Intra-day Precision Research Articles

A Highly Sensitive Triple Quad LC-MS/MS Method Development and Validation for the Determination of Bexicaserin (LP352) in Human Plasma and Urine Matrices.

Bexicaserin is a highly selective agonist at the 5-HT2c receptor in clinical development for the treatment of seizures associated with developmental and epileptic encephalopathies (DEEs). We report an LC-MS/MS method for the quantitative estimation of bexicaserin in human plasma and urine. The sample preparation involves the extraction of bexicaserin and internal standard (13CD2-bexicaserin; IS) from 150 μL plasma and 50 μL urine using a solid phase extraction method. The chromatographic separation of bexicaserin and IS was achieved on a Poroshell EC-C18 column using 5 min gradient program. The calibration curve ranged from 0.1 to 100 ng/mL in plasma and 1.0 to 1000 ng/mL in urine. Intraday and interday precision and accuracy, linearity, matrix effect, extraction recovery, carry-over, dilution integrity, a battery of stability studies, and incurred sample reanalysis were performed for bexicaserin in both plasma and urine. The intraday and interday accuracy and precision were well within the acceptable limits in both plasma and urine matrices. Stability studies in plasma and urine showed that bexicaserin was stable on bench for 24 h, in autosampler over 54 h, five freeze-thaw cycles, and long-term storage at -20°C and -80°C for > 368 days. The validated methods were successfully applied in clinical study.

Accelerating isotope dilution LC-MS-based desmosine quantification for estimating elastin turnover.

Circulating total desmosine, representing endogenous systemic elastin degradation activity, is an emerging biomarker for mortality risk in several diseases and aging. However, the existing analytical method takes more than 23 hours to complete, limiting its potential applications. The objective of this study was to shorten the turnover time of a stable isotope dilution liquid chromatogram mass spectrometry-based desmosine assay. Plasma samples were analyzed using acid hydrolysis followed by solid-phase extraction and LC-MS. Two approaches to reduce assay time were tested: microwave-assisted acid hydrolysis and direct injection following solid-phase extraction. The combination of acid hydrolysis at 180°C for 8 minutes and a low-volume elution design for solid-phase extraction reduced the overall assay time to ~ 30 minutes. The assay was validated with intra-day precision and accuracy ranging from 4% to 14%, and -7% to 9%, respectively, while inter-day precision and accuracy were 0% to 9% and 1% to 3%, respectively. The assay was tested in a cohort of patients with acute aortic dissection and control subjects, where desmosine concentrations were approximately three-fold higher in patients. These results demonstrated that rapid desmosine analysis can be achieved with the use of both microwave-assisted hydrolysis and streamlined solid-phase extraction.

Unraveling the in vivo pharmacokinetic behavior of mPEG5-NH2 polymer in rats by UHPLC-MS/MS assay.

As an important chemical reagent, methoxy polyethylene glycol amine (mPEG-NH2) is widely used in biomedical field. Unraveling the pharmacokinetic behavior of mPEG-NH2 polymers is essential for revealing the toxicity and efficiency of mPEG-NH2 related drug delivery systems. In this study, a simple analytical assay based on mass spectrometry (MS) was first established and validated for quantification of mPEG5-NH2 in biological matrix. The multiple reaction monitoring (MRM) transitions at m/z 252.2 (precursor ions)→87.7 (fragment ions) and m/z 371.2 (precursor ions)→89.2 (fragment ions) were chosen to determine mPEG5-NH2 and OH-PEG8-OH, respectively. The UHPLC-MS/MS assay showed excellent linearity over the range of 0.01-10μg/mL. Intra-day and inter-day accuracies and precisions of the assay were all within±6.44%. The analytical assay was successfully applied to reveal the in vivo pharmacokinetic behavior of mPEG5-NH2 in rats.

Development and validation of a sensitive LC-MS/MS assay for determination of upadacitinib in human plasma and its application in patients with inflammatory bowel disease.

Upadacitinib is a selective Janus kinase (JAK) 1 inhibitor approved by the Food and Drug Administration for the treatment of moderate-to-severe inflammatory bowel disease (IBD). We aimed to establish and validate a method for determining Upadacitinib in patients with IBD by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The mobile phase was 0.1 % formic acid: acetonitrile (35:65, v/v) at a flow rate of 0.40 mL/min. Upadacitinib and its internal standard Upadacitinib 15N, d2 were separated by a Waters Xbridge BEH C18 column (4.6 × 100 mm, 2.5 μm) and subjected to mass analysis using positive electrospray ionization (ESI). The calibration range of Upadacitinib was 0.5-200 ng/mL with the correlation coefficient r2 ≥ 0.99. Accuracies ranged from -9.48 % ~ 8.27 % and the inter- and intra-day precisions were less than 15 % for all analytes in quality control samples. There was no significant matrix effect. The range of extraction recoveries was 87.53-93.47 % for all analytes. Twenty-one plasma samples were obtained from the sixth affiliated hospital of Sun Yat-sen University. The median plasma concentration of Upadacitinib was 7.32 (0.56-26.78) ng/mL. This newly developed method is sensitive, simple, and successfully applied in determining Upadacitinib in IBD patients to provide reference for safe and effective drug administration in clinical practice.

Fast and Sensitive Swab-Based Bioluminescent Detection Method for Meat and Chicken Microbiological Contamination Using Amydetes vivianii Firefly Luciferase

New faster and more sensitive detection methods are required for food microbiological contamination quality control throughout the entire handling process. The adenosine triphosphate (ATP) bioluminescence detection method based on the firefly luciferin–luciferase system has been a widely used approach for decades due to its practicality, efficiency, and rapidity. The luciferase of the Amydetes vivianii firefly, cloned and produced in our laboratory, displays a high bioluminescence and stability, showing desirable properties for ATP assays. Using this enzyme, in this study we developed and validated a swab-based ATP detection method for meat, chicken, and milk, allowing us to distinguish between contaminated and non-contaminated forms of these foods. This method demonstrated robust interday and intraday precision, an accuracy of 70–71% across different matrices, and a limit of detection of 1.0 × 104 cps for the dilutions and 2.7 × 103 cps for the swabs, fulfilling all validation criteria and ensuring reliability for routine applications. Except for milk, which has very low endogenous ATP levels due to pasteurization, thus requiring sample pre-processing, the method allowed us to luminometrically detect ATP on the surface of meat and chicken in less than an hour using an assay solution. The method showed higher sensitivity compared to an available commercial kit due to its intense signal, with a remarkable ~22-fold increase in luminescence intensity when comparing the highest ATP concentration of Amydetes luciferase with a commercially available luciferase, allowing for detecting microbiological contamination at lower levels or using less sensitive luminometers.

Simultaneous extraction of flavonoids and saponins from functional food via basic deep eutectic solvent-assisted mechanochemical extraction.

An efficient, green, and novel basic deep eutectic solvent (DES)-assisted mechanochemical extraction method, combined with ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, is proposed for simultaneous extraction and determination of target compounds from functional food. Several parameters are investigated using single-factor optimization, and response surface methodology is employed to determine the optimum extraction conditions. The conditions include a grinding time of 316.29s, 83.72μL of DES (K2CO3 and glycerol), and vortex time of 2.408min. The developed method provides good linearity (R2>0.9958), stable intraday and interday precision ranging from 0.07% to 3.31%, high sensitivity (LODs <20.83ng/mL), and acceptable accuracy with recoveries of 83.75%-102.02%. Moreover, satisfactory greenness (0.71) and practicality (72.5) are achieved in terms of the Analytical Greenness metric and the blue applicability grade index. The proposed method notably contributes to the simultaneous extraction and detection of flavonoids and saponins in astragali radix.

Simultaneous Quantification of Biogenic Amines and their Metabolites in Mice Tissue by Combining Ultraviolet and Integrated Pulsed Amperometric Detectors.

We developed a reversed-phased high-performance liquid chromatographic method combining ultraviolet detection and integrated pulsed amperometric detection for the simultaneous quantification of dopamine, 5-hydroxyindolacetic acid, homovanillic acid, serotonin, 3,4-dihydroxyphenylacetic acid, norepinephrine and epinephrine. All target components were completely separated in a C18 column with isocratic elution of 5% acetonitrile solution containing 8mM HClO4 and 0.20mM 1-octanesulfonic acid as an ion pairing reagent. This method showed limits of detection of 0.03-0.1ng and limits of quantification of 0.10-0.3ng with linear regression coefficients of 0.9998-1.0000. All inter-day and intra-day precision values were below 9.58%, and the average recoveries were 93.71-109.82% for mouse striatum samples. The mean levels of the seven components in striatal brain tissue in a mouse model of Parkinson's disease decreased by 2-12 times compared to those of a control group. In particular, the decrease in dopamine, 5-hydroxyindolacetic acid and 3,4-dihydroxyphenylacetic acid was statistically significant (P < 0.05). Orthogonal partial least squares discriminant analysis confirmed that the seven components are useful biomarkers. The significance of the developed method lies in its ability to simultaneously analyze seven biogenic amines related to Parkinson's disease by combining two detectors, offering a simple and cost-effective approach for clinical and biological labs.

Determination of Saccharides in Cottonseed Processing Waste Liquid and By-Products by High Performance Liquid Chromatography with Evaporative Light Scattering Detection (HPLC-ELSD)

A method utilizing high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) has been developed and validated for the simultaneous determination of raffinose, glucose, sucrose, and stachyose in waste liquid from cottonseed processing and in byproducts including cottonseed meal. The separation was performed using a carbohydrate ES column (250 × 4.6 mm, 5 μm) and isocratic elution with acetonitrile and water (70:30, v/v). The temperature of the column oven was maintained at 35 °C. The drift tube temperature for evaporative light scattering detection (ELSD) was operated 40 °C, while the nitrogen flow was set to 1.5 L·min−1. The saccharides were separated by HPLC-ELSD within 20 min. The coefficients of determination (R 2 = 0.9986–0.9999) indicate that there was a linear relationship between saccharides and the response. The limits of detection were 0.004–0.023 mg·mL−1 and the quantification range was from 0.013 to 0.080 mg·mL−1. The intraday and interday precision were from 0.05 to 0.55% and 0.20 to 0.58%, respectively. The recovery was from 102.75 ± 2.03% to 106.76 ± 2.09% and the relative standard deviations were 1.87 to 1.98%. The method exhibited good stability in determining four saccharides in cottonseed processing waste liquid and the resultant byproduct cottonseed meal. This method exhibits high separation efficiency and sensitivity, excellent precision, and simplicity, which allows determination of the high value-added product, raffinose, in cottonseed processing waste liquid and meal.

Carbonyl compounds responsive 2,4-dinitrophenylhydrazine doped polydimethylsiloxane based membrane as alternative derivatizing strategy prior to in-tube solid phase microextraction coupled with capillary liquid chromatography analysis.

In this work, a DNPH doped PDMS based membrane was developed to facilitate carbonyl compound derivatization. This membrane delivers DNPH in presence of carbonyl compounds to form hydrazones. Subsequently, the resulting hydrazones are preconcentrated, separated and detected by in-tube solid phase microextraction (IT-SPME) coupled on-line with capillary liquid chromatography (CapLC) with Uv-Vis diode array detection (DAD). The method proposed allowed the determination of the analytes up to 200 µg/L. The limits of detection were between 0.1 and 3.3 µg/L and intra-day precision and inter-day precision were lower than 7 % and 21 %. The applicability of the strategy proposed was tested in environmental (waters and PM10) and biological samples (saliva). Samples were satisfactorily analysed with recoveries in the range of 68 %-80 %. The main advantages of the proposed strategy were the increment of DNPH stability, a reduction in the solutions and wastes required for the analysis and protection of the chromatographic column minimizing the excess of DNPH, thus providing a more sustainable analytical method to estimate carbonyl compounds.

Simultaneous stereoisomeric preconcentration and determination of alkaloids and stereoisomers in Uncariae ramulus cum uncis using field enhanced sample injection and dynamic pH junction sweeping method by cyclodextrin electrokinetic chromatography.

A rapid and sensitive enrichment technique, field enhanced sample injection-dynamic pH junction-sweeping (FESI-DypH-sweeping) was successfully developed for the simultaneous separation and concentration of alkaloids and stereoisomers of Uncariae ramulus cum uncis (UR) by cyclodextrin electrokinetic chromatography (CDEKC) with diode array detection system. The sample was prepared in a low-conductivity (FESI), low-pH (DypH) sample matrix (4 mM phosphate buffer, 3% methanol, pH=3), and the background electrolyte (BGE) consisted of a high-conductivity, high-pH buffer (40 mM phosphate buffer, pH 7.0, 8 mg/mL carboxymethyl-β-cyclodextrin, and 30% methanol). The crucial parameters influencing separation and enrichment efficiency were systematically optimized using single variable method. Under the optimum conditions, the method provided satisfactory resolution for seven alkaloids within 18 minutes, achieving enrichment factors ranging from 25 to 78 folds. The limits of detections (LODs) ranged from 0.04 to 0.12 μg/mL and limits of quantifications (LOQs) ranged from 0.10 and 0.40 μg/mL respectively. Intra-day and inter-day precision, expressed as relative standard deviations, were all below 3.5%.

Development and Validation of the RP-HPLC Method for Dexamethasone Sodium Phosphate Determination in Nasal Chitosan Microsphere Preparations

The purpose of this work was to provide a robust, sensitive, accurate, and straightforward analytical method for measuring dexamethasone sodium phosphate (DSP) in chitosan microspheres preparedn using the spray drying method. DSP was quantitatively analyzed using RP-HPLC with an ultraviolet detector at 254 nm, a mobile phase that contained a mixture of acetonitrile and 0.1% sodium dihydrogen phosphate monohydrate (50:50) operating isocratically at a flow rate of 1.0 mL/min, and a stationary phase that was a C18 PrincetonSPHER-100 C18-QB 100A HPLC Column (250 × 4.6 mm, 5 um). The ICH recommendations were followed in the validation of the analytical method. DSP had a retention duration of 2.899 minutes and a tailing factor of 0.827. The RP-HPLC method was linear (R = 0.9992) in the 15-60 ug/mL concentration range. The limits of quantitation (LOQ) and detection (LOD) were 4.425 ug/mL and 1.327 ug/mL, respectively. The relative standard deviations for the intra-day and inter-day precisions were 0.057-0.876% and 0.780-0.949%, respectively. The recovery percentages at 50, 100, and 200% concentration levels were within the 99.269-100.980% range. The validated method has been successfully applied to determine DSP entrapment efficiency in chitosan microspheres. A linear, sensitive, accurate, precise, and robust technique of determining DSP in chitosan microsphere preparations is offered by the established RP-HPLC method.

Comparison of three different in vitro digestion methods for carbohydrates.

Spectrophotometer method, ELISA, and High-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) method have been widely used to quantify and characterize the glucose released from rice after in vitro digestion. Despite this, the results of the three methods may not be comparable. This work investigated the limitation of detection (LOD) and quantification (LOQ) of the glucose released after in vitro rice digestion. Intra-day and inter-day precision were evaluated through analyzing the contents of rapidly digestible starch (RDS), DS (RDS + slowly digestible starch (SDS)), and total starch (TS). Tests of accuracies were also conducted. The results demonstrated that all three methods showed good linear correlations, i.e., y = 0.3417x + 0.0799 (r2 = 0.994), y = 0.1719x + 0.0900 (r2 = 0.993), and y = 58078x + 6039.4 (r2 = 0.999), respectively. Concerning the LOD and LOQ, HPAEC-PAD showed the lowest 0.07 and 0.24mg/L, followed by the Spectrophotometer of 2.72 and 9.07mg/L. ELISA showed the highest 9.95 and 33.16mg/L. The recoveries of these methods ranged from 98.62% to 100.56%, 99.05% to 102.72%, and 96.71% to 103.27%, respectively. HPAEC-PAD method had lower contents of RDS, DS, and TS, indicating it may be poor. Combining above factors, Spectrophotometer was chosen and used in digestion of biscuits and steamed buns. RS of biscuits was the highest, possibly due to the formation of lipid-starch complex. Additionally, steamed buns had the highest RDS. These observations suggested that Spectrophotometer method was suitable for accurately and reproducibly analyzing carbohydrate digestion in different food systems.

A multi-residue method based on solid phase extraction followed by HPLC-HRMS/MS analysis for the determination of dyes and additives released from polyester fibres after degradation.

Microfibres released from textiles are one of the most common types of microplastics (MPs) found in the environment. Whether they are synthetic or natural, they can undergo degradation in different environmental matrices. This may result in the leaching of a variety of chemicals, mainly textile dyes and additives of high toxicity that need to be regulated. A novel method was developed, validated and applied to identify and quantify these types of compounds present in a hydrolytic alkaline degradation solution (neutralized), but it can also be used in similar laboratory-simulated solutions, and seawater. The employed solution was utilized in an accelerated degradation simulation of two different polyester (PES) fibre types. Thirteen compounds were extracted and quantified using a solid-phase extraction protocol followed by HPLC-HRMS/MS. Intra-day (Intra-R) and inter-day (Inter-R) precision ranged from 0.02 to 6.23 % and 0.08 to 8.85 %, respectively, while linearity (R2) values were >0.9980. The limits of detection (LOD=0.7- 3.3 ng mL-1) and quantification (LOQ=0.5- 10 ng mL-1) were determined for the proposed method. Good recoveries were obtained for all compounds studied (65-120 %), while matrix effects ranged from -6 to 30 %, depending on the analyte. Ten compounds were detected and quantified in the degradation solution of the two different polyester fibres, with three (benzothiazole, 4-nitrophenol, 2,6-dichloro-4-nitroaniline) being PES type specific, while the rest were found in both types. A non-target analysis allowed the identification of a wider range of possible leachates (55 compounds in positive ion mode and 24 in negative).

Development and validation of a quantification method for direct oral anticoagulants from capillary blood using volumetric absorptive microsampling and online SPE-LC-MS.

The number of prescriptions for new direct oral anticoagulants (DOACs) apixaban, edoxaban, rivaroxaban and dabigatran has increased exponentially in recent years, increasingly replacing the old gold standard, vitamin-K-antagonists. Due to their wide therapeutic range, therapeutic drug monitoring (TDM) is not required, although it has been proven that this could significantly reduce side effects. In order to develop a cost-efficient and simple method for the simultaneous detection of the DOACs and phenprocoumon, a new technology for sample preparation from capillary blood in the ambulant sector named VAMS® was integrated and an LC-MS detector with on-line solid phase extraction (SPE) applying a Turboflow HTLC CycloneTM 1.0x50 mm column was used. The mobile phase consisted of methanol with water (3/97 v/v) and 0.1% ammonia solution with a flow rate of 2.5mL/min. For the chromatographic separation, a Phenomenex LTD Kinetex 2.6µm C18 100Å, 100x3.0mm column with a flow rate of 0.3mL/min in gradient mode was utilized. The mobile phase consisted of acetonitrile, water and formic acid (A: 10:90:0.1 v/v and B: 95:05:0.1 v/v). The method was fully validated in the therapeutic range of the substances according to current guidelines. The LLOQ ranged from 3.5µg/L for rivaroxaban to 88µg/L for phenprocoumon and the intra-day and inter-day precision was less than 13% and 12%, while the accuracy was within a range of 85.7-113% and 88.7-106%, respectively. Samples could be stored in the Mitra® devices for at least seven days at room temperature except of dabigatran. Because the Mitras® were used, exactly 10µL of blood could be drawn and no significant haematocrit effect was observed. A reliable, simple and cost-effective extraction and analysis LC-MS method could be developed and validated. This method is therefore applicable in ambulatory care.

A non-derivatized high-performance liquid chromatography method for simultaneous quantification of hydroxyl acids and amino acids in ammonolysis reactions.

A non-derivatized high-performance liquid chromatographic (HPLC) method was developed for the simultaneous quantification of hydroxyl acids and their amination products in ammonolysis reaction mixtures. By optimizing the mobile phase composition and pH (0.04 M KH2PO4-5% methanol, pH = 2.7), complete separation of hydroxyl acids and their amination products was achieved, using lactic acid as a model substrate. Lactic acid exhibited excellent linearity within the range of 0.02 to 25 mM (R2 = 0.99968), with a relative standard deviation (RSD, n = 6) of 3.51% and a recovery ratio between 96.10% and 102.23%. Similarly, alanine exhibited good linearity from 0.02 to 50 mM (R2 = 0.99999) with an RSD of 4.04% and recovery ratios between 97.30% and 100.03%. Both analytes demonstrated good intra-day precision, with RSDs of 4.23% and 1.18%, respectively. The substrate universality tests confirmed the method's ability to rapidly and effectively separate other hydroxyl acids and their corresponding amino acids in ammonolysis reactions. The results of quantitative comparison with AQC, PITC, and OPA derivatization also proved the method to be a reliable approach for the individual or simultaneous quantification of hydroxyl acids and amino acids, offering essential support for monitoring the conversion ratio and selectivity in hydroxyl acid amination processes. Moreover, it provides valuable guidance for optimizing the amination reaction of hydroxyl groups, potentially extending catalytic advantages to other related reactions.

Optimization of HPLC method for metanephrine and normetanephrine detection in urine: Enhancing diagnostic precision for pheochromocytoma.

Catecholamines and their metabolites play critical physiological roles in the human body. Paragangliomas and pheochromocytomas are rare adrenal tumors that significantly alter catecholamine metabolism, particularly the concentrations of metanephrine (MN) and normetanephrine (NMN). This study presents the development and validation of a rapid and straightforward analytical method using reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with a photodiode array (PDA) detector for quantifying MN and NMN in 24-h urine samples. Sample preparation involved adding 1 mL of urine to a tube containing the internal standard 3-methoxy-4-hydroxy benzylamine hydrochloride (MHBA) and a 2 g/L solution of 2-aminoethyl-diphenylborinate. After vortex mixing and centrifugation, ethyl acetate was used for extraction, and the organic layer was dried under nitrogen at 50-60 °C before reconstitution in the mobile phase. Chromatographic separation was achieved on an RP C-18 column with an isocratic flow of the mobile phase (sodium dihydrogen phosphate, citric acid monohydrate, acetonitrile, and sodium octyl sulfate). Detection was performed at 347 nm, with peak identification based on standard retention times. The method was validated for linearity (10-2000 ng/mL), recovery, sensitivity, precision, accuracy, selectivity, carryover, stability, and dilution effects. It showed a strong correlation coefficient (>0.99) and accuracy within ± 15 %. Inter- and intra-day precision confirmed the method reliability. This validated technique is suitable for clinical and research applications involving catecholamine metabolite screening.

Highly Sensitive LC-MS/MS Method for Determination of Dexamethasone in Rat Plasma and Brain Tissue: An Application to Pharmacokinetic Study in Rats.

A highly sensitive and rapid LC-MS/MS method was developed and validated for the quantification of dexamethasone in rat plasma and brain tissue. Protein precipitation method was used for sample preparation. The separation of dexamethasone and the IS (labetalol) was achieved on an Atlantis dC18 column using an isocratic mobile phase (10 mM ammonium formate and acetonitrile, 25/75, v/v) delivered at 0.7 mL/min flow-rate. Dexamethasone and the IS were eluted at 1.03 and 1.06 min, respectively. The MS/MS transitions monitored were m/z 393.100 → 373.100 (dexamethasone) and 329.100 → 91.100 (IS). Method validation was performed as per FDA guidelines and all parameters met the acceptance criteria. The assay was validated with a quantification range of 0.05-1046 ng/mL in both matrices. The intraday and interday precision for were in the range of 2.62-7.28 and 2.76%-6.98% and 2.24-6.85 and 2.97%-6.37%, in plasma and brain tissue, respectively. Dexamethasone was stable in a series of stability conditions in both matrices. Post-intravenous administration to rats, dexamethasone concentrations in plasma and brain tissue were quantifiable up to 24 and 10 h, respectively. Dexamethasone half-life was ~2.30 h. Dexamethasone exhibited low clearance and moderate volume of distribution in plasma but in brain tissue the clearance and volume of distribution were high.

Simultaneous Determination of Vitamins A and E and Their Generated Metabolites in Human Serum by LC-MS/MS.

In the context of personalized and precision medicine, simultaneous monitoring of different forms of vitamins A and E and their metabolites could help us better understand the status of vitamins A and E in the body. The aim of this study was to establish a method for simultaneous determination of 13 kinds of vitamins A and E and their metabolites in human serum. Serum samples were directly detected by LC-MS/MS after deproteinization. Chromatographic and mass spectrometry parameters were optimized to achieve good separation and sensitivity for these analytes, especially for isomers. Finally, all analytes were effectively separated on Kinetex biphenyl stationary phase. The method covered a large profile of vitamins A and E and their metabolites in a run time only of 10 min. Good linearities were achieved in the quantitative range for each analyte with the correlation coefficients higher than 0.9916. The recoveries were in the range of 78.8%-111.6% with the intraday and interday precisions within 9.6%. This method was simple, sensitive, and accurate and had been successfully applied for the determination of vitamins A and E and their metabolites in human serum samples and could provide technical support for clinical nutritional evaluation of these vitamins.

Development and Validation of an Analytical Method for 2-Thiouracil, 4-Thiouracil, and 6-Methyl-2-thiouracil in Bovine Urine: A Method for Monitoring Inspections in Beef Exports to the European Union

Thiouracil (2-thiouracil) is a thyrostat used to promote weight gain in cattle. However, its use is prohibited within the European Union (EU), necessitating the monitoring of its presence in bovine urine for beef exports to the EU. In this study, we present the development and validation of a quantitative method for the determination of 2-thiouracil, 4-thiouracil, and 6-methyl-2-thiouracil in bovine urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method involves stabilizing the analytes by adding hydrochloric acid and ethylenediaminetetraacetic acid to the sample, followed by derivatization with 3-iodobenzyl bromide, cleanup with a divinylbenzene-N-vinylpyrrolidone copolymer cartridge, and subsequent LC-MS/MS analysis. The developed method was validated for determination of 2-thiouracil, 4-thiouracil, and 6-methyl-2-thiouracil in bovine urine at a concentration of 10 μg/L. The trueness ranged from 94 to 97%, with intra-day precisions below 5% and inter-day precisions below 8%. No chromatographic interference was observed near the analytes' retention times. This analytical method is particularly valuable because it can determine whether 2-thiouracil was illicitly administered or ingested via feed containing plants of the Brassicaceae family, by confirming the presence of 6-methyl-2-thiouracil or 4-thiouracil alongside 2-thiouracil in bovine urine.

Development, validation, and clinical application of LC-MS/MS method for simultaneous determination of ibrutinib, zanubrutinib, orelabrutinib, acalabrutinib, and their active metabolites in patients with B-cell lymphoma.

Bruton's tyrosine kinase inhibitors (BTKis) exhibit significant interindividual pharmacokinetics, making therapeutic drug monitoring (TDM) a promising approach for personalized therapy. However, simultaneous quantification of multiple BTKis poses technical challenges. A unified protocol for BTKis detection would be clinically desirable. Herein, we developed and validated a novel LC-MS/MS method for the simultaneous analysis of four BTKis including ibrutinib (IBR), zanubrutinib (ZAN), orelabrutinib (ORE), and acalabrutinib (ACB) and active metabolite of IBR and ACB (DIH and ACBM, respectively) in human plasma. The samples were prepared by liquid-liquid extraction using tert-butyl methyl ether. Ibrutinb-d4 (IS) was used as an internal standard. Chromatographic separation was obtained on an XBridge C18 column and connected to an LC-30AD system coupled to an API 4000+ mass spectrometer. The mobile phase comprised 10 mM ammonium acetate containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. The optimized multiple reaction monitoring transitions of m/z 441.4 → 138.3, 475.4 → 304.2, 472.5 → 455.5, 428.3 → 411.5, 466.1 → 372.2, 482.2 → 388.4, and 445.5 → 142.5 were selected to inspect IBR, DIH, ZAN, ORE, ACB, ACBM, and IS, respectively. The method exhibited linearity from 1 to 1000 ng/mL (r > 0.99) for all analytes, with intra-day and inter-day precision of 1.8 to 9.7% and accuracy below 15%. Recovery ranged from 90.4 to 113.6%, and matrix effect varied from 89.3 to 111.0%. All compounds demonstrated stability under relevant conditions. Application of the method to 57 blood samples from 18 patients demonstrated high interpatient variability, with ORE plasma concentrations ranging from 25.6 to 89.9%. The validated LC-MS/MS method provides a feasible, specific, and rapid approach for quantification of BTKis in clinical settings. Simultaneous determination of four BTKis and their metabolites in a single extraction process and chromatographic run reduces analysis time, cost, and resources. The observed variability among individuals highlights the value of TDM for personalized treatment.