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.     

Concurrent Determination of Tigecycline,Tetracyclines and Their 4-Epimer Derivatives in Chicken Muscle Isolated from a Reversed-Phase Chromatography System Using Tandem Mass Spectrometry

A quantitative and qualitative method using a high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) detection approach was developed and validated for the analysis of tigecycline, four tetracyclines and their three 4-epimer derivatives in chicken muscle. Samples were extracted repeatedly with 0.1 mol/L Na₂EDTA–McIlvaine buffer solution. After vortexing, centrifugation, solid-phase extraction, evaporation and reconstitution, the aliquots were separated using a C8 reversed-phase column (50 mm × 2.1 mm, 5 µm) with a binary solvent system consisting of methanol and 0.01 mol/L trichloroacetic acid aqueous solution. The typical validation parameters were evaluated in accordance with the acceptance criteria detailed in the guidelines of the EU Commission Decision 2002/657/EC and the U.S. Food and Drug Administration Bioanalytical Method Validation 05/24/18. The matrix-matched calibration curve was linear over the concentration range from the limit of quantitation (LOQ) to 400 μg/kg for doxycycline, and the calibration graphs for tetracycline, chlortetracycline, oxytetracycline, their 4-epimer derivatives and tigecycline showed a good linear relationship within the concentration range from the LOQ to 200 μg/kg. The limits of detection (LODs) for the eight targets were in the range of 0.06 to 0.09 μg/kg, and the recoveries from the fortified blank samples were in the range of 89% to 98%. The within-run precision and between-run precision, which were expressed as the relative standard deviations, were less than 5.0% and 6.9%, respectively. The applicability was successfully demonstrated through the determination of residues in 72 commercial chicken samples purchased from different sources. This approach provides a novel option for the detection of residues in animal-derived food safety monitoring.     

Magnetic Molecularly Imprinted Polymers for the Rapid and Selective Extraction and Detection of Methotrexatein Serum by HPLC-UV Analysis

In this work, novel selective recognition materials, namely magnetic molecularly imprinted polymers (MMIPs), were prepared. The recognition materials were used as pretreatment materials for magnetic molecularly imprinted solid-phase extraction (MSPE) to achieve the efficient adsorption, selective recognition, and rapid magnetic separation of methotrexate (MTX) in the patients’ plasma. This method was combined with high-performance liquid chromatography–ultraviolet detection (HPLC–UV) to achieve accurate and rapid detection of the plasma MTX concentration, providing a new method for the clinical detection and monitoring of the MTX concentration. The MMIPs for the selective adsorption of MTX were prepared by the sol–gel method. The materials were characterized by transmission electron microscopy, Fourier transform-infrared spectrometry, X-ray diffractometry, and X-ray photoelectron spectrometry. The MTX adsorption properties of the MMIPs were evaluated using static, dynamic, and selective adsorption experiments. On this basis, the extraction conditions were optimized systematically. The adsorption capacity of MMIPs for MTX was 39.56 mgg⁻¹, the imprinting factor was 9.40, and the adsorption equilibrium time was 60 min. The optimal extraction conditions were as follows: the amount of MMIP was 100 mg, the loading time was 120 min, the leachate was 8:2 (v/v) water–methanol, the eluent was 4:1 (v/v) methanol–acetic acid, and the elution time was 60 min. MTX was linear in the range of 0.00005–0.25 mg mL⁻¹, and the detection limit was 12.51 ng mL⁻¹. The accuracy of the MSPE–HPLC–UV method for MTX detection was excellent, and the result was consistent with that of a drug concentration analyzer.     

Preparation of Antihypertensive Drugs in Biological Matrix with Solvent Front Position Extraction for LC–MS/MS Analysis

Solvent front position extraction procedure was used to prepare biological samples containing selected antihypertensive drugs (ramipril, lercanidipine, indapamide, valsartan, hydrochlorothiazide, perindopril, and nebivolol). Substances separated from the biological matrix components (bovine serum albumin) were quantified by means of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Sample preparation process was performed with the use of a prototype horizontal chamber with a moving pipette driven by a 3D printer mechanism enabling a controlled eluent flow velocity. Application of this device was advantageous for simultaneous preparation of several samples for further quantitative analysis, with a synchronized reduction of manual operations and solvent consumption. Quantitative results obtained for the majority of the investigated antihypertensive drugs in a complex biological matrix were satisfactory. The values of the %RSD were around 5% for six of the seven substances (with the exception of indapamide). The method exhibits a suitable accuracy (the relative error percentage was below 10% for most drugs). The values of LOD and LOQ were in the range of 1.19 µg/L–8.53 µg/L and 3.61 µg/L–25.8 µg/L, respectively.     

Development and Validation of the LC–MS/MS Method for Determination of 130 Natural and Synthetic Cannabinoids in Cannabis Oil

Dietary supplements are widely available products used by millions of people around the world. Unfortunately, the procedure of adding pharmaceutical and psychoactive substances has recently been observed, in order to increase the effectiveness of supplements in the form of hemp oils. For this reason, it is extremely important to develop analytical methods for the detection of substances prohibited in dietary supplements and food products. In the present study, using the LC–MS/MS technique, an innovative method for the detection and quantification of 117 synthetic cannabinoids and 13 natural cannabinoids in dietary supplements and food products in the form of oils during one 13-min chromatographic run was developed. Each method was fully validated by characterization of the following parameters: The limit of detection was set to 0.1 ng/mL (100 µg/g, 0.01%). The limit of quantification ranged from 0.05 ng/mL to 50 ng/mL. The criteria assumed for systematic error caused by methodological bias (±20%) resulting from the recovery of analytes after the extraction process, as well as the coefficient of variation (CV) (≤20%), were met for all 130 tested compounds. The positive results of the validation confirmed that the developed methods met the requirements related to the adequacy of their application in a given scope. Additionally, methods developed using the LC–MS/MS technique were verified via proficiency tests. The developed analytical procedure was successfully used in the analysis of hemp oils and capsules containing them in the studied dietary supplements.     

Quantification of Paeoniflorin by Fully Validated LC–MS/MS Method: Its Application to Pharmacokinetic Interaction between Paeoniflorin and Verapamil

A rapid, sensitive, and specific LC-MS/MS method was developed and fully validated for the detection of paeoniflorin only in rat plasma, and applied to pharmacokinetic studies, including intravenous, multi-dose oral and combined administrations with verapamil. In this study, tolbutamide was used as the internal standard, and the protein precipitation extraction method, using acetonitrile as the extraction agent, was used for the sample preparation. Subsequently, the supernatant samples were analyzed on a Phenomenex Gemini^® NX-C18 column with a flow rate of 1.0 mL/min in a gradient elution procedure. In the extracted rat plasma, the method exhibited high sensitivity (LLOQ of 1.0 ng/mL) upon selecting ammonium adduct ions ([M+NH₄]⁺) as the precursor ions and good linearity over the concentration range of 1.0–2000 ng/mL, with correlation coefficients >0.99. The intra- and inter-batch accuracy RE% values were within ±8.2%, and the precision RSD% values were ≤8.1% and ≤10.0%, respectively. The results show that the method can be successfully applied to quantitate paeoniflorin in biological samples. Additionally, paeoniflorin is subsequently confirmed to be the substrate of the P-gp transporter in vivo and in vitro for the first time, which would be necessary and beneficial to investigate the clinical safety and efficacy of PF with other drugs in the treatment of rheumatoid arthritis.     

Determination of Dapoxetine Hydrochloride in Human Plasma by HPLC–MS/MS and Its Application in a Bioequivalence Study

Dapoxetine is used for the treatment of premature ejaculation. The present study developed an HPLC–MS/MS method to determine the levels of dapoxetine in human plasma processed using simple protein precipitation. Dapoxetine-d₇ was selected as the internal standard. The established method was performed using a mass spectrometer equipped with an electrospray ionization source in multiple positive ion reactions to monitor the mode using the precursor-to-product ion transitions of m/z 306.2–157.2 and m/z 313.2–164.2 for dapoxetine-d7 and dapoxetine, respectively. The method was evaluated based on its selectivity, linearity, limit of quantification, precision, accuracy, matrix effects, dilution integrity, stability, and extraction recovery. As a result of the model used in the present study, the validated linear ranges of dapoxetine were determined to be 2.00~1000 ng/mL in plasma, and the selectivity, precision, accuracy, dilution integrity, stability, and extraction recovery met the accepted standard. No matrix interference was observed. The method was successfully validated and applied to pharmacokinetic studies in healthy Chinese volunteers during the fasting and postprandial periods, respectively.     

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.     

Multiresidue Pesticide Analysis in Tea Using GC–MS/MS to Determine 12 Pesticide Residues (GB 2763-2021)

Pesticides are widely used on tea plants, and pesticide residues are of significant concern to consumers. The National Food Safety Standard Maximum Residue Limits for Pesticides in Food (GB 2763-2021) was recently amended. However, detection methods for pesticides newly added to the list of residues in beverages have not yet been established. For that reason, this study developed a solid-phase extraction (SPE) and gas chromatography–tandem mass spectrometry (GC–MS/MS) method for determining the residues of 12 pesticides, including four newly added, in black and green tea. Sample preparation processes (sample extraction, SPE clean-up, elution solvent, and elution volume) were optimized to monitor these residues reliably. Multiple reaction monitoring (MRM) was used for GC–MS/MS electron impact (EI) mode determination. Finally, satisfactory recoveries (70.7–113.0% for green tea and 72.0–99.1% for black tea) were achieved at three concentrations (10 μg/kg, 20 μg/kg, and 100 μg/kg). The LOQs were 0.04–8.69 μg/kg, and the LODs were 0.01–3.14 μg/kg. This study provides a reliable and sensitive workflow for determining 12 pesticide residues in tea, filling a gap in the newly revised National Standards.     

Chromatographic Determination of the Mycotoxin Patulin in 219 Chinese Tea Samples and Implications for Human Health

Patulin (PAT) is a mycotoxin, with several acute, chronic, and cellular level toxic effects, produced by various fungi. A limit for PAT in food of has been set by authorities to guarantee food safety. Research on PAT in tea has been very limited although tea is the second largest beverage in the world. In this paper, HPLC−DAD and GC−MS methods for analysis of PAT in different tea products, such as non-fermented (green tea), partially fermented (oolong tea, white tea, yellow tea), completely fermented (black tea), and post-fermented (dark tea and Pu-erh tea) teas were developed. The methods showed good selectivity with regard to tea pigments and 5-hydroxymethylfurfural (5-HMF) and a recovery of 90–102% for PAT at a 10–100 ppb spiking level. Limit of detection (LOD) and limit of quantification (LOQ) in tea were 1.5 ng/g and 5.0 ng/g for HPLC−UV, and 0.25 ng/g and 0.83 ng/g for GC−MS. HPLC was simpler and more robust, while GC−MS showed higher sensitivity and selectivity. GC−MS was used to validate the HPLC−UV method and prove its accuracy. The PAT content of 219 Chinese tea samples was investigated. Most tea samples contained less than 10 ng/g, ten more than 10 ng/g and two more than 50 ng/g. The results imply that tea products in China are safe with regard to their PAT content. Even an extreme daily consumption of 25 g of the tea with the highest PAT content (124 ng/g), translates to an intake of only 3 μg/person/day, which is still an order of magnitude below the maximum allowed daily intake of 30 µg for an adult.     

Development of a Quantitative Method for Detection of Multiclass Veterinary Drugs in Feed Using Modified QuPPe Extraction and LC–MS/MS

A method was developed for the rapid and quantitative analysis of 30 veterinary drugs belonging to 17 classes (amphenicols (1), anthelmintics (1), cephalosporins (4), coccidiostats (1), lincosamides (1), macrolide (1), nitroimidazole (1), penicillins (3), phenylhydrazines (1), polypeptides (1), pyrethrins (1), quinolones (5), sulfonamides (3), tetracycline (3), neuroleptic agents (1), triazene trypanocidal agents (1), other. (1)) in feeds. The proposed method with a modified Quick Polar Pesticides (QuPPe) sample preparation was validated for the determination of 30 veterinary drugs in feed samples by liquid chromatography triple-quadrupole mass spectrometry (LC–MS/MS). The sample was extracted with methanol containing 1% acetic acid and purified by dispersive solid-phase extraction (d-SPE) with C18. Good linearity (r² ≥ 0.98) was observed, and the LOQ values ranged from 10 to 200 µg/kg. Average recoveries ranged from 70.8 to 118.4%, and the relative standard deviation was ≤ 18.7%. This validated method was used in the determination of 30 veterinary drugs in 142 feed samples obtained from South Korea. The results show that lincomycin was present in only one of the tested feed samples, although it was detected at a value lower than the LOQ. In conclusion, this multi-residue method can be used for screening through the detection and quantitation of residual multiclass veterinary drugs in feed samples.     

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.     

Dried Plasma Spot Based LC–MS/MS Method for Monitoring of Meropenem in the Blood of Treated Patients

Meropenem (MER) is widely used to treat complicated and serious infections. Therapeutic drug monitoring (TDM) provides a valid clinical tool to avoid suboptimal concentrations and dose–related adverse reactions. However, TDM seems to face challenges since the limited stability of MER in plasma makes transport difficult between clinics and laboratories. Dried plasma spot (DPS) sampling is an attractive but underutilized method for TDM that has the desired features of easy collection, storage, and transport, and overcomes known hematocrit (HCT) issues in dried blood spot (DBS) analysis. This study was designed to investigate a DPS–based liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for quantification of MER. The method was developed and validated for DPS and wet plasma samples. Calibration curves were linear (R² > 0.995) over the concentration range of 0.5–50 µg/mL. Overall accuracy and precision did not exceed 15% and no significant matrix effect was observed. MER has been more stable in DPS than in wet plasma samples. A comparison of DPS and wet plasma concentrations was assessed in 32 patients treated with MER. The results showed that there was no significant difference between the two methods. So the DPS method developed in this study is appropriate and practical for the monitor of MER in the daily clinical laboratory practice.     

Quantification of Zonisamide in Dried Blood Spots and Dried Plasma Spots by UPLC–MS/MS: Application to Clinical Practice

In this research, a UHPLC–MS/MS method was developed and validated for the determination of zonisamide in dried plasma spots (DPS) and dried blood spots (DBS). Detection of zonisamide and internal standard, 1-(2,3-dichlorphenyl)piperazine, was carried out in ESI+ mode by monitoring two MRM transitions per analyte. Total run time, less than 2.5 min, was achieved using Acquity UPLC BEH Amide (2.1 × 100 mm, 1.7 µm particle size) column with mobile phase comprising acetonitrile–water (85:15%, v/v) with 0.075% formic acid. The flow rate was 0.225 mL/min, the column temperature was 30 °C and the injection volume was 3 µL. Desolvation temperature, desolvation gas flow rate, ion source temperature and cone gas flow rate were set by the IntelliStart software tool in combination with tuning. All of the Guthrie cards were scanned, and DPS/DBS areas were determined by the image processing tool. The influence of hematocrit values (20–60%) on accuracy and precision was evaluated to determine the range within which method for DBSs is free from Hct or dependency is within acceptable limits. The validated method was applied to the determination of zonisamide levels in DPS and DBS samples obtained from patients confirming its suitability for clinical application. Finally, the distribution of zonisamide into the red blood cells was estimated by correlating its DPS and DBS levels.     

High-Throughput Analysis of Amino Acids for Protein Quantification in Plant and Animal-Derived Samples Using High Resolution Mass Spectrometry

Current methods for measuring the abundance of proteogenic amino acids in plants require derivatisation, extended run times, very sensitive pH adjustments of the protein hydrolysates, and the use of buffers in the chromatographic phases. Here, we describe a fast liquid chromatography–mass spectrometry (LC–MS) method for the determination of amino acids that requires only three steps: hydrolysis, neutralisation, and sample dilution with a borate buffer solution for pH and retention time stability. The method shows excellent repeatability (repeated consecutive injections) and reproducibility (repeated hydrolysis) in the amino acid content, peak area, and retention time for all the standard amino acids. The chromatographic run time is 20 min with a reproducibility and repeatability of <1% for the retention time and <11% for the peak area of the BSA and quality control (QC) lentil samples. The reproducibility of the total protein levels in the hydrolysis batches 1–4 was <12% for the BSA and the lentil samples. The level of detection on column was below 0.1 µM for most amino acids (mean 0.017 µM).     

Assessment of Tissue Distribution and Metabolism of MP1, a Novel Pyrrolomycin,in Mice Using a Validated LC-MS/MS Method

MP1 is a novel marinopyrrole analogue with activity in MYCN amplified neuroblastoma cell lines. A rapid, selective, and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of MP1 in mouse plasma. Analyte separation was achieved using a Waters Acquity UPLC^®BEH C18 column (1.7 µm, 100 × 2.1 mm). Mobile phase consisted of 0.1% acetic acid in water (10%) and methanol (90%) at a total flow rate of 0.25 mL/min. The mass spectrometer was operated at unit resolution in the multiple reaction monitoring (MRM) mode, using precursor ion > product ion transitions of 324.10 > 168.30 m/z for MP1 and 411.95 > 224.15 m/z for PL-3. The MS/MS response was linear over the concentration range from 0.2–500 ng/mL for MP1, correlation coefficient (r²) of 0.988. Precision (% RSD) and accuracy (% bias) were within the acceptable limits as per FDA guidelines. MP1 was stable under storage and laboratory handling conditions. The validated method was successfully applied to assess the solubility, in-vitro metabolism, plasma protein binding, and bio-distribution studies of MP1.     

Eco-Friendly,Simple, Fast,and Sensitive UPLC-MS/MS Method for Determination of Pexidartinib in Plasma and Its Application to Metabolic Stability

Pexidartinib is the first drug approved by the U.S. Food and Drug Administration specifically to treat the rare joint tumor tenosynovial giant cell tumor. In the current study, a validated, selective, and sensitive UPLC-MS/MS assay was developed for the quantitative determination of pexidartinib in plasma samples using gifitinib as an internal standard (IS). Pexidartinib and IS were extracted by liquid-liquid extraction using methyl tert-butyl ether and separated on an acquity BEH C₁₈ column kept at 40 °C using a mobile phase of 0.1% formic acid in acetonitrile: 0.1% formic acid in de-ionized water (70:30). The flow rate was 0.25 mL/min. Multiple reaction monitoring (MRM) was operated in electrospray (ESI)-positive mode at the ion transition of 418.06 > 165.0 for the analyte and 447.09 > 128.0 for the IS. FDA guidance for bioanalytical method validation was followed in method validation. The linearity of the established UPLC-MS/MS assay ranged from 0.5 to 1000 ng/mL with r > 0.999 with a limit of quantitation of 0.5 ng/mL. Moreover, the metabolic stability of pexidartinib in liver microsomes was estimated.     

A Comparative Study on Analysis of Ginsenosides in American Ginseng Root Residue by HPLC-DAD-ESI-MS and UPLC-HRMS-MS/MS

Ginseng (Panax quinquefolius), a popular herbal and nutritional supplement consumed worldwide, has been demonstrated to possess vital biological activities, which can be attributed to the presence of ginsenosides. However, the presence of ginsenosides in ginseng root residue, a by-product obtained during processing of ginseng beverage, remains unexplored. The objectives of this study were to develop a high-performance liquid chromatography-photodiode array detection-mass spectrometry (HPLC-DAD-ESI-MS) and an ultra-high-performance-liquid-chromatography-tandem mass spectrometry (UPLC-HRMS-MS/MS) method for the comparison of ginsenoside analysis in ginseng root residue. Results showed that by employing a Supelco Ascentis Express C18 column (150 × 4.6 mm ID, particle size 2.7 μm) and a gradient mobile phase of deionized water and acetonitrile with a flow rate at 1 mL/min and detection at 205 nm, a total of 10 ginsenosides, including internal standard saikosaponin A, were separated within 18 min and detected by HPLC-DAD-ESI-MS. Whereas with UPLC-HRMS-MS/MS, all the 10 ginsenosides were separated within six minutes by using an Acquity UPLC BEH C18 column (50 × 2.1 mm ID, particle size 1.7 μm, 130 Å) and a gradient mobile phase of ammonium acetate and acetonitrile with column temperature at 50 °C, flow rate at 0.4 mL/min and detection by selected reaction monitoring (SRM) mode. High accuracy and precision was shown, with limit of quantitation (LOQ) ranging from 0.2–1.9 μg/g for HPLC-DAD-ESI-MS and 0.269–6.640 ng/g for UPLC-HRMS-MS/MS. The contents of nine ginsenosides in the ginseng root residue ranged from <LOQ-26.39 mg/g by HPLC-DAD-ESI-MS and <LOQ-21.25 mg/g by UPLC-HRMS-MS/MS, with a total amount of 38.37 and 34.71 mg/g, respectively.     

A Rapid and Sensitive LC−MS/MS Method for the Quantitation of Physalin A with Special Consideration to Chemical Stability in Rat Plasma: Application to a Pharmacokinetic Study

Physalin A is a promising natural product with excellent anti-inflammatory and anti-tumor activities. However, the pharmacokinetic profile of physalin A is still unclear. In this study, a rapid and sensitive analytical method based on LC–MS/MS for the quantitation of physalin A in rat plasma with special consideration to its chemical stability was developed and validated. To avoid the degradation of physalin A, the separation of plasma was conducted at 4 °C directly after the blood samples were collected. Meanwhile, plasma samples were immediately precipitated with acetonitrile containing tolbutamide (internal standard, IS) and the pH of the supernatant was adjusted to 1.5 with formic acid. Chromatographic separation of physalin A and IS was achieved on an ACQUITY UPLC BEH-C18 column (2.1 × 50 mm, 1.7 μm) using 0.1% formic acid and acetonitrile as mobile phase delivered at 0.3 mL/min in a gradient elution mode. Physalin A and IS were detected through negative ion electrospray ionization in multiple reaction monitoring (MRM) mode. The MS/MS ion transitions for physalin A and IS were m/z 525.1–148.9 and m/z 269.8–169.9, respectively. The developed method showed good linearity over the range of 2.00–400 ng/mL. This method was successfully applied to the pharmacokinetic study of physalin A in rats following its intragastric administration and the findings were beneficial for future studies of physalin A.     

Method of Glyphosate,AMPA, and Glufosinate Ammonium Determination in Beebread by Liquid Chromatography—Tandem Mass Spectrometry after Molecularly Imprinted Solid-Phase Extraction

The aim of this study was to develop a method for the determination of glyphosate, its metabolite aminomethylphosphonic acid (AMPA), and glufosinate ammonium residues in beebread samples, which could then be used to assess bees’ exposure to their residues. The complexity of beebread’s matrix, combined with the specific properties of glyphosate itself, required careful selection and optimization of each analysis step. The use of molecularly imprinted solid-phase extraction (MIP-SPE) by AFFINIMIP glyphosate as an initial clean-up step significantly eliminated matrix components and ensured an efficient derivatization step. Colorless beebread extracts were derivatized by the addition of 9-fluorenylmethyl chloroformate (FMOC-Cl). After derivatization, in order to remove FMOC-OH and residual borate buffer, a solid-phase extraction (SPE) clean-up step using Oasis HLB was carried out. Instrumental analysis was performed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The method was validated according to the SANTE/11312/2021 guideline at concentrations of 5, 10, and 100 µg/kg, and satisfactory recovery (trueness) values (76–111%) and precision (RSDr) ≤ 18% were obtained. The limit of quantification (LOQ) was 5 µg/kg for AMPA and glufosinate ammonium and 10 µg/kg for glyphosate. The method was positively verified by the international proficiency test. Analysis of beebread samples showed the method’s usefulness in practice. The developed method could be a reliable tool for the assessment of beebread’s contamination with residues of glyphosate, its metabolite AMPA, and glufosinate ammonium.