Journal of Chromatography B最新文献

β-propiolactone (BPL) is an alkylating agent used for inactivation of biological samples such as vaccines. Due to its known carcinogenic properties, complete hydrolysis of BPL is essential, and the detection of trace amounts is crucial. In this study a novel High-Performance Liquid Chromatography-Ultraviolet (HPLC-UV) method was developed. Rhodamine B hydrazide (RBH) was synthesized and utilized as a derivatizing reagent to react with BPL. The reaction was optimized in a weak acidic solution, resulting in a high yield. The separation of the RBH-derivatized BPL was achieved on a C8 column and detected by a UV detector at a wavelength of 560 nm. The method's validation demonstrated a high linearity (r2 > 0.99) over a concentration range of 0.5–50 µg/mL, with detection and quantification limits of 0.17 µg/mL and 0.5 µg/mL, respectively. The average recovery of samples was 85.20 % with a relative standard deviation (RSD) of 1.75 %. This method was successfully applied for BPL residue analysis in inactivated COVID-19 vaccines. This novel derivatization method offers a promising solution for monitoring BPL residues in the vaccine production process for quality control purposes and compliance with regulatory standards.

The isoelectric focusing has realized various improvements, including the protocols and creation of mIEF (microcolumn isoelectric focusing) instruments with excellent sensitivity for screening of diabetes and beta thalassemia. However, the problem of manual sample loading and hydration for the mIEF limits the operational capacity for stably detecting and quantitating most abnormal hemoglobin (Hb). Herein, we provided a high stable sample loading protocol for analysis of alpha thalassemia and Hb variants. In contrast to the previous volume of 20 μl, a 100 µl blood sample solution in this protocol was optimized with mixture of 6.4–7.5 and 3–10 pH carrier ampholytes, pI markers and loaded for 30 mins IPG microcolumn hydration. The hydrated microcolumn was then automatically loaded onto the mIEF chip array to which CH₃COOH and NH₄OH act as anodic and cathodic solutions. Lastly, the IEF was run for 9 mins. Hb H, Barts, A_1c, F, A₂ and CS were simultaneously separated and focused with higher resolution and sensitivity in quantifying H and Barts as low as 0.6 and 0.5 % respectively. Accordingly, there was an enhanced stability and linearity with a rapid assay time of 45 secs per sample. Moreover, analysis showed a fitting linear relationship with conventional technology at R² = 0.9803 for H and R² = 0.9728 for Barts thereby indicating greater accuracy confirmed by the AUC. Hence, the developed protocol could simply be employed for high stable and throughput batch sample loading of hydration, and accurate separation and quantitation of Hb variants for alpha and beta thalassemia.

An imidacloprid colloidal gold immunochromatographic strip was developed in this work, and systematic analytical conditions were deeply investigated. The test strips were used for rapid screening of imidacloprid residues in Chinese herbal medicines. The performance of the colloidal gold test strips was investigated by using five selected Chinese herbal medicines (malt, Coix seed, lotus seed, dried ginger and honeysuckle). As a result, the developed imidacloprid colloidal gold immunochromatographic test strips could be used for rapid screening of imidacloprid residues in 60 kinds of different herbs (including 26 kinds of root/rhizome medicines, 20 kinds of seed/fruit/pericarp medicines, 11 kinds of flower/leaf/whole herb medicines, and 3 kinds of bark/aboveground issues of herb medicines), and the cut-off value was 50 μg/kg. The development of this method can achieve the goal of on-site, rapid and low-cost screening of imidacloprid residues in different herbs, which is of great significance for the quality assurance of herbs.

Microbial volatile organic compounds (MVOCs) are thought to play a key role in the interactions between mycoparasitic fungi, such as the biocontrol agent Trichoderma atroviride (T. atroviride), and their environment. However, the analysis of MVOC emissions from fungal samples is challenging because of low analyte concentrations, typically in the ppb_V-range, and the complex chemical nature of biological samples. In a recent study using proton transfer reaction-time of flight-mass spectrometry (PTR-ToF-MS) to determine MVOC emissions from T. atroviride, many product ions were unspecific, as they could arise from a large number of possible analytes. The aim of the present study was to determine whether fast gas chromatography (fast-GC) coupled to PTR-ToF-MS could be used to overcome this issue and constitute a suitable on-line, near real-time method to identify and quantify fungal MVOC emissions in the ppb_V-to-ppm_V regime. Using gas standards of eleven MVOCs known to be emitted by T. atroviride such as 6-amyl-α-pyrone (6-PP), 2-pentylfuran, 1-octen-3-ol, 2-heptanone, 3-octanone, 2-methyl-1-propanol, 2-pentanone, 3-methyl-1-butanol, 3-methylbutanal, acetone and ethanol, we developed a fast-GC method with a total runtime of 180 s which significantly enhances the analytical specificity of PTR-ToF-MS compared to conventional PTR-ToF-MS without fast-GC separation. Limits of detection were on the order of 0.1–4 ppb_V. The increased analytical specificity demonstrated notable benefits, especially for MVOCs having partially overlapping distributions of product ions when analyzed directly using PTR-ToF-MS.

In order to demonstrate the applicability of the analytical method, we analysed T. atroviride samples in four biological replicates twice daily over a duration of five days. Using the fast-GC method, nine out of the eleven MVOC species considered in this study in the headspace of T. atroviride could be identified and quantified and their time evolution over the five-day incubation period determined. The measured volume mixing ratios (VMRs) ranged from single-digit ppb_V (2-pentylfuran) up to few ppm_V (6-PP and ethanol), with the other compounds in the 10-to-100-ppb_V range (1-octen-3-ol, 2-heptanone, 2-methyl-1-propanol, 3-methyl-1-butanol, 3-methylbutanal and acetone).

Our results suggest that fast-GC-PTR-ToF-MS is a method well-suited for the analysis of gas-phase samples of biological origin, including but not limited to (mycoparasitic) fungi, in a wide range of VMRs from sub-ppb_V to few-ppm_V.

Amiodarone and mexiletine are used for ventricular arrhythmias, for which a combination therapy of both anti-arrhythmic drugs (AADs) is not uncommon. Therapeutic drug monitoring (TDM) can be beneficial for clinical guidance of therapy, especially to correctly identify adverse events. Desethylamiodarone, the active metabolite of amiodarone, accumulates over time and is associated with serious adverse events. Therefore, simultaneous TDM for amiodarone, desethylamiodarone and mexiletine is advantageous in clinical practice. The presented LC-MS/MS method was validated for selectivity, matrix effect, linearity, accuracy, precision, carry-over and stability. The method was continuously evaluated during eight months of clinical use. The method was shown to be linear within the measured range of 0.1 to 10 mg/L for each component. The matrix effect was considered negligible. No interfering responses were found for amiodarone, desethylamiodarone and the isotopic-labeled internal standards. A constant and reproducible within-run contribution of 45.3 %, originating from the system, was identified for mexiletine. The systemic contribution to the peak area of the lowest quantifiable concentration of mexiletine affected the selectivity and carry-over effect measurements. Multiple measurements showed that regression adjusted concentrations were accurate and reproducible, indicating calibration correction was applicable. Sample stability was found to be within limits for all storage conditions and freeze–thaw cycles. Furthermore, long-term method evaluation with external controls resulted in stable measurements with a percentage coefficient of variance between 1.3 % and 6.3 %. The presented practical and reliable method is applicable for clinical TDM and will allow clinical practitioners to guide drug therapy of amiodarone and mexiletine.

Metalloproteins binding with trace elements play a crucial role in biological processes and on the contrary, those binding with exogenous heavy metals have adverse effects. However, the methods for rapid, high sensitivity and simultaneous analysis of these metalloproteins are still lacking. In this study, a fast method for simultaneously determination of both essential and toxic metal-containing proteins was developed by coupling size exclusion chromatography (SEC) with inductively coupled plasma tandem mass spectrometry (ICP-MS/MS). After optimization of the separation and detection conditions, seven metalloproteins with different molecular weight (from 16.0 to 443.0 kDa) were successfully separated within 10 min and the proteins containing iron (Fe), copper (Cu), zinc (Zn), iodine (I) and lead (Pb) elements could be simultaneously detected with the use of oxygen as the collision gas in ICP-MS/MS. Accordingly, the linear relationship between log molecular weight and retention time was established to estimate the molecular weight of unknown proteins. Thus, the trace metal and toxic metal containing proteins could be detected in a single run with high sensitivity (detection limits in the range of 0.0020–2.5 μg/mL) and good repeatability (relative standard deviations lower than 4.5 %). This method was then successfully used to analyze metal (e.g., Pb, Zn, Cu and Fe) binding proteins in the blood of Pb-intoxicated patients, and the results showed a negative correlation between the contents of zinc and lead binding proteins, which was identified to contain hemoglobin subunit. In summary, this work provided a rapid and sensitive tool for screening metal containing proteins in large number of biological samples.

Milk is one of the most widely consumed foods globally. To protect consumer interests, it is essential to establish an analytical method to detect the degree of heating in milk. A novel approach using nano liquid chromatography-orbitrap fusion mass spectrometer was developed for screening and identifing thermally sensitive peptides markers in the milk heating process (below 100 °C). This method integrates untargeted proteomics and chemometric tools to analyze protein quantitation data from differently heat-treated milk. Thirteen potential markers were screened out and identified, and further confirmed using by standard substances. Then, the accurate concentrations of 13 potential markers determined by isotope-dilution ultra-performance liquid chromatography-tandem triple quadrupole mass spectrometry were further mining the highly specific and thermally sensitive peptides markers. And Four peptides—INLFDTPLETQYVR, FELLGCELNGCTEPLGLK, QFQFIQVAGR, and GEADALNLDGGYIYTAGK—were selected as marker peptides to differentiate normal pasteurized milk from overheated pasteurized milk. The concentrations of INLFDTPLETQYVR ranges from 150 ± 11 µg/L to 350 ± 23 µg/L, while the concentrations of FELLGCELNGCTEPLGLK ranges from 40 ± 5 µg/L to 92 ± 3 µg/L, can distinguish normal pasteurized milk from overheated pasteurized milk. QFQFIQVAGR indicates overheated pasteurized milk at 230 ± 21 µg/L, and GEADALNLDGGYIYTAGK signifies 750 ± 43 µg/L. This study provides new insights for distinguishing overheated pasteurized milk.

An integrated method combining solid-phase extraction (SPE) with ultra-performance liquid tandem mass spectrometry (UPLC-MS/MS) has been established for quantifying bacitracin (BTC), bacitracin zinc (BZ), and bacitracin methylene disalicylate (BMD) in animal feed. A pretreatment procedure that can effectively, quickly, and simultaneously extract and purify BTC, BZ, or BMD in feed was developed for the first time through the optimization of extraction and SPE conditions. After extraction with acetonitrile + methanol + 15 % ammonia solution (1:1:1, v:v:v) and dilution with EDTA solution (1.5 mmol/L, pH 7.0), a SPE procedure was carried out with C₁₈ cartridge. Following LC-MS/MS analysis utilized a Waters Peptide BEH C₁₈ column with a gradient elution of 0.1 % formic acid in water/acetonitrile with. This method demonstrated a strong linear correlation (R² > 0.9980) across a 0.01–1.0 mg/L concentration span, based on a matrix-matched standard curve. Satisfactory recoveries of BTC (bacitracin A, B₁, B₂, and B₃), BZ, and BMD in different feeds were obtained from 80.7 % to 108.4 %, with relative standard deviations below 15.7 %. Low limits of quantification ranging within 7.2–20 μg/kg were achieved for bacitracin A, B₁, B₂, and B₃. This method provided an effective and reliable detection method to prevent the addition of BTC and different BTC formulations in feeds.

Diabetic nephropathy (DN) remains the primary cause of end-stage renal disease (ESRD), warranting equal attention and separate analysis of glomerular, tubular, and interstitial lesions in its diagnosis and intervention. This study aims to identify the specific proteomics characteristics of DN, and assess changes in the biological processes associated with DN. 5 patients with DN and 5 healthy kidney transplant donor control individuals were selected for analysis. The proteomic characteristics of glomeruli, renal tubules, and renal interstitial tissue obtained through laser capture microscopy (LCM) were studied using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Significantly, the expression of multiple heat shock proteins (HSPs), tubulins, and heterogeneous nuclear ribonucleoproteins (hnRNPs) in glomeruli and tubules was significantly reduced. Differentially expressed proteins (DEPs) in the glomerulus showed significant enrichment in pathways related to cell junctions and cell movement, including the regulation of actin cytoskeleton and tight junction. DEPs in renal tubules were significantly enriched in glucose metabolism-related pathways, such as glucose metabolism, glycolysis/gluconeogenesis, and the citric acid cycle. Moreover, the glycolysis/gluconeogenesis pathway was a co-enrichment pathway in both DN glomeruli and tubules. Notably, ACTB emerged as the most crucial protein in the protein–protein interaction (PPI) analysis of DEPs in both glomeruli and renal tubules. In this study, we delve into the unique proteomic characteristics of each sub-region of renal tissue. This enhances our understanding of the potential pathophysiological changes in DN, particularly the potential involvement of glycolysis metabolic disorder, glomerular cytoskeleton and cell junctions. These insights are crucial for further research into the identification of disease biomarkers and the pathogenesis of DN.

Peganum harmala L., a traditional medicinal plant in China, is renowned for its significant alkaloid content in seeds and roots exhibiting a wide range of pharmacological activities, including antidepressant, antiseptic, and antiviral. However, the volatile composition of the herb remained unclear. Apart from that, the extraction of volatile compounds through essential oil presents challenges due to the low yield and the degradation of volatile active compounds at high temperatures. This study used multiple sample preparation methods including headspace (HS), needle trap device (NTD), and liquid–liquid extraction (LLE) coupled with gas chromatography-mass spectrometry (GC–MS) to analyze the volatile compounds from the areal part of P. harmala L.. A total of 93 compounds were identified with NTD facilitating the first detection of harmine among the volatile organic compounds. Through network pharmacology and protein interaction analysis, the compounds’ potential therapeutic targets of the compounds were explored, and 23 key targets were obtained (AKT1, ALB, PTGS2, MAOA, etc). KEGG pathway enrichment analysis indicated significant involvement in neuroactive ligand-receptor interactions and serotonergic synapses. The results enhanced the understanding of P. harmala’s pharmacological mechanisms and supported its ethnopharmacological use.