxin in Takifugu obscurus Based on Solid-Phase Microextraction Coupled with Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

Pufferfish is nutritious and delicious, but the tetrodotoxin (TTX) that may exist in its body poses a serious safety hazard. It is important to use scientific and effective methods to detect the TTX in pufferfish, but most of the existing methods require complex pre-treatment steps and have sample lethality. The solid-phase microextraction (SPME) technology can be used for in vivo detection due to its advantages such as no solvent demand, simple operation, and fast detection speed. In this study, the GO-PAN@PNE SPME fibers were made via a dipping method, and their extraction effect was verified in the TTX aqueous and spiked fish. The established method has good reproducibility, and the limit of detection of TTX in pufferfish was 32 ng·g⁻¹, and the limit of quantitation was 150 ng·g⁻¹, which can meet the detection needs of pufferfish for safe consumption. This method was used to in vivo detect the Takifugu obscurus exposed to the TTX, to determine the content of TTX in the pufferfish muscle. The detection method established in this study can relatively quickly and easily realize the in vivo detection of TTX in the pufferfish, which can provide theoretical support for improvement in the food safety level of the pufferfish.     

Fast Screening of Biomembrane-Permeable Compounds in Herbal Medicines Using Bubble-Generating Magnetic Liposomes Coupled with LC–MS

A novel strategy based on the use of bionic membrane camouflaged magnetic particles and LC–MS was developed to quickly screen the biomembrane-permeable compounds in herbal medicines. The bionic membrane was constructed by bubble-generating magnetic liposomes loaded with NH₄HCO₃ (BMLs). The lipid bilayer structure of the liposomes enabled BMLs to capture biomembrane-permeable compounds from a herbal extract. The BMLs carrying the compounds were then separated from the extract by a magnetic field. Upon heat treatment, NH₄HCO₃ rapidly decomposed to form CO₂ bubbles within the liposomal bilayer, and the captured compounds were released from BMLs and analyzed by LC–MS. Jinlingzi San (JLZS), which contains various natural ingredients, was chosen to assess the feasibility of the proposed method. As a result, nine potential permeable compounds captured by BMLs were identified for the first time. Moreover, an in vivo animal study found that most of the compounds screened out by the proposed method were absorbed into the blood. The study provides a powerful tool for rapid and simultaneous prediction of multiple biomembrane-permeable components.     

UPLC–Q–TOF–MS/MS Analysis of Phenolic Compounds from the Fruit of Cephalostachyum fuchsianum Gamble and Their Antioxidant and Cytoprotective Activities

Bamboo is a widely distributed graminaceous plant in China and is a potential source of bioactive substances. Incidentally, bamboo’s fruit is rich in phytochemicals such as polyphenols and flavonoids, which are significant to human health. In this study, we identified the phenolic compounds of the fruit and investigated the antioxidant activities of Cephalostachyum fuchsianum Gamble (CFG) fruit polyphenols with in vitro and in vivo tests for the first time. UPLC–Q–TOF–MS/MS analysis results showed that the fruit contained 43 phenolic compounds, including 7 hydroxybenzoic acids, 12 flavonoids, 7 coumarins, 10 hydroxycinnamic acids, 1 terpenoid, and 5 lignans. The TPC of SP extracts was higher than that of IBPs extracts in FP and FF. The SP extracts in FP showed better antioxidant activities in vitro compared to those in FF. In addition, polyphenols from CFG fruits protected against H₂O₂-induced oxidative damage in HepG2 cells, and the protective effect of polyphenols in FP was superior to that in FF. The analysis results showed that CFG fruit has great potential in exploiting natural chemical substances, which can provide valuable pieces of information for the further development and utilization of CFG.     

Simultaneous Analysis of Fenpropimorph and Fenpropimorph Acid in Six Different Livestock Products Using a Single-Sample Preparation Method Followed by Liquid Chromatography–Tandem Mass Spectrometry

Pesticides in livestock products must be measured to ensure food safety. We developed a single-sample preparation method followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) for simultaneous determination of fenpropimorph and fenpropimorph acid in six different livestock products. The extraction method was a modification of the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method and was validated according to the CODEX guidelines. The matrix-matched calibration curves for fenpropimorph and fenpropimorph acid exhibited good linearity, with coefficients of determination (R² values) higher than 0.998. The limit of detection (LOD) and the limit of quantitation (LOQ) were 1.25 and 5.0 µg kg⁻¹, respectively. The average recovery values ranged from 61.5% to 97.1% for samples fortified to the LOQ, 2 × LOQ, and 10 × LOQ. The method fully complied with the CODEX guidelines and was successfully applied to real samples obtained from domestic markets.     

Eco-Friendly UPLC–MS/MS Method for Determination of a Fostamatinib Metabolite,Tamatinib, in Plasma: Pharmacokinetic Application in Rats

Fostamatinib is a prodrug of the active metabolite tamatinib, which is a spleen tyrosine kinase (Syk) inhibitor used in the treatment of primary chronic adult immune thrombocytopenia and rheumatoid arthritis. A highly sensitive, rapid, reliable, and green method was developed and validated using ultra-performance liquid chromatography and tandem mass spectrometry (UPLC–MS/MS) for quantification of tamatinib in rat plasma. Ibrutinib was used as internal standard and liquid–liquid extraction was applied using tert-butyl methyl ether. The analyte was separated on an Acquity^TM CSH C₁₈ (2.1 mm × 100 mm, 1.7 µm) column using mobile phase consisting of 10 mM ammonium acetate and acetonitrile (10:90) and the flow rate was 0.25 mL/min. Electrospray ionization (ESI) was carried out in positive mode. Quantitation of tamatinib and the IS was performed using multiple reaction monitoring mode with precursor-to-product transitions of m/z 471.1 > 122.0 and m/z 441.1 > 84.0, respectively. The calibration range was 0.1–1000.0 ng/mL and the linearity of the method was ≥0.997. The developed method greenness was investigated. All principal parameters for the method, including linearity, accuracy, precision, recovery, and stability, were within acceptable ranges. Tamatinib pharmacokinetic study in rats was successfully carried out using the developed method.