Lower Limit Of Quantification Research Articles

A Smartphone Operated Intelligent Stochastic Miniplatform for On-Site Screening of Milk for Fast Determination of Ochratoxin A

Ochratoxine A is a very stable mycotoxin which cannot be destroyed below 250°C. Found in grains, coffee, grapes, wine, milk, and meat, it can produce kidney damage and cancer. Therefore, a smartphone operated intellingent stochastic miniplatform was designed, characterised, and validated for the on-site screening of milk for fast determination of ochratoxine A. (Z)-N-[2-(4-hydroxyphenyl) ethyl]octadec-9-enamide was used as modifier of a gold matrix for a screen printed stochastic sensor used as sensing tool in the design of the miniplatform. A wide linear concentration range (1.0 × 10−15 − 1.0 × 10−7 mol l‒1) and a low limit of quantification of 1 fmol l‒1 were achieved for the assay of ochratoxin A. Recovery values higher than 99.00% were obtained for ochratoxine A, when the miniplatform was used for the screening of cow’s milk and vegetarian milk.

Elevated Hepatitis B virus RNA levels in hepatocellular carcinoma patients compared to cirrhotic individuals: A propensity score matched analysis.

To delineate the levels of serum Hepatitis B virus (HBV) RNA in patients with HBV-related hepatocellular carcinoma (HCC) and study comparisons with those of individuals afflicted with cirrhosis. Adult patients diagnosed with HBV-related cirrhosis or HCC (initial diagnosis) were enrolled in the cross-sectional study. Serum HBV DNA level was quantified through a real-time polymerase chain reaction assay with a lower limit of quantification (LLQ) of 20 IU/ml. Additionally, serum HBV RNA was quantified employing RNA real-time fluorescence thermostatic amplification detection technology with LLQ of 100 copies/ml. Propensity score matching (PSM) was conducted to ensure balance in between-group confounders. A total of 187 patients (47 with HCC and 140 with cirrhosis) were recruited, among whom 140 (74.9%) had undergone antiviral therapy prior to their inclusion, with varying durations. Serum HBV RNA was detectable in 89.4% of HCC patients at the time of carcinoma diagnosis. After PSM, individuals with HCC exhibited significantly elevated levels of serum HBV DNA and HBV RNA compared to those with cirrhosis (median lgHBV RNA 3.1 vs 2.0 copies/ml, P = 0.001). Subgroup analysis, including 38 patients who exhibited ultrasensitive HBV DNA negativity, revealed similar results (median lgHBV RNA 3.0 vs 0.0 copies/ml, P < 0.001). Serum HBV RNA levels were significantly higher in HBV-related HCC patients compared to cirrhotic patients. The presence of serum HBV RNA positivity or elevated levels was associated with the onset of HCC.

Randomized, double-blind, phase 1a single-ascending dose and food effect studies assessing safety and pharmacokinetics of EC5026 in healthy volunteers.

Chronic pain represents a significant unmet medical need, affecting one-fifth of the U.S. population. EC5026 is a small molecule inhibitor of the enzyme soluble epoxide hydrolase (sEH) which is being developed as a novel non-opioid, non-NSAID analgesic. EC5026 prolongs the action of epoxy fatty acids, endogenous analgesic lipid mediators that are rapidly metabolized by sEH. We evaluated the safety and pharmacokinetic profile of EC5026 in two phase I trials, a single-ascending dose (SAD) study and a fed-fasted study. The SAD study evaluated EC5026 doses ranging from 0.5 to 24 mg in healthy volunteers. EC5026 was well tolerated. No treatment-emergent adverse events were considered related to EC5026. No apparent treatment- or dose-related trends in laboratory results, vital signs, physical examinations, or electrocardiograms were observed. A linear, near-dose-proportional increase in exposure was observed with progressive doses in the SAD study; plasma exposure was below or near the lower limit of quantification after 0.5-2 mg doses. Mean half-lives ranged from 41.8 to 59.1 h. for doses of 8-24 mg, supporting a once-daily dosing regimen. In the fed-fasted study using 8 mg EC5026 tablets, higher peak concentrations (66%) and total exposures (53%) were observed under the fed condition. Plasma concentrations declined in a monoexponential manner with mean half-lives of 59.5 h. in the fed state and 66.9 h. in the fasted state. Future clinical trials using EC5026 for the treatment of pain are justified based on the favorable outcomes from both clinical trials along with preclinical evidence of analgesic activity.

Generic Enrichment Method for Liquid Chromatography-Multiple Reaction Monitoring-Mass Spectrometry Assay for Quantitative Measurement of Biological Therapeutics in Serum.

The study aims to leverage the capabilities of Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry (LC-MRM), a key technique in quantifying therapeutic proteins in pharmacokinetic studies. The focus is on demonstrating an enrichment method using ProteoMiner beads, which can be integrated with LC-MRM to detect low-abundance biotherapeutics in serum, such as monoclonal antibodies and gene therapy products. The ProteoMiner enrichment method was employed and integrated with LC-MRM. The lower limit of quantification of serum drug substance concentrations was compared with that achievable with immuno-enrichment. The method used commercially available reagents, eliminating the need for assay-specific antibodies and reducing potential bias and development time. The ProteoMiner enrichment method showed comparable performance to immuno-enrichment, meeting traditional assay requirements in terms of precision, accuracy, and specificity. The ProteoMiner enrichment method, when combined with LC-MRM, offers a reliable and efficient alternative to immuno-enrichment for detecting and quantifying low-abundance biotherapeutics in serum. This approach, which uses commercially available reagents, can eliminate the bias and time associated with the development of assay-specific antibodies. It holds significant potential for accelerating pharmacokinetic analysis in both early and late stages of pharmaceutical development.

Occurrence and health risk assessment of PFAS and possible precursors: a case study in a drinking water treatment plant and bottled water (south Catalonia, Spain).

The presence of PFAS in drinking water may pose a serious threat to human health. This study aims to determine the levels of these compounds and their precursors in water samples from a drinking water treatment plant (DWTP) located in l'Ampolla (Spain) and to assess their fate. Additionally, ten Spanish bottled waters were analyzed to compare the occurrence of PFAS in the mentioned matrices and in drinking water. Off-line solid phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was applied to determine 26 PFAS and PFAS precursors after a total oxidizable precursor assay. The analytical method presents low quantification limits (0.25-5ng/L). A total PFAS concentration of up to 16ng/L in all the DWTP samples was obtained, and 6:2 FTS was the only precursor detected. Results are close to the quantification limits, resulting in a high degree of uncertainty, and for this, it is difficult to evaluate the DWTP PFAS removal efficiency. Regarding bottled water, total PFAS concentration found was up to 12ng/L in one of ten samples, with no precursors detected. Exposure assessment revealed that there is no risk associated with the ingestion of the samples analyzed. Moreover, there were no differences in terms of risk between drinking water from l'Ampolla DWTP and bottled water.

Efficient extraction and HPLC determination of sulfonamide residues in milk samples using acylated graphene oxide-functionalized polyurethane sponges: A tailored approach for efficient analyte recovery

This study reports a novel surface modification of polyurethane sponge (PUS) with acylated graphene oxide (AGO), yielding a versatile sorbent material (AGO@PUS) for the efficient extraction of sulfonamide residues from milk samples. The AGO@PUS combines the extractive properties of AGO with the advantages of PUS, enabling easy, fast, cost-effective, and efficient sample preparation. The material was characterized using FTIR, SEM, XRD and the extraction protocol was optimized for the determination of three sulfonamides (SGD, SDS, and SDZ) by HPLC-UV. The proposed method exhibited excellent linearity (0.01–1000 µg mL−1), low limits of detection (0.04–0.06 µg mL−1) and quantification (0.13–0.21 µg mL−1), and high coefficients of determination (R2 = 0.993–0.997). The maximum recovery rate reached 99.63 %, demonstrating the exceptional sorption capacity of AGO@PUS. Notably, the clear extracts obtained with this material highlight its potential for efficient sample preparation and analysis.

Exploiting the Applicability of Polytetrahydrofuran-Modified Polyester for the Fabric Phase Sorptive Extraction of Doxycycline from Human Urine.

In this report, a polytetrahydrofuran-coated polyester fabric phase sorptive extraction (FPSE) for the determination of doxycycline in human urine was described. The sol-gel polytetrahydrofuran sorbent proved to be superior against other sol-gel coated cellulose and polyester membranes tested. The effect of the extraction parameters including membrane surface area, sample pH and volume, salt concentration, extraction time, stirring rate, etc., on the extraction efficiency of the analyte was studied using the "one-factor-at-a-time" (OFAT) and Box-Behnken design approaches. The analytical method proposed was validated in compliance with FDA guidelines for bioanalytical procedures. The method was linear in the determination range of 100-5000 ng/mL with the determination coefficient of 0.9953. The limit of detection (LOD) and the lower limit of quantification for doxycycline was 17 and 100 ng/mL, respectively. The relative recoveries for intra-day and inter-day studies ranged from 98.5-112.2% and 89.6-96.8%, respectively. The relative standard deviation was lower than 14.7% in all cases, exhibiting good precision. The sol-gel polytetrahydrofuran-modified FPSE membranes were reusable for at least 30 times. The greenness of the developed method was evaluated using Sample Preparation Metric of Sustainability (SPMS) and Blue Applicability Grade Index (BAGI) metric tools. Finally, the analytical scheme was successfully employed for the quantitation of urinary doxycycline collected at various time points following the administration of doxycycline-containing tablets.

Novel Technique for Simultaneous Ethylene Glycol and Its Metabolites Determination in Human Whole Blood and Urine Samples Using GC-QqQ-MS/MS.

Toxicological analyses often necessitate the identification of compounds belonging to diverse functional groups. For GC-MS analyses, derivatization of compounds belonging to different functional groups can pose a challenge and requires the development of comprehensive methods of analysis. One example could be ethylene glycol, whose widespread use is related to possible unintentional or suicidal intoxications. This fact clearly indicates the need to develop sensitive methods for the determination of ethylene glycol and its metabolites in biological material, as only such complex analysis allows for proper toxicological expertise. A simultaneous GC-QqQ-MS/MS method for the determination of ethylene glycol together with its metabolites, glyoxal and glycolic acid, as well as the detection of glyoxylic acid and oxalic acid, was developed and fully validated. A novel approach for simultaneous derivatization of substances from different groups (alcohols, aldehydes, and carboxylic acids) was established. Sample preparation included the addition of three internal standards (BHB-d4, ethylene glycol-d4 and methylglyoxal), precipitation with acetonitrile and subsequent derivatization with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), as well as pentafluorophenylhydrazine (PFPH). Detection was carried out with the use of triple quadrupole mass spectrometer. The ionization method was electron impact, and quantitative analysis was carried out in multiple reaction monitoring mode. The lower limit of quantification was 1 μg/mL, 0.1 μg/mL, and 500 μg/mL for ethylene glycol, glyoxal, and glycolic acid, respectively. The presented method was applied in three authentic postmortem cases of ethylene glycol intoxication.

Metabolic signatures of metabolites of the purine degradation pathway in human plasma using HILIC UHPLC–HRMS

The metabolic disorders in the purine degradation pathway have proven to be closely associated with several human diseases. However, the etiology is not yet fully understood. Profile assay of purine intermediates and uric acid involved in the metabolic pathway can provide additional insight into the nature and severity of related diseases. Purine metabolites are endogenous chemicals with high hydrophilicity, polarity, and similar structures, thus there is a great need for a specific method to quantify them directly in biological fluids with a short running time. Herein, eight purine degradation pathway metabolites, including xanthine, hypoxanthine, guanine, xanthosine, inosine, guanosine, adenosine and uric acid, in human plasma were quantitatively measured using hydrophilic interaction chromatography-tandem high-resolution mass spectrometry (HILIC-HRMS) in a short running time of 10 min. The method was systematically validated for specificity, linearity of the calibration curve, the limit of detection, the limit of quantification, the lower limit of quantification, precision, accuracy, extraction recovery, matrix effect, and stability. The results showed that the method was linear (R2 > 0.99), accurate (the intra- and inter-day recoveries of all analytes ranged from 90.0 % to 110.0 %), and precise (the intra- and inter-day precisions were less than 6.7 % and 8.9 %, respectively) with the lower limits of quantification ranging from 3 to 10,000 ng/mL. The extraction recoveries and matrix effects were repeatable and stable. All the analytes were stable in the autosampler and could be subject to three freeze-thaw cycles. The developed method was ultimately applied to 100 plasma specimens from healthy individuals. The results showed that the concentrations of different purine metabolites varied dramatically in plasma specimens. Diet and body mass index (BMI) were the most significant factors determining purine levels, followed by drinking and sex. Age, smoking and bedtime showed a very weak correlation with purine metabolism. The findings of the present work reveal the characteristics of purine metabolism in human plasma under non-pathological conditions. The results also highlight the factors that can cause changes in purine metabolism, which are useful in developing effective treatment strategies for metabolic disorders of purines, particularly for those caused by lifestyle factors.

Improving the Sensitivity of Protein Quantification by Immunoaffinity Liquid Chromatography─Triple Quadrupole Mass Spectrometry Using an Iterative Transition Summing Technique.

The desire to reach ever-diminishing lower limits of quantification (LLOQ) to probe changes in low abundance protein targets has led to enormous progress in sample preparation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrumentation. To maximize signal and reduce noise, many approaches have been employed, including specific immunoaffinity (IA) enrichment and reducing the LC flow to the nanoflow (nLC) level; however, additional sensitivity gains may still be required. Recently, a technique termed "echo summing" has been described for small-molecular-weight analytes on a triple quadrupole (QqQ) MS where multiple iterations of the same, single selected reaction monitoring (SRM) transition are collected, summed, and integrated, yielding significant analyte dependent signal-to-noise (S/N) improvements. Herein, the direct applicability of echo summing to protein quantification by sequential IA combined with nLC-MS/MS (IA-nLC-MS/MS) is described for a beta nerve growth factor (NGF) and a soluble asialoglycoprotein receptor (sASGPR) assay from human serum. Five iterations of echo summing outperformed traditional collection in relative average accuracy (-1.5 ± 7.7 vs -41.7 ± 10.7% bias) and precision (7.8 vs 18.4% coefficient of variation (CV)) of the low-end quality control (QC) sample (N = 4) for NGF and improved functional sensitivity of serially diluted serum QC samples (N = 5 each population) approximately 2-fold (1.96 and 2.00-fold) for two peptides of sASGPR. Echo summing also extended the minimum quantifiable QC level for sASGPR 4-fold lower. Similar gains are believed to be achievable for most protein IA-nLC-MS/MS assays.

Quantitative determination of phenazepam and its active metabolite in human blood plasma at different extraction procedures

Introduction. The presence of the active metabolite (3-hydroxyphenazepam, 3-OH-PHEN), the wide interindividual variability of the therapeutic effect of phenazepam (PHEN), as well as its active moiety in the blood, determine the relevance of therapeutic drug monitoring (TDM). To do this, the researcher must have an express analytical technique with a wide analytical range, a low limit of quantification (LLOQ), and with robustness with different sample preparation methods.Aim. Development and validation of quantitative methods for PHEN and 3-OH-PHEN in human blood plasma with different sample preparation methods.Materials and methods. The determination of PHEN and 3-OH-PHEN has been performed by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Solid-phase extraction (SPE) and liquid extraction with support (SLE) otherwise called liquid-liquid extraction in the solid phase were used for sample preparation. Metoprolol was utilized as an internal standard (IS). Gradient elution profile between mobile phase A (0.2 % aqueous formic acid) and B (100 % acetonitrile) has been used. Column: Hypersil GOLD® C18, 50 × 2.1 mm, 3.5 μm.Results and discussion. Two methods have been developed for the quantitative determination of PHEN and 3-OH-PHEN in human blood plasma using different sample preparation methods: SPE and SLE. The conditions of chromatographic separation and mass spectrometric detection of the analytes are selected. The following validation characteristics were determined for both methods: selectivity, calibration curve, accuracy, precision, degree of extraction, LLOQ, carry-over effect, matrix factor, stability of standard solutions and analyte in the matrix.Conclusion. The validation results of the developed methods meet the established criteria, which allows them to be used for the quantitative determination of PHEN and 3-OH-PHEN in human blood plasma. The wide analytical range for both methods 1–1000.00 ng/ml allows the use them for pharmacokinetics and bioequivalence studies, as well as in toxicology.

Efficient Green Solvent Extraction and Bioassay Studies of Favipiravir in Rat Dried Blood Spots Using Ionic Liquid-Based Dispersive Liquid-Liquid Microextraction (IL-DLLME) Coupled with HPLC-PDA: Application to Bioequivalence Studies

Background: Favipiravir is an antiviral drug having pyrazine group moiety. It is a reliable and an efficient green solvent, and a highly recoverable bio-sampling method is required for it. It is widely used in COVID-19 treatment for the prevention of the spread of viral infections in the body Objective: This proposed green solvent (ionic liquid)-based bioassay study aimed to quantify favipiravir in rat-dried blood spots using Dispersive Liquid-liquid Microextraction (IL-DLLME). Methods: The proposed bioassay separation was achieved through an isocratic elution mode using a hybrid silica-based ODS column (250 × 4.6 mm; 5 μm), a column temperature of 25°C, an injection volume of 10 μL, a flow rate of 1.0 mL/min, and a detector wavelength set at 310 nm. The run time was less than 10 minutes. Mobile phase was delivered with acetonitrile, methanol, and 10 mM Na2HPO4 at pH 4.0 ± 0.5, (15: 20: 65, v/v/v). In a microtube, 50 mL of Ionic Liquid (IL), 500 μL of disperser ACN, 50 μL of 10% NaCl, and IS (20 ng mL-1) were added to perform the Dried Blood Spot (DBS) sample extraction methodology. The advantages of the proposed methodology have been found to include minimum hematocrit effect and an adequate blood volume for testing, easy transportation, and significant extraction recovery. The sample analysis has been carried out using HPLC-PDA with oseltamivir used as an internal standard. Results: We have investigated the important variables, i.e., salt concentration (10% NaCl, analyte recovery being higher) and disperser solvent [50μL of BMIHP plus 500 μL of ACN (v/v) yielding the highest recovery], in the extraction process. Extraction Recovery (ER) and Enrichment Factor (EF) were also evaluated using the IL-DLLME method. The calibration curve examined a range of 0.5-150 μg/mL, with a lower Limit of Quantification (LOQ) being 0.5 μg/mL in QC as well as calibration samples, respectively. Conclusion: Significant bioanalytical validation has been performed and all the parameters have been evaluated systematically as per bioanalytical method validation protocols, i.e., US FDA- 2018 guidelines. The following analytical parameters have been covered: standard curve, limit of quantification, range, recovery, stability, accuracy, precision, sensitivity, ER, EF, and selectivity. The developed bioassay method has been successfully applied in the pharmacokinetic studies of rats and successfully applied in bulk drugs.

Nano Fe-Al-bi-MOF-based dispersive micro solid phase extraction of diazinon, acetochlor, hexaconazole, oxadiazon, fenitrothion, haloxyfop-R-methyl, diniconazole, and tebuconazole from fruit juices

This research presents an analytical technique based on nano Fe-Al-bi-MOF designed for the extraction and preconcentration of some pesticides from fruit juices. The MOF was synthesized using iron and aluminum salts along with 1,4-benzenedicarboxylic acid. The obtained sorbent was subjected to a series of characterization techniques to accurately determine the phase and characteristics of the material. The MOF (mg amount) was added in the fruit juices for extracting the targeted pesticides. The mixture was agitated using a vortex mixer to enhance the analytes sorption onto the sorbent, followed by centrifugation to facilitate settling down the analyte-loaded particles. Subsequently, the sorbent particles were subjected to elute with mL-volume of acetonitrile. Ultimately, the resulting eluate was mixed with a µL-volume of 1,2-dibromoethane and rapidly injected into deionized water. The milky solution underwent the process of centrifugation and sedimentation in order to separate the organic phase. An appropriate portion of it was injected into a gas chromatograph equipped with a flame ionization detector. Low limits of detection (0.53–2.59 µg L−1) and quantification (1.75–8.57 µg L−1), high enrichment factors (295−350), appreciable extraction recoveries (59–70 %), and the use of three-dimensional nano MOF in the analytical method development are the highlights of the research.

Metabolic profiling and pharmacokinetic studies of alkamides, a pair of cis-trans isomers N-isobutyl-2E,4E,8Z,10E/Z-dodecatetraenamide, from Asari Radix et Rhizoma by UHPLC-Q/TOF-MS and UHPLC-MS/MS

Cis-trans isomers of N-isobutyl-2E,4E,8Z,10E-dodecatetraenamide (DDA-E) and N-isobutyl-2E,4E,8Z,10Z-dodecatetraenamide (DDA-Z) are representative alkamides with numbness of tongue, anti-inflammatory and analgesic activities of Asari Radix et Rhizoma. However, their respective metabolic pathways and pharmacokinetic behaviors are still unknown. This study aim to investigate the metabolism of the two alkamides in vitro and in vivo using ultra-high-performance liquid chromatography-quadruple-time-of-flight mass spectrometry. Furthermore, a rapid, sensitive, and selective ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed to quantify DDA-E/Z in rat plasma. Results indicated that DDA-E and DDA-Z showed significant differences in metabolism and pharmacokinetics. Across all samples, 24 metabolites of DDA-E and 21 metabolites of DDA-Z were detected. A variety of pathways were involved in the production of these metabolites, mainly hydroxylation and oxidation. The linear range of DDA-E/Z was 1–2500 ng/mL (R2 = 0.9984), and the lowest quantification limit was 1 ng/mL. Precision, accuracy, extraction recovery, matrix effect, and stability of DDA-E/Z were within acceptable limits. Pharmacokinetic research was conducted using male Sprague-Dawley rats receiving intravenous (1 mg/kg) or intragastric (40 mg/kg) administration of DDA-E or DDA-Z solution. There was a calculated absolute bioavailability of 15.67 % for DDA-E and 4.83 % for DDA-Z when consumed orally. The apparent volume of distribution of intravenous and intragastric administrations were 4.44 ± 0.41 L/kg and 5.18 ± 0.67 L/kg for DDA-E, and 1.56 ± 1.66 L/kg and 2.35 ± 0.42 L/kg for DDA-Z. The maximal plasma concentrations of DDA-E and DDA-Z were 599.84 ± 149.92 nM and 422.09 ± 69.17 nM, and the time to maximum peak were 4.33 ± 3.51 h and 0.70 ± 1.12 h, respectively. In conclusion, in subsequent pharmacodynamics and safety evaluation studies, great attention should be paid to the metabolic characteristics and pharmacokinetic differences between DDA-E and DDA-Z.

Cortisone in saliva of pigs: validation of a new assay and changes after thermal stress

BackgroundCortisone is derived from cortisol through the action of the enzyme 11β-hydroxysteroid dehydrogenase type II, and it has gained importance in recent years as a biomarker of stress. This study aimed to develop and validate an assay for the measurement of cortisone in pig saliva and evaluate whether its concentration varies in stressful situations. For this purpose, a specific immunoassay was developed and validated analytically, and a study was performed to evaluate whether cortisone concentrations in saliva can vary under heat stress conditions.ResultsThe assay proved to be accurate, reliable, and sensitive for the measurement of cortisone in pig saliva. The limit of detection of the assay was set at 0.006 ng/ml, and the lower limit of quantification was 0.023 ng/ml. It also correlated significantly with the results obtained by LC‒MS/MS (P = 0.003; r = 0.64). In addition, the cortisone concentration in animals subjected to prolonged heat stress decreased significantly 15 days after treatment (P < 0.0001).ConclusionsAccording to these results, cortisone measured by this assay could be used as a tool for the non-invasive evaluation of thermal stress in pig saliva.

Pharmacokinetics and Bioavailability Study of a Novel Smoothened Inhibitor TPB15 for Treatment of Triple-Negative Breast Cancer in Rats by High Performance Liquid Chromatography-Mass Spectrometry.

Smoothened (SMO), a key component of the hedgehog signaling pathway, represents a therapeutic target for triple negative breast cancer(TNBC), yet the chemotherapy response rate in TNBC patients is only 40-50%, underscoring the urgent need for the development of novel drugs to effectively treat this condition. The novel compound TPB15, an SMO inhibitor derived from [1,2,4] triazolo [4,3-α] pyridines, demonstrated superior anti-TNBC activity and lower toxicity compared to the first SMO inhibitor vismodegib in both in vitro and in vivo. However, the compound's pharmacokinetic properties remain unclear. The present work aims to develop a simple HPLC-MS/MS method to profile the pharmacokinetics and bioavailability of TPB15 in rats as a ground work for further clinical research. Separation was performed on an Agilent ZORBAX StableBond C18 column by gradient elution using acetonitrile and 0.1% formic acid as mobile phase at a flow rate of 0.3mL/min. Multiple reaction monitoring(MRM) in positive mode with the transitions of m/z 454.2 → 100.0, 248.1 → 121.1 was employed to determine TPB15 and internal standard tinidazole, respectively. The specificity, intra- and inter- day precision and accuracy, extraction recovery, stability, matrix effect, dilution integrity and carryover of the method was validated. The pharmacokinetics and bioavailability studyof TPB15 were carried out on rats through intravenous injection at the dose of 5 mg/kg and oral gavage at the dose of 25 mg/kg, and the pharmacokinetics parameters were calculated by the non-compartment analysis using the pharmacokinetics software DAS 2.1.1. The values of specificity, intra- and inter- day precision and accuracy, extraction recovery, stability, matrix effect, dilution integrity and carryover satisfied the acceptable limits. The lower limit of quantification of this method was 10 ng/mL with a linear range of 10-2000ng/mL. The validated method was then applied to pharmacokinetics and bioavailability studies in rat by dosing with gavage (25mg/kg) and intravenous injection(5mg/kg), and the oral bioavailability of TBP15 in rat was calculated as 16.4 ± 3.5%. The pharmacokinetic parameters were calculated as following: maximum of plasma concentration (Cmax) (PO: 2787.17 ± 279.45µg/L), Time to maximum plasma concentration (Tmax) (PO: 4.20 ± 0.90 h), the area under the concentration-time curve 0 to time (AUC0-t) (PO: 17,373.03 ± 2585.18 ng/mL·h, IV: 21,129.79 ± 3360.84ng/mL·h), the area under the concentration-time curve 0 to infinity (AUC0-∞) (PO: 17,443.85 ± 2597.63 ng/mL·h, IV: 17,443.85 ± 2597.63ng/mL·h), terminal elimination half-life (t1/2) (PO: 7.26 ± 2.16h, IV: 4.78 ± 1.09h). TPB15, a promising candidate for treating TNBC, hasdemonstrated outstanding efficacy and safety in vitro and in vivo. This study established a simple, sensitive, and rapid HPLC-MS/MS bioanalytical method, developed and validated in accordance with FDA and EMA guidelines, for conducting pharmacokinetic and bioavailability studies of TPB15. The results revealed a favorable pharmacokinetic profile owing to its long t1/2. Nevertheless, the next phase of research should include formulation screening to enhance bioavailability, as well as clinical trials, metabolism pathway analysis, and assessment of potential drug-drug interactions.

Development of Zeolite Imidazole Framework-Based Adsorbent for Effective Microextraction and Preconcentration of Histamine in Food Samples.

This study is the first to focus on the preconcentration and determination of histamine (HIS) in food samples using zeolite imidazole frameworks (ZIFs) on a solid-phase microextraction (SPME) platform. ZIF was developed on a polypropylene hollow fiber (PPHF) substrate (ZIF@PPHF) and characterized. The extraction performance was optimized by adjusting several parameters, including pH, contact time for adsorption, and desorption conditions. Under the optimized conditions, a wide linear dynamic range (0.05-250 mg/L) with high R2 values (0.9989), low limit of detection (0.019 mg/L), and low limit of quantification (0.050 mg/L) were determined as analytical figures of merit. Additionally, a reusability study confirmed that ZIF@PPHF preconcentrated 83% of the HIS up to the fourth cycle. The developed method was used to preconcentrate HIS in fish and cheese samples. The spiked real samples confirmed the validity and accuracy of this method. The percentage mean recoveries ± relative standard deviation (% RSD, n = 3) at the concentration levels of 5, 10, and 50 mg/L of HIS and the sample amount of 5 g for intra- and inter days ranged from 97 ± 1.10 to 102.80 ± 0.90 and from 96.40 ± 1.82 to 103.40 ± 0.79, respectively. The results suggest that the analytical method validation parameters were acceptable, indicating the repeatability and sensitivity of the method.

Phosphatidylethanol is a promising tool for screening alcohol consumption during pregnancy.

Prenatal alcohol exposure (PAE) is one of the leading causes of preventable developmental disabilities. A lack of objective screening methods results in an under-recognition of the phenomenon. Phosphatidylethanol (PEth) is a specific ethanol biomarker that reveals alcohol intake up to several weeks after alcohol use. So far, PEth has mostly been a tool for detecting moderate and heavy drinking. With lower PEth cut-offs, revealing even minor prenatal alcohol consumption is possible. We aimed to find out if a sensitive method for PEth analysis would give additional information about PAE and to assess the cut-off value for a positive alcohol result in prenatal screening. The study was an observational study of 3000 anonymous blood samples collected from the Helsinki University Hospital Diagnostic Center between June and September 2023. The Finnish Red Cross Blood Service received the samples originally for blood group typing and antibody screening as part of the prenatal blood screening program. We developed a sensitive PEth 16:0/18:1 analysis method using ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) equipment after liquid-liquid extraction of PEth from whole blood. The lower limit of quantification was 1 ng/mL. PEth was ≥2 ng/mL in 5.2% of the cases, ≥8 ng/mL in 2.0%, and ≥20 ng/mL in 1.0%. The detection time of PEth can be several weeks, especially with low PEth concentrations and after heavy alcohol consumption. It remained unknown whether the positive PEth tests resulted from drinking deliberately during pregnancy or before pregnancy recognition. We suggest adding PEth 16:0/18:1 to a routine prenatal blood screening program with a cut-off of 2 ng/mL-and in positive cases, clinical evaluation and retesting in 2-4 weeks. In clinical settings, information on gestational week and alcohol consumption before pregnancy is relevant and needs to be considered when interpreting low PEth concentrations.

Quantitative analysis of nonsteroidal anti-inflammatory drugs in dried blood spot from mountain ultra-trail runners. Contribution of pharmacokinetic models for the interpretation of the results.

Monitoring of drug use in athletes is of interest both for health and competition-related issues. Considering the advantages of Dried Blood Sampling (low invasiveness, easy sampling, long term storage), we have validated a quantitative LC-MS/HRMS method for the screening of 16 nonsteroidal anti-inflammatory drugs. For all drugs, accuracy and imprecision were within 15% for the 3 levels of quality control and lower than 20% for the lower limit of quantification. Application was performed from samples obtained for Ultra-Trail du Mont-Blanc® 2021 and 2022. A focus on ibuprofen and its metabolites (hydroxyibuprofen, carboxyibuprofen, ibuprofen glucuronide and hydroxyibuprofen glucuronide) was made because the results showed that it was the most detected nonsteroidal anti-inflammatory drug. Further, an interpretation of the ibuprofen concentrations was proposed either from experimental data obtained after an intake of ibuprofen by 10 control subjects, or from a pharmacokinetic modelling and simulations. Depending on the analytical performances of the method, we proposed possible detection windows for ibuprofen in runners. The pharmacokinetic model made it possible to consider two scenarios with and without modification of the total clearance of ibuprofen linked to a modification of the pharmacokinetics of the drugs due to the practice of a long and intense physical activity.

Pharmacokinetics of two triterpenoid saponins and three flavonoids in Astragalus membranaceus leaves by UHPLC-MS/MS

Astragalus membranaceus (A. membranaceus) leaves can be used both as a medicine and food material. Their main chemical components are flavonoids and triterpenoid saponins. The pharmacokinetics of A. membranaceus leaves are rarely reported in the literature. This study aimed to investigate the pharmacokinetics of five major bioactive components of A. membranaceus leaves [rhamnocitrin 3-glucoside (RCG), tiliroside (TIL), rhamnocitrin 3-neohesperidoside (RNH), huangqiyenin R (HuR), and huangqiyenin I (HuI)]. Simultaneously using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. The extract of A. membranaceus leaves was administered orally to rats, and the rat plasma was subjected to a fast, sensitive, and specific UHPLC-MS/MS method. Butylparaben served as the internal standard. The plasma samples were pretreated using isopropanol/ethyl acetate (1:1, v/v) liquid–liquid extraction. Chromatographic separations were performed at a flow rate of 0.3 mL/min on a Waters ACQUITY HSS T3 Column (2.1 mm × 100 mm, 1.8 μm) using mobile phases of 0.1 % formic acid/water and 0.1 % formic acid/acetonitrile. Mass spectrometry detection was performed using an electrospray ionization ion source in the negative-ion mode and the multiple reaction monitoring mode. All analytes had an intraday and interday relative standard deviation of less than 14.10 %. The range of accuracy was –11.94–6.920 % and –15.22–5.800 %. The lower limits of quantification for RCG, TIL, RNH, HuR, HuI was 10.24, 10.27, 10.12, 5.137, and 5.841 ng/mL, respectively. The criteria were met by stability, matrix effects, and extraction recovery. The pharmacokinetic parameters of A. membranaceus leaf extract were ultimately obtained using this analytical method. The study provides a theoretical basis for future pharmacological research, clinical application, and development of healthy food from A. membranaceus leaves.