Journal of Chromatography B最新文献

Dried Blood Spots (DBS) revolutionize therapeutic drug monitoring using LC-MS for the precise quantification of cardiovascular drugs (CDs), enabling personalized treatment adapted to patient-specific pharmacokinetics with minimal invasiveness. This study aims to achieve simultaneous quantification of eight CDs in DBS, overcoming physicochemical challenges. A two-step protein precipitation method was used for simple and precise sample preparation. The drugs were analyzed using LC-MS/MS in ESI positive-ion mode, showing high sensitivity and linearity, with a correlation coefficient (r²) exceeding 0.999, after being separated on a reversed-phase chromatography by gradient elution of DW-acetonitrile containing 0.1 % formic acid + 2 mM ammonium formate. The validation results indicate good selectivity, with no observed matrix effect and carry-over. The intra- and inter-day accuracy and precision were within 6 % for most drugs, except for digoxin and deslanoside at low therapeutic levels where the variation was within 20 %. Stability tests confirmed suitable DBS handling and storage conditions, indicating drug stability for at least 30 days at room temperature. The analysis of whole spot has demonstrated remarkable precision and reliability in all target drugs. The analysis of 3 mm internal diameter discs, punched in and out of DBS, presumed to contain 3 µL of blood, showed acceptable accuracy for most drugs, with less polar drugs like digoxin and deslanoside showing lower accuracy, indicating a need for further correction due to non-uniform drug distribution. Consequently, the developed LC-MS/MS method enables the quantification of multiple CDs in a single DBS analysis, while suggesting the potential for accuracy-based analysis.

C. madagascariensis, an unexplored species of Burseraceae is used by local population for the management of inflammation and throat pain. The disease alleviation by this plant could be due to the presence of rich repository of active compounds with various pharmacological importances. In this study, therefore, the profiling of metabolites and isolation of active compounds of C. madagascariensis was performed. Furthermore, the ethanol, ethyl acetate extracts and a selected active compound was subjected for in vitro and in vivo anti-inflammatory activities. Metabolomic analysis identified and quantified 116 metabolites from leaves, young stem and gum-resins of C. madagascariensis (Burseraceae) followed by multivariate PCA analysis. NMR, GC–MS and HPLC were used to analyze primary and secondary metabolites. Subsequently, five main isolated compounds were identified as trimethoxy tetrahydrobenzo dioxolo isochromene (TTDI), butyl phenol, butyl propionate phenol, germacrone and β-elemenone. Amongst them, TTDI was found to be a novel compound. Hence, a process was developed to obtain the enriched fraction of TTDI in ethanol and ethyl acetate extracts of leaves. Furthermore, TTDI and extracts were subjected for their in vitro anti-inflammatory activity in LPS sensitized murine splenocytes. The results showed that TTDI and both extracts significantly suppressed the levels of pro-inflammatorycytokines (TNF-α, IFN-γ). Interestingly, the suppression of pro-inflammatory cytokines was evenmore significant by the similar concentration of TTDI when compared with colchicine. However, the level of anti-inflammatory cytokine (IL-10) was found to be unchanged. Additionally, in vivo anti-inflammatory study revealed a significant reduction in carrageenan induced paw edema by TTDI and both the extracts. In the docking study, TTDI was more active than colchicine with strong binding affinity to COX-2, PLA2, and 5β reductase. Our results highlighted that the presence of metabolites with medicinal and nutraceutical importance in C. madagascariensis, could provide opportunities for the development of a new plant-based therapeutics for inflammation.

OPC-61815 is an intravenous formulation vasopressin antagonist designed to treat heart failure patients, especially who have difficulty in oral intake. Tolvaptan together with DM-4103 and DM-4107 are considered as the major metabolites of OPC-61815 biotransformed in the liver via cytochrome P450 (CYP) 3A. An efficient and robust ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of OPC-61815 and its three metabolites in human plasma was developed and fully validated. To our best knowledge, it was the first published method that simultaneously quantified all of these four analytes in only one run. Simple and rapid sample preparation procedure and very short UPLC-MS/MS run time (3.5 min) offered OPC-61815 and its metabolites relatively high throughput detection, which was greatly beneficial to further clinical bio-sample analysis. The method showed good linearity and sufficient sensitivity in the range of 2.00–1000 ng/mL with a low limit of quantitation (2.00 ng/mL) for each analyte. For samples with concentrations above 1000 ng/mL, 100-fold dilution with blank plasma before sample preparation was accepted. High precision and accuracy, high selectivity and satisfactory recovery of this method were demonstrated. For all of the four analytes, no significant matrix effect or carry-over was observed. The stability of analytes and internal standards under different conditions were evaluated to ensure they were stable during the whole period of storage, preparation and detection. Also, re-injection reproducibility was investigated. In addition, the conversion test showed that almost no OPC-61815 converted into DM-4103 and DM-4107 during sample processing, while attention should be paid to the concentration difference between OPC-61815 and tolvaptan in bioanalysis. The developed UPLC-MS/MS method was successfully applied to an open, single and multiple dose administration phase I trial for monitoring the pharmacokinetics of OPC-61815. This work provided a promising way for further pharmacokinetic study of OPC-61815.

This study established a method to prepare and detect OPs adducts on butyrylcholinesterase (BChE) and human serum albumin (HSA). OPs (methyl paraoxon, ethyl paraoxon, methyl parathion, parathion) were incubated with BChE or HSA in vitro, and the adducts of OPs-BChE or OPs-HSA were prepared and qualitatively analyzed by ultra-performance liquid chromatography data-dependent high-resolution tandem mass spectrometry (UPLC-ddHRMS/MS). The amounts of BChE and HSA in the incubating systems were varied and the resulting amounts of the adducts were determined using linear regression. OPs-BChE in the blood were isolated by immunomagnetic separation (IMS), and then digested into the OPs-nonapeptide adduct by pepsin. The proteins in the remaining blood plasma were precipitated and digested by pronase to OPs-tyrosines(OPs-Tyr), which were quantified by UPLC-ddHRMS/MS. 4 OPs-nonapeptides and 4 OPs-Tyr adducts were obtained through the process above. The relative mass deviation of incubated adducts between the actual and theoretical exact masses was less than 10 ppm, and further confirmed by fragmentation mass spectra analysis. Calibration curves were linear for all adducts with a coefficient of determination value (R²) ≥0.995. The limits of detection (LOD) and limits of quantification (LOQ) for adducts detected by MS ranged from 0.05 to 1.0 ng/mL, and from 0.1 to 2.0 ng/mL, respectively. The recovery percentages for adducts ranged from 76.1 % to 107.1 %, matrix effects ranged from 83.4 % to 102.1 %. The inter-day and intra-day precision were 6.1–10.1 % and 6.9–12.9 % for adducts. This study provides a new reference method for the detection of organophosphorus pesticide poisoning. In addition, two blood samples with organophosphorus poisoning were tested by the designed method, and the corresponding adducts were detected in both samples.

Interest is increasing in the use of different liquid chromatography techniques coupled online to mass spectrometry for the quantification of platinum anticancer drugs in human plasma to inform cancer chemotherapy. We developed, validated and studied the application of a method for quantification of intact oxaliplatin in human plasma using ultra high performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (UHPLC-ICP-MS). Plasma samples were processed instantly after collection from patients to preserve oxaliplatin speciation by methanol-deproteinization, and storage of diluted supernatants (plasma:methanol 1:2 v/v) at −80 °C. UHPLC separation of intact oxaliplatin and internal standard (carboplatin) was achieved using a C18 column and linear gradient mobile phase (Mobile phase A: water-methanol (97:3 v/v), 0.075 mM sodium dodecyl sulfate, 9.79 nM thallium adjusted to pH 2.5 with trifluoromethanesulfonic acid; Mobile phase B: 100 % methanol (v/v)) with ICP-MS detection to monitor platinum and thallium at m/z 195 and 205, respectively. The limit of quantification was 50 nM in methanol-deproteinized diluted plasma (1:2 v/v). Linearity was established for calibration standards ranging from 50 to 500 nM made in methanol-deproteinized diluted plasma (1:2 v/v), and for dilution of higher concentration samples in blank matrix containing internal standard (final dilution 1:29 v/v). Intra-day and inter-day accuracy ranged from 96.8 to 103 % of nominal concentration and precision from 0.62 to 2.49 % coefficient of variation. Recovery was complete and a matrix effect confirmed the requirement for matrix-matched standards. Intact oxaliplatin was stable during storage for at least 473 days, and during analysis, in methanol-deproteinized diluted plasma (1:2 v/v). The method was applied to determining the plasma concentrations of intact oxaliplatin in patients undergoing cancer chemotherapy, and studies of oxaliplatin degradation in vitro. This improved method based on UHPLC-ICP-MS will allow more specific, efficient and reliable quantification of intact oxaliplatin in human plasma.

The production of biogenic amines (BAs), which are markers of both quality and safety in fish and fishery products, is influenced by the harvesting technique, handling, and other operations including those carried out on board the vessel. Scombroid dark-meat fish (e.g. tuna) are the fish species most frequently linked to histamine poisoning. The most commonly found BAs in fish are histamine, tyramine, putrescine, and cadaverine, which are produced when microbes decarboxylate the corresponding free amino acids. In this study, a rapid and cost-effective HILIC-MS/MS method was developed and validated for the determination of putrescine, cadaverine, histamine and tyramine in tuna samples. A simple sample preparation procedure was followed using the solvent mixture MeOH/H₂O (50/50, v/v), 0.1 % acetic acid for protein precipitation and analyte extraction. Intra- and inter-day accuracy, expressed as %Recovery (%R), ranged from 88.0 % (Cad) to 102.7 % (Tyr) and from 85.0 % (Cad) to 99.8 % (Tyr), respectively. Intra- and inter-day precision, expressed as %Relative Standard Deviation (%RSD), ranged from 0.4 % (Tyr, Put) to 3.3 % (His) and from 0.7 % (Tyr) to 5.0 % (Cad), respectively. Limits of detection (LOD) and quantification (LOQ) varied from 0.0009 to 0.0940 mg/kg and from 0.0030 mg/kg to 0.3100 mg/kg, respectively, depending on the analyte. Regarding the potential toxic effects linked to biogenic amines in foods, samples examined in this study showed no risk. The proposed method is an important analytical tool for routine analysis of BAs in fish products.

The field of recombinant adeno-associated virus (rAAV) gene therapy has attracted increasing attention over decades. Within the ongoing challenges of rAAV manufacturing, the co-production of impurities, such as empty and partial capsids containing no or truncated transgenes, poses a significant challenge. Due to their potential impact on drug efficacy and clinical safety, it is imperative to conduct comprehensive monitoring and characterization of these impurities prior to the release of the final gene therapy product. Nevertheless, existing analytical techniques encounter notable limitations, encompassing low throughput, long turnaround times, high sample consumption, and/or complicated data analysis. Chromatography-based analytical methods are recognized for their current Good Manufacturing Practice (cGMP) alignment, high repeatability, reproducibility, low limit of detection, and rapid turnaround times. Despite these advantages, current anion exchange high pressure liquid chromatography (AEX-HPLC) methods struggle with baseline separation of partial capsids from full and empty capsids, resulting in inaccurate full-to-empty capsid ratio, as partial capsids are obscured within peaks corresponding to empty and full capsids. In this study, we present a unique analytical AEX method designed to characterize not only empty and full capsids but also partial capsids. This method utilizes continuous N-Rich chromatography with recycling between two identical AEX columns for the accumulation and isolation of partial capsids. The development process is comprehensively discussed, covering the preparation of reference materials representing full (rAAV-LacZ), partial (rAAV-GFP), and empty (rAAV-empty) capsids, N-rich method development, fraction analysis, determination of fluorescence response factors between capsid variants, and validation through comparison with other comparative techniques.

Racecadotril, an anti-secretory medication, has been used as an adjuvant in an oral rehydration therapy for children experiencing severe diarrhea. Racecadotril is quickly converted to thiorphan, an active metabolite, after oral treatment, which mediates all subsequent activities. An efficient and rapid liquid chromatography-tandem mass spectrometry method was developed and fully validated to measure thiorphan in human plasma, using thiorphan-d7 as an internal standard. The extraction method used was protein precipitation while chromatographic separation was achieved using InertSil CN-3 (50 × 2.1 mm, 5 µm column). The assay was linear over the concentration range of 1–200 ng/ml with correlation coefficients of ≥0.9991. The intra- and inter-day precisions were less than 10.0 % for all concentrations investigated. 0.02 % aqueous formic acid and methanol (30:70 v: v) were used as mobile phases, with an analysis time of less than 1 min. This method proved stable under several conditions. The developed method worked well in a three-period pharmacokinetic bioequivalence study after a single oral administration of 100 mg racecadotril to 15 healthy Jordanian volunteers under fasting conditions.

Cortex Morin Radicis (CMR) is the dried root bark of Morus alba. L. It has a variety of effects such as antibacterial, anti-tumour, treatment of cardiovascular diseases or upper respiratory tract disease and so on. The pursuit for drugs selected from Cortex Mori Radicis having improved therapeutic efficacy necessitates increasing research on new assays for screening bioactive compounds with multi-targets. In this work, we applied immobilized β₁-AR and β₂-AR as the stationary phase in chromatographic column to screen bioactive compounds from Cortex Morin Radicis. Specific ligands of the two receptors (e.g. esmolol, metoprolol, atenolol, salbutamol, methoxyphenamine, tulobuterol and clorprenaline) were utilized to characterize the specificity and bioactivity of the columns. We used high performance affinity chromatography coupled with ESI−MS to screen targeted compounds of Cortex Morin Radicis. By zonal elution, we identified morin as a bioactive compound simultaneously binding to β₁-AR and β₂-AR. The compound exhibited the association constants of 3.10 × 10⁴ and 2.60 × 10⁴ M⁻¹ on the β₁-AR and β₂-AR column. On these sites, the dissociation rate constants were calculated to be 0.131 and 0.097 s⁻¹. Molecular docking indicated that the binding of morin to the two receptors occurred on Asp200, Asp121, and Val122 of β₁-AR, Asn312, Thr110, Asp113, Tyr316, Gly90, Phe193, Ile309, and Trp109 of β₂-AR. Likewise, mulberroside C was identified as the bioactive compound binding to β₂-AR. The association constants and dissociation rate constants were calculated to be 1.08 × 10⁴ M⁻¹ and 0.900 s⁻¹. Molecular docking also indicated that mulberroside C could bind to β₂-AR receptor on its agonist site. Taking together, we demonstrated that the chromatographic strategy to identify bioactive natural products based on the β₁-AR and β₂-AR immobilization, has potential for screening bioactive compounds with multi-targets from complex matrices including traditional Chinese medicines.

Lipidomics is focusing on the screening of lipid species in complex mixtures using mass spectrometry-based approaches. In this work, we aim to enhance the intestinal lipidome coverage within the Oligo-Mouse-Microbiota (OMM¹²) colonized mouse model by testing eight mobile phase conditions on five reversed-phase columns. Our selected mobile phase modifiers included two ammonium salts, two concentrations, and the addition of respective acids at 0.1 %. We compared two columns with hybrid surface technology, two with ethylene bridged hybrid technology and one with core–shell particles. Best performance was attained for standards and intestinal lipidome, using either ammonium formate or acetate in ESI(+) or ammonium acetate in ESI(−) for all column technologies. Notably, a concentration of 5 mM ammonium salt showed optimal results for both modes, while the addition of acids had a negligible effect on lipid ionization efficiency. The HST BEH C18 column improved peak width and tailing factor parameters compared to other technologies. We achieved the highest lipid count in colon and ileum content, including ceramides, phosphatidylethanolamines and phosphatidylcholines, when using 5 mM ammonium acetate in ESI(−). Conversely, in ESI(+) 5 mM ammonium formate demonstrated superior coverage for diacylglycerols and triacylglycerols.