Application of Proteomics Technology Based on LC-MS Combined with Western Blotting and Co-IP in Antiviral Innate Immunity.

As an interferon-stimulating factor protein, STING plays a role in the response and downstream liaison in antiviral natural immunity. Upon viral invasion, the immediate response of STING protein leads to a series of changes in downstream proteins, which ultimately leads to an antiviral immune response in the form of proinflammatory cytokines and type I interferons, thus triggering an innate immune response, an adaptive immune response in vivo, and long-term protection of the host. In the field of antiviral natural immunity, it is particularly important to rigorously and sequentially probe the dynamic changes in the antiviral natural immunity connector protein STING caused by the entire anti-inflammatory and anti-pathway mechanism and the differences in upstream and downstream proteins. Traditionally, proteomics technology has been validated by detecting proteins in a 2D platform, for which it is difficult to sensitively identify changes in the nature and abundance of target proteins. With the development of mass spectrometry (MS) technology, MS-based proteomics has made important contributions to characterizing the dynamic changes in the natural immune proteome induced by viral infections. MS analytical techniques have several advantages, such as high throughput, rapidity, sensitivity, accuracy, and automation. The most common techniques for detecting complex proteomes are liquid chromatography (LC) and mass spectrometry (MS). LC-MS (Liquid Chromatography-Mass Spectrometry), which combines the physical separation capability of LC and the mass analysis capability of MS, is a powerful technique mainly used for analyzing the proteome of cells, tissues, and body fluids. To explore the combination of traditional proteomics techniques such as Western blotting, Co-IP (co-Immunoprecipitation), and the latest LC-MS methods to probe the anti-inflammatory pathway and the differential changes in upstream and downstream proteins induced by the antiviral natural immune junction protein STING.

Characterising the urinary acylcarnitine and amino acid profiles of HIV/TB co-infection, using LC-MS metabolomics.

INTRODUCTION: The human immunodeficiency virus (HIV) and tuberculosis (TB) co-infection presents significant challenges due to the complex interplay between these diseases, leading to exacerbated metabolic disturbances. Understanding these metabolic profiles is crucial for improving diagnostic and therapeutic approaches. OBJECTIVE: This study aimed to characterise the urinary acylcarnitine and amino acid profiles, including 5-hydroxyindoleacetic acid (5-HIAA), in patients co-infected with HIV and TB using targeted liquid chromatography mass spectrometry (LC-MS) metabolomics. METHODS: Urine samples, categorised into HIV, TB, HIV/TB co-infected, and healthy controls, were analysed using HPLC-MS/MS. Statistical analyses included one-way ANOVA and a Kruskal-Wallis test to determine significant differences in the acylcarnitine and amino acid profiles between groups. RESULTS: The study revealed significant metabolic alterations, especially in TB and co-infected groups. Elevated levels of medium-chain acylcarnitines indicated increased fatty acid oxidation, commonly associated with cachexia in TB. Altered amino acid profiles suggested disruptions in protein and glucose metabolism, indicating a shift towards diabetes-like metabolic states. Notably, TB was identified as a primary driver of these changes, affecting protein turnover, and impacting energy metabolism in co-infected patients. CONCLUSION: The metabolic profiling of HIV/TB co-infection highlights the profound impact of TB on metabolic pathways, which may exacerbate the clinical complexities of co-infection. Understanding these metabolic disruptions can guide the development of targeted treatments and improve management strategies, ultimately enhancing the clinical outcomes for these patients. Further research is required to validate these findings and explore their implications in larger, diverse populations.

Revealing Changes in Celecoxib Nanostructured Lipid Carrier's Bioavailability Using Hyaluronic Acid as an Enhancer by HPLC-MS/MS.

Purpose: Oral drug administration is the most common and convenient route, offering good patient compliance but drug solubility limits oral applications. Celecoxib, an insoluble drug, requires continuous high-dose oral administration, which may increase cardiovascular risk. The nanostructured lipid carriers prepared from drugs and lipid excipients can effectively improve drug bioavailability, reduce drug dosage, and lower the risk of adverse reactions. Methods: In this study, we prepared hyaluronic acid-modified celecoxib nanostructured lipid carriers (HA-NLCs) to improve the bioavailability of celecoxib and reduce or prevent adverse drug reactions. Meanwhile, we successfully constructed a set of FDA-compliant biological sample test methods to investigate the pharmacokinetics of HA-NLCs in rats. Results: The pharmacokinetic analysis confirmed that HA-NLCs significantly enhanced drug absorption, resulting in an AUC0-t 1.54 times higher than the reference formulation (Celebrex®). Moreover, compared with unmodified nanostructured lipid carriers (CXB-NLCs), HA-NLCs enhance the retention time and improve the drug's half-life in vivo. Conclusion: HA-NLCs significantly increased the bioavailability of celecoxib. The addition of hyaluronic acid prolonged the drug's in vivo duration of action and reduced the risk of cardiovascular adverse effects associated with the frequent administration of oral celecoxib.

Software-assisted automated detection and identification of "unknown" analogues of benzodiazepines in liquid chromatography mass spectrometry analysis.

RATIONALE: Benzodiazepines (BZDs) construct a large group of psychoactive drugs acting as depressants of the central nervous system (CNS) and used in medicine as sedatives and anxiolytic and antiepileptic agents. The illicit use of these materials is a worldwide problem, and for many years, part of the benzodiazepines have been abused as rape drugs. For example, flunitrazepam (Rohypnol) is most commonly linked by media reports to drug-facilitated sexual assaults, more commonly referred to as "date rape." Furthermore, there are growing concerns for other misuses of these drugs. Over the last few years, there was an increase in the number, type, and availability of new psychoactive substances (NPS) belonging to the benzodiazepine group, challenging standard forensic labs to fully identify the chemical structure of new, unknown benzodiazepines. METHODS: This work demonstrates a new application of the automated tool for the detection and identification of benzodiazepine analogues using high-resolution-accurate-mass LC-MS analysis, followed by "Compound Discoverer" (CD) software data processing, to automatically detect various benzodiazepine analogues by picking peaks and compare them to in silico calculated modifications made on a predefined basic backbone. Subsequently, a complete structural elucidation for the proposed molecular formula is obtained by MS/MS data analysis of the suspected component. RESULTS: This method was found to be useful for the automated detection and putative identification of a series of nine "unknown" benzodiazepine analogues, at concentrations in the low ng/mL range. CONCLUSIONS: We hereby present a general demonstration of this powerful tool for the forensic community in the detection and identification of hazardous unknown compounds.

Optimized LC-MS/MS methods for quantifying antibody-drug conjugate payloads in cell culture media containing phenol red.

Aim: Phenol red is commonly used in cell culture media, but can be detrimental to bioanalysis of in vitro samples as it may impact instrument reliability. Many researchers do their final stage of culture in 'phenol red free' media, but in collaborative work this is not always feasible.Materials & methods: A comparison was made between typical extraction methods to reduce phenol red matrix interferences, including organic solvent precipitation and solid phase extraction.Results: The final method was demonstrated to be precise and accurate for the measurement of a target analyte by LC-MS/MS, and was applied to an in vitro ADC deconjugation study.Conclusion: This method allows for for continued bioanalytical support of in vitro models used in drug development.

[Box: see text].

High-coverage identification of hydroxyl compounds based on pyridine derivatization-assisted liquid chromatography mass spectrometry.

Hydroxyl compounds are widely present in plants and play essential roles in plant growth and development. High-coverage detection of hydroxyl compounds is crucial for understanding the physiological processes of plants. Despite the prevalence of chemical derivatization-assisted liquid chromatography-high resolution mass spectrometry (CD-LC-HRMS) in high-coverage detection of compounds with diverse functional groups, the confident identification of these compounds after derivatization remains a significant challenge. Herein, a novel method was developed for the identification of pyridine (PY)-derivatized hydroxyl compounds by comparing the MS/MS similarity of derivatized and corresponding underivatized compounds. Fragmentation rules of standards were summarized, and theoretical calculations have demonstrated the MS/MS similarity of PY-derivatized hydroxyl compounds with their underivatized counterparts. The effectiveness of the developed method was demonstrated by identifying PY-derivatized authentic standards. A total of 90 hydroxyl compounds were putatively identified in maize using the proposed method. This method can significantly enhance ionization efficiency with minimal impact on the quality of the MS/MS spectra, enabling the effective utilization of mass spectra databases for the identification of hydroxyl compounds.

Analysis of total microcystins by Lemieux oxidation and liquid chromatography-mass spectrometry in fish and mussels tissues: Optimization and comparison of protocols.

Microcystins (MCs) can be detected in various matrices in two forms: a freely extractable fraction and a total (free and covalently protein-bound) fraction. Although the majority of MCs analyses are limited to the free fraction, they do not allow the analysis of all MCs variants or protein-bound forms. Other methods, known as total MCs analysis methods, enable simultaneous analysis of all MCs variants, as well as bound forms, which may be a major form of toxin accumulation in organisms. Among these techniques, the chemical oxidation method (e.g. Lemieux) allows the detection of total forms of MC (and nodularins) by oxidizing the common part to all MC and nodularins, and analyzing the resultant MMPB product (2-methyl-3-methoxy-4-phenylbutyric acid). However, the execution of this method in the context of health monitoring is challenging due to the variability of the protocols, the recoveries obtained with these protocols, and the important matrix effects associated with the method. The objectives of this study were i) to optimize an existing protocol of chemical oxidation "Lemieux1" on fresh fish fillet matrices, ii) to compare two existing protocols ("Lemieux1" and "Lemieux2"), and iii) apply Lemieux oxidation to fish fillets and livers naturally contaminated with MCs-producing cyanobacteria and to freshwater mussels contaminated with MCs in laboratories. Optimization of the "Lemieux1" protocol, in particular in the oxidation and SPE (solid phase extraction) steps improved the method's yields on the fresh fish fillet matrix (from <5 % to around 40 %). Moreover, several quantification methods have been compared through various calibration techniques (solvent calibration curve, matrix-matched calibration curve, oxidized MC-LR calibration curve and also by testing the addition of d3-MMPB as an internal standard). Comparison with the "Lemieux2" protocol showed the best results on the same matrix, with yields of around 65 %. MMPB was analyzed using this "Lemieux 2" protocol, in livers of carps sampled during an episode of cyanobacteria proliferation, at concentrations ranging from 17.9 to 27.5 µg/kg MMPB and at concentrations ranging from 50 to 2890 µg/kg MMPB in freshwater mussels laboratory contaminated to MCs.

Metabolomics analysis of patients with Schistosoma japonicum infection based on UPLC-MS method.

BACKGROUND: Schistosomiasis is still one of the most serious parasitic diseases. Evidence showed that the metabolite profile in serum can potentially act as a marker for parasitic disease diagnosis and evaluate disease progression and prognosis. However, the serum metabolome in patients with Schistosoma japonicum infection is not well defined. In this study, we investigated the metabolite profiles of patients with chronic and with advanced S. japonicum infection. METHODS: The sera of 33 chronic S. japonicum patients, 15 patients with advanced schistosomiasis and 17 healthy volunteers were collected. Samples were extracted for metabolites and analyzed with ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). RESULTS: We observed significant differences in metabolite profiles in positive and negative ion modes between patients with advanced and chronic S. japonicum infection. In patients with chronic S. japonicum infection, 199 metabolites were significantly upregulated while 207 metabolites were downregulated in advanced infection. These differential metabolites were mainly concentrated in steroid hormone biosynthesis, cholesterol metabolism and bile secretion pathways. We also found that certain bile acid levels were significantly upregulated in the progression from chronic to advanced S. japonicum infection. In receiver operator characteristic (ROC) analysis, we identified three metabolites with area under the curve (AUC) > 0.8, including glycocholic (GCA), glycochenodeoxycholate (GCDCA) and taurochenodeoxycholic acid (TCDCA) concentrated in cholesterol metabolism, biliary secretion and primary bile acid biosynthesis. CONCLUSIONS: This study provides evidence that GCA, GCDCA and TCDCA can potentially act as novel metabolite biomarkers to distinguish patients in different stages of S. japonicum infection. This study will contribute to the understanding of the metabolite mechanisms of the transition from chronic to advanced S. japonicum infection, although more studies are needed to validate this potential role and explore the underlying mechanisms.

A multianalyte LC-MS/MS method for accurate quantification of Nitrosamines in Olmesartan tablets.

This research summaries the development, optimization and validation of liquid chromatography tandem mass spectrometric (LC-MS/MS) method for concurrent measurement of seven nitrosamines viz; NDMA, NDEA, NDIPA, NDPA, NEIPA, NMPA & NMBA in Olmesartan tablet. Controlling these nitrosamines at trace levels is imperative for ensuring the safety of drug substances and products for consumption. Various regulatory authorities stress the significance of utilizing highly sensitive analytical methods to precisely measure nitrosamines at trace levels. The method applied effective chromatographic separation and optimized parameters for mass spectrometric detection. Detection was carried out using APCI positive ion mode. Chromatographic separation was achieved using a Thermo Accucore PFP column (150 mm x 4.6 mm, 2.6 µ), with a simple gradient elution of mobile phase consisting of 0.1 % formic acid in water (mobile phase A) and methanol (mobile phase B). The total run time was 20 min, with a flow rate of 0.800 mL/min. The method was validated according to the International Council on Harmonisation (ICH Q2 (R2)) guidelines. The established method demonstrated excellent linearity (R2> 0.99) and sensitivity for all the nitrosamines. Detection and quantification limits were sufficiently low for trace nitrosamine levels having good S/N ratio. The method showed good accuracy in Olmesartan tablet samples, with recoveries ranges between 80 % to 120 %. The new analytical approach has exceptional repeatability and reliability, making it possible to precisely quantify the levels of seven nitrosamines in Olmesartan medoxomil tablets in a single analytical run.

A validated UPLC-MS/MS method for the quantification of immunosuppressive drugs in peripheral blood mononuclear cells using liquid-liquid extraction with low temperature purification without complex pretreatment steps.

Immunosuppressive drugs (ISDs) are given to avoid the allograft rejection after transplantation. The concentrations of ISDs should be closely monitored owing to their wide inter-individual variability in its pharmacokinetics and narrow therapeutic window. Currently, the whole blood concentration measurement is the major approach of therapeutic drug monitoring of clinical ISDs in organ transplantation. Its correlation with the efficacy of ISDs remains elusive. While the acute rejection after transplantation may occur even when whole-blood ISDs concentrations are within the target range. Since the site of action of ISDs are within the lymphocyte, direct measurement of drug exposure in target cells may more accurately reflect the clinical efficacy of ISDs. Although several methods have been developed for the peripheral blood mononuclear cells (PBMCs) extraction and drug concentration measurement, the complex pre-processing has limited the study of the relationship between intracellular ISDs concentrations and the occurrence of rejection. In this study, the extraction of ISDs in PBMCs was carried out by the liquid-liquid extraction with low temperature purification, without centrifugation. The lower limit of quantitation were 0.2 ng/mL for cyclosporine A, tacrolimus and sirolimus, 1.0 ng/mL for mycophenolic acid, and the within-run and between-run coefficient of variations were both less than 12.4 %. The calibration curves of mycophenolic acid had a linear range (ng/mL): 1.0-128.0 (r2 = 0.9992). The calibration curves of other three ISDs had a linear range (ng/mL): 0.2-20.48 (r2 > 0.9956). A total of 157 clinical samples were analyzed by the UPLC-MS/MS for ISDs concentration in blood or plasma ([ISD]blood or plasma) and the concentration within PBMCs ([ISD]PBMC). Although there was strong association between [ISD]PBMC and [ISD]blood or plasma, the large discrepancies between concentration within [ISD]blood or plasma and [ISD]PBMC were observed in a small proportion of clinical samples. The developed method with short analysis time and little amounts of blood sample can be successfully applied to therapeutic drug monitoring of ISDs in PBMCs for analysis of large numbers of clinical samples and is helpful to explore the clinical value of ISDs concentration in PBMCs.

Bioactivity of silverleaf nightshade (Solanum elaeagnifolium Cav.) berries parts against Galleria mellonella and Erwinia carotovora and LC-MS chemical profile of its potential extract.

Natural products received much attention as an environmentally beneficial solution for pest management. Therefore, the extracts of invasive silverleaf nightshade (Solanum elaeagnifolium Cav.) weeds using their berries parts (seeds, peels and mucilage) supported by bioassay-guided fractionation were tested against both the greater wax moth (Galleria mellonella) and Erwinia carotovora pv. carotovora causes of the blackleg of potatoes. The seeds and peels of S. elaeagnifolium were successively extracted by maceration using dichloromethane (DCM), ethyl acetate (EtOAc), and ethanol (EtOH), respectively. While, its mucilage was extracted using EtOAc. The successive EtOH extract of the plant seeds had promising inhibition efficacy and the best minimal inhibition concentration (MIC) of 50 µg/ml against E. Carotovora amongst other extracts (DCM and EtOAc of the plant berries parts). Depending on dose response activity, EtOH extract had G. mellonella larval mortality and pupal duration rates (LC50; 198.30 and LC95; 1294.73 µg/ml), respectively. Additionally, this EtOH extract of seeds was fractionated using preparative TLC to three characteristic bands. The insecticidal and bacterial activities of these isolated bands (SEA, SEB, and SEC) were evaluated at a dose of 100 µg/ml, causing mortality by 48.48, 62.63 and 92.93% (G. mellonella larvae) and inhibition by 15.22, 0.00 and 31.66 mm (E. carotovora), respectively. Moreover, the separated major three bands were tentatively identified using LC-ESI-MS analysis revealing the presence of two phenolic acids; chlorogenic acid (SEA) and dicaffeoyl quinic acid (SEB) in addition to one steroidal saponin (SEC) annotated as borassoside E or yamoscin. Finally, the plant seeds' successive EtOH extract as well as its active constituents, exhibited potential broad-spectrum activity and the ability to participate in future pest management initiatives. A field study is also recommended to validate its bio-efficacy against selected pests and to develop its formulations.

4-Vinylpyridine copolymers for improved LC-MS tryptophan and kynurenine determination in human serum.

Tryptophan (an essential amino acid) and its clinically important metabolite-kynurenine contribute to several fundamental biological processes and methods that allow their determination in biological samples are in demand. The novelty of the work was a demonstration of the utility of two polymers: 4-vinylpyridine crosslinked with trimethylolpropane trimethacrylate (poly(4VP-co-TRIM)) or 1,4-dimethacryloyloxybenzene (poly(4VP-co-14DMB))-in terms of human serum clean-up for simultaneous LC-MS determination of tryptophan and kynurenine. The goal was to achieve a reduction of the matrix effect, which is responsible for signal suppression, with minimal capture of analytes. The adsorption properties of the polymeric beads were studied by evaluating the adsorption kinetics and isotherms in model matrices. Therefore, the adsorption capacities of both molecules were not efficient, the tested 4-vinylpyridine-based copolymers have shown great promise (especially poly(4VP-co-TRIM)) as sorbents for serum clean-up. In the model human serum matrix, poly(4VP-co-TRIM) provided good recoveries of tryptophan and kynurenine (76% and 87%, respectively) and allowed for the reduction of the matrix effect. Performances of both copolymers were compared to those of commercially available sorbents (octadecylsilane, activated charcoal, and primary secondary amine).

Development of UPLC-MS/MS method for studying the pharmacokinetic interactions of fuzuloparib with curcumin in rats.

Fuzuloparib is a novel orally bioactive poly-ADP-ribose polymerase inhibitor (PARPi), which was approved by the Chinese Regulatory Agency (CRA) in 2020 for the treatment of platinum-sensitive recurrent ovarian, fallopian tube, and primary peritoneal cancers. This study firstly presents a rapid and accurate ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for analyzing the levels of fuzuloparib and its major metabolite (SHR165202), and to investigate drug-drug interaction between fuzuloparib and curcumin in vitro and in vivo studies. After protein precipitation with acetonitrile, mobile phase consisted of acetonitrile and 0.1 % formic acid with a gradient elution was used to successfully separate fuzuloparib, SHR165202 and talazoparib (internal standard, IS). The results indicated that fuzuloparib and SHR165202 had good linearity over the calibration range of 2-50 ng/mL and 1-20 ng/mL, respectively. The precision, accuracy, stability, matrix effect, and extraction recovery required for methodological validation all complied with the requirements of the Bioanalytical Method Validation Guidelines. In vitro microsome incubation experiments, curcumin exhibited inhibitory effect on fuzuloparib in both rat liver microsomes (RLM) and human liver microsomes (HLM) with half-maximal inhibitory concentration (IC50) value of 10.54 µM and 47.64 µM, respectively, and the corresponding mechanism was non-competitive. Furthermore, the inhibitory mechanism of curcumin on fuzuloparib was validated through molecular docking. In pharmacokinetic experiments in rats, curcumin significantly altered the plasma exposure of fuzuloparib, resulting in significant increases in AUC(0-t) and Cmax of fuzuloparib and a significant decrease in CLz/F. Moreover, the metabolite SHR165202 showed significant increases in AUC(0-t), AUC(0-∞), Tmax and Cmax and a significant decrease in CLz/F. This further supports the notion that curcumin could inhibit the metabolism of fuzuloparib. Therefore, when co-administering fuzuloparib and curcumin in clinic, it is recommended to monitor plasma levels of fuzuloparib and pay close attention to adverse effects. If necessary, the dose of fuzuloparib needs to be reduced.

An innovative UPLC-MS/MS method for the quantitation and pharmacokinetics of eupafolin in rat plasma.

In this experiment, a rapid and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology was established and validated for the quantitation and pharmacokinetic analysis of eupafolin in rat plasma, utilizing licochalcone B as internal standard (IS). After liquid-liquid extraction of the analyte samples by ethyl acetate, chromatographic separation was achieved using a UPLC HSS T3 column under gradient elution conditions, with the mobile phase consisting of acetonitrile and water (with 0.1 % formic acid). Eupafolin was quantified by multiple reaction monitoring (MRM) in electrospray positive-ion mode (ESI+), employing the mass transition m/z 315.2 â†’ 300.3 for eupafolin and m/z 285.4 â†’ 270.3 for IS. Eupafolin demonstrated excellent linear relationship (r > 0.99) over the concentration range of 1.25-1250 ng/mL, with the lower limit of quantification (LLOQ) of the UPLC-MS/MS assay determined as 1.25 ng/mL. Method validation followed the bioanalytical method validation criteria outlined by the FDA. The accuracy of eupafolin ranged from 86.7 % to 111.2 %, and the precision was less than 12 %. The matrix effect was observed at 92.8 %-98.6 %, while the recoveries exceeded 83.2 %. The established UPLC-MS/MS assay was successfully employed for the pharmacokinetic evaluation of eupafolin in rats. The half-lives (t1/2z) were determined to be 1.4 ± 0.4 h and 2.5 ± 1.4 h for intravenous and oral administration, respectively. Notably, the bioavailability of eupafolin was relatively low (8.3 %). The optimized UPLC-MS/MS technology showed highly sensitive, selective, and effective, rendering it suitable for the pharmacokinetics of eupafolin in preclinical practice.

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.

BACKGROUND AND OBJECTIVES: 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. METHODS: 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 0.3 mL/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  study of 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. RESULTS: 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 10-2000 ng/mL. The validated method was then applied to pharmacokinetics and bioavailability studies in rat by dosing with gavage (25 mg/kg) and intravenous injection(5 mg/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 ± 279.45 µg/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 ± 3360.84 ng/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 ± 2597.63 ng/mL·h), terminal elimination half-life (t1/2) (PO: 7.26 ± 2.16 h, IV: 4.78 ± 1.09 h). CONCLUSIONS: TPB15, a promising candidate for treating TNBC, has demonstrated 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.

Charge Variants Characterization of Co-Formulated Antibodies by Three-Dimensional Liquid Chromatography-Mass Spectrometry.

Co-formulated antibodies can bring clinical benefits to patients by combining two or more antibodies in a single dosage form. However, the quality analysis of co-formulated antibodies raises additional challenges, compared to individual antibodies, due to the need for accurate analysis of multiple antibodies in one solution. It is extremely difficult to effectively separate the charge variants of the two co-formulated antibodies using one ion exchange chromatography (IEC) method because of their similar characteristics. In this study, a novel method was developed for the charge variants characterization of co-formulated antibodies using three-dimensional liquid chromatography-mass spectrometry (3D-LC-MS). Hydrophobic interaction chromatography (HIC) was used as the first dimension to separate and collect the two co-formulated antibodies. The two collections were then injected into the second-dimension IEC separately for charge variants separation and analysis. Subsequently, the separated charge variants underwent on-line desalting in the third-dimension reverse-phase chromatography (RPC) and subsequent mass spectroscopy analysis. The novel method could simultaneously provide a charge variants ratio and post-translational modification (PTM) data for the two co-formulated antibodies. Therefore, it could be used for release testing and stability studies of co-formulated antibodies, making up for the shortcomings of the existing approaches. It was the first time that charge variants of co-formulated antibodies were characterized by the 3D-LC-MS method, to the best of our knowledge.

A sensitive, expandable AQC-based LC-MS/MS method to measure amino metabolites and sphingolipids in cell and serum samples.

Sphingolipids are a major lipid species found in all eukaryotes. Among structurally complex and diversified lipids, sphingoid bases have been heavily linked to various metabolic diseases. However, most current LC-MS-based methods lack the sensitivity to detect low-abundant sphingoid bases. The 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization reagent, which efficiently forms covalent bonds with amino groups, has been widely used for amino acid detection. Nevertheless, the commonly used reverse-phase HPLC method for amino acid analysis is not suitable for amphipathic sphingolipids. To address this issue, we report a robust reverse-phase HPLC-MS/MS method capable of separating and detecting hydrophilic amino acids and sphingoid bases in a single run with high sensitivity. This method is also inclusive of other amino metabolites with an expandable target list. We tested this method under various conditions and samples, demonstrating its high reproducibility and sensitivity. Using this approach, we systematically analyzed human serum samples from healthy individuals, dyslipidemia, and type II diabetes mellitus (T2DM) patients, respectively. Two sphingolipids and five amino acids were identified with significant differences between the control and T2DM groups, highlighting the potential of this method in clinical studies.

Validation of a method for the determination of Aderamastat (FP-025) in K2EDTA human plasma by LC-MS/MS.

Aderamastat (FP-025) is a small molecule, selective matrix metalloproteinase (MMP)-12 inhibitor, under development for respiratory conditions which may include chronic inflammatory airway diseases and pulmonary fibrosis. To support evaluation of the pharmacokinetic parameters of Aderamastat in humans, we developed and validated a high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analytical method for the quantification of Aderamastat in human plasma. This assay was validated in compliance with the Food and Drug Administration (FDA) Good Laboratory Practice Regulations (GLP) and European Medicines Agency (EMA) guidelines. K2EDTA human plasma samples were spiked with internal standard, processed by liquid-liquid extraction, and analyzed using reversed-phase HPLC with Turbo Ion Spray® MS/MS detection. Separation was done using a chromatographic gradient on 5 µm C6-Phenyl 110 Å, 50*2 mm analytical column at a temperature of 35 °C. The LC-MS/MS bioanalytical method, developed by QPS Taiwan to determine the concentration of Aderamastat in K2EDTA human plasma, was successfully validated with respect to linearity, sensitivity, accuracy, precision, dilution, selectivity, hemolyzed plasma, lipemic plasma, batch size, recovery, matrix effect, and carry-over. These data indicate that the method for determination of Aderamastat concentrations in human K2EDTA plasma can be used in pharmacokinetics studies and subsequent clinical trials with Aderamastat. Authors declare that, this novel data is not published and not under consideration for publication by another journal than this journal. All data will be made available on request.

LC-MS analysis of chiral amino acids in human urine reveals D-amino acids as potential biomarkers for colorectal cancer.

Colorectal cancer (CRC) is a common malignant tumor in the gastrointestinal tract. Changes in amino acid metabolites have been implicated in tumorigenesis and disease progression. Biomarkers on the basis of chiral amino acids, especially D-amino acids, have not been established for early diagnosis of CRC. Quantification of chiral amino acids, especially very low concentrations of endogenous D-amino acids, is technically challenging. We report here the quantification of L- and D-amino acids in urine samples collected from 115 CRC patients and 155 healthy volunteers, using an improved method. The method of chiral labeling, liquid chromatography, and tandem mass spectrometry enabled separation and detection of 28 amino acids (14 L-amino acids, 13 D-amino acids and Gly). Orthogonal partial least squares discriminant analysis identified 14 targeted variables among these chiral amino acids that distinguished the CRC from the healthy controls. Binary logistic regression analysis revealed that D-α-aminobutyric acid (D-AABA), L-alanine (L-Ala), D-alanine (D-Ala), D-glutamine (D-Gln) and D-serine (D-Ser) could be potential biomarkers for CRC. A receiver operating characteristic curve analysis of combined multi-variables contributed to an area under the curve (AUC) of 0.995 with 98.3 % sensitivity and 96.8 % specificity. A model constructed with D-AABA, D-Ala, D-Gln, and D-Ser achieved an AUC of 0.988, indicating important contributions of D-amino acids to the association with CRC. Further analysis also demonstrated that the metabolic aberration was associated with age and the development of CRC, D-methionine (D-Met) was decreased in CRC patients with age over 50, and D/L-Gln in patients at stage IV was higher than patients at stage I. This study provides the signature of D-amino acids in urine samples and offers a promising strategy for developing non-invasive diagnosis of CRC.

Analysis of Flutianil and OC56635 Residues in Hemp Cannabis Matrices by LC-MS/MS.

In this paper, we present analytical methodologies for the determination of the thiazolidine fungicide flutianil (trade name GATTEN) and its primary metabolite OC56635 in hemp cannabis matrices. A total of nine crop matrices were tested: whole seed, fiber, flower buds, hemp hearts, hemp seed oil, hemp meal, hemp flour, ethanol extracted CBD resin (CBD-E), and supercritical CO2 extracted CBD resin (CBD-C). Processing of the CBD-E and CBD-C crop fractions was carried out in-house using methods detailed herein. Field sample analysis utilized sequential extractions, stacked solid phase extraction (SPE) column cleanups, and evaporation to prepare the samples for LC-MS/MS quantitation. Method validations for each fraction were carried out using untreated hemp matrices over a minimum of three levels, with lowest levels of method validation (LLMV) of 0.010 µg/g for all fractions except the CBD resins, for which LLMV was 0.020 µg/g. Flutianil-treated samples from nine field sites were collected from several crop production regions and analyzed to determine the distribution of incurred flutianil and OC56635 residues within the different hemp matrices. This data was generated in support of nationwide registration with the United States Environmental Protection Agency (USEPA).