nqiu Yu

Optimization of mobile phase for the determination of Esomeprazole and related compounds and investigation of stress degradation by LC–MS

In this study, the objective was to investigate the degradation behavior of Esomeprazole under different recommended stress conditions according to International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use [1] by HPLC. Our research showed that the effect of mobile phase species on separation was significant for the determination of Esomeprazole and its related compounds. Successful separation of the drug from its related impurities and degradation products formed under different stress conditions was achieved using ammonium acetate buffer/ACN by a gradient elution. Compared with phosphate buffer/ACN, ammonium acetate buffer/ACN under same pH and gradient showed a great improvement in resolution due to the change of elution order. The drug was subjected to stress conditions including acidic, alkaline, oxidative, photolytic, and thermal conditions. Extensive degradation occurred in acidic and oxidative conditions, while mild degradation was observed in alkaline and photolytic conditions. Besides, it turned out the drug was extremely stable under thermal condition. The stability-indicating LC-UV method was validated with respect to linearity, precision, accuracy, specificity, and robustness. The LC-MS method was also adopted for the characterization of degradation products. Based on the m/z values and fragmentation patterns, the degradation pathway of the drug has been proposed.

Simultaneous determination of six toxic alkaloids in human plasma and urine using capillary zone electrophoresis coupled to time‐of‐flight mass spectrometry

A novel capillary zone electrophoresis separation coupled to electro spray ionization time-of-flight mass spectrometry method was developed for the simultaneous analysis of six toxic alkaloids: brucine, strychnine, atropine sulfate, anisodamine hydrobromide, scopolamine hydrobromide and anisodine hydrobromide in human plasma and urine. To obtain optimal sensitivity, a solid-phase extraction method using Oasis MCX cartridges (1 mL, 30 mg; Waters, USA) for the pretreatment of samples was used. All compounds were separated by capillary zone electrophoresis at 25 kV within 12 min in an uncoated fused-silica capillary of 75 μm id × 100 cm and were detected by time-of-flight mass spectrometry. This method was validated with regard to precision, accuracy, sensitivity, linear range, limit of detection (LOD), and limit of quantification (LOQ). In the plasma and urine samples, the linear calibration curves were obtained over the range of 0.50-100 ng/mL. The LOD and LOQ were 0.2-0.5 ng/mL and 0.5-1.0 ng/mL, respectively. The intra- and interday precision was better than 12% and 13%, respectively. Electrophoretic peaks could be identified by mass analysis.

Exploration of Candidate Biomarkers for Human Psoriasis Based on GC-MS Serum Metabolomics.

Recent studies have shown that dysregulated metabolic pathways are linked to psoriasis pathogenesis. However, an extensive, unbiased metabolic analysis in patients with psoriasis has not been completely explored. The metabolome represents the end products of proteomics or cellular processes that may be closely associated with the pathogenesis of psoriasis.

Metabolomics investigation of cutaneous T cell lymphoma based on UHPLC-QTOF/MS.

OBJECTIVES The identification of cutaneous T cell lymphoma (CTCL) biomarkers may serve as a predictor of disease progression and treatment response. The aim of this study was to map potential biomarkers in CTCL plasma. DESIGN AND METHODS Plasma metabolic perturbations between CTCL cases and healthy individuals were investigated using metabolomics and ultrahigh performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). RESULTS Principal component analysis (PCA) of the spectra showed clear metabolic changes between the two groups. Thirty six potential biomarkers associated with CTCL were found. CONCLUSIONS Based on PCA, several biomarkers were determined and further identified by LC/MS/MS analysis. All of these could be potential early markers of CTCL. In addition, we established that heparin as anticoagulant has better pre-treatment results than EDTA with the UHPLC-QTOF/MS approach.

A development of LC–MS method combining ultrafiltration and lyophilization for determination of r-RGD-Hirudin in human serum

A reliable and validated LC-MS method was established for determination of r-RGD-Hirudin in human serum. Ultrafiltration was used instead of liquid-liquid extraction or solid phase extraction for water solubility drug r-RGD-Hirudin extraction. Freeze drying was used for concentration. The experiment conditions, including pre-processing procedure and LC-MS, have been investigated and optimized. Comparing with reported assays, the current method showed significant improvement in specificity, linearity, precision and sensitivity. This method has been successfully applied in clinical research of r-RGD-Hirudin.

Cross-platform metabolomics investigating the intracellular metabolic alterations of HaCaT cells exposed to phenanthrene

Phenanthrene (Phe) is one of the most abundant Polycyclic aromatic hydrocarbons (PAHs) contamination from various ambient sources, which has a tremendous impact on public health. However, our knowledge regarding its effects on skin remains limited. In this study, we investigated the metabolite profiling of the human keratinocytes HaCaT cells after Phe exposure to understand the toxic effects of Phe exposure on skin. To obtain a broad picture of metabolome with various hydrophilicity, a cross-platform approach with GC-MS and UHPLC-QTOF-MS has been employed. Data were analyzed by multivariate statistical analysis and samples were separated successfully using supervised PLS-DA models. It was shown that the impacts of Phe exposure on HaCaT cells were both dose-related and time-related. A total of 48 Phe-regulated metabolites were identified and among which 19 were confirmed by reference standards. By pathway analysis, amino acid metabolism, glutathione metabolism and glycerophospholipid metabolism were highlighted as the major metabolic pathways disturbed by Phe. Furthermore, it was found that the mechanisms included a reduced amino pool and a reduced antioxidant status. Overall, these results aid in improving understanding of the dermal toxicology related to Phe, and demonstrate this cross-platform approach is suitable for metabolomics researches on HaCaT cells.

A sensitive method for digoxin determination using formate-adduct ion based on the effect of ionization enhancement in liquid chromatograph-mass spectrometer.

A sensitive and rapid method based on formate-adduct ion detection was developed and fully validated for digoxin determination in rat plasma. For LC/MS/MS detection with formate-adducts as precursor ions, transitions of m/z 825.5→779.9 for digoxin and m/z 809.5→763.4 for the internal standard (digitoxin) were monitored in negative mode. To investigate the impact of formic acid on the mass response and method sensitivity, a formic acid concentration range of 0-0.1% (0, 0.0005%, 0.002%, 0.01%, 0.1%, v/v) was evaluated. A concentration of 0.002% gave the highest sensitivity, which was 16- to 18-fold higher than deprotonated ions, and was designated as the contribution giving the strongest ionization enhancement and adduction. A number of parameters were then varied in order to optimize the method, and a limit of quantitation (LOQ) at 0.2 ng/mL was reached with an injection volume of 5 μL, a total run time of 3 min, and 0.1 mL of rat plasma. A calibration curve was plotted over the range 0.2-50 ng/mL (R(2)=0.9998), and the method was successfully applied to study pharmacokinetics in rat following a single oral administration of digoxin (0.05 mg/kg). Four additional steroid saponins (digitoxin, deslanoside, ginsenoside Rg1 and Rb1) were investigated to assess the impact of formic acid on the mass response of steroid saponins. Compounds with a conjugated lactonic ring in their structures such as digoxin, digitoxin and deslanoside tended to form stable formate-adduct ions more easily. The LC/MS/MS method developed here is therefore well suited for the quantification of steroid saponins that are difficult to deprotonate using other MS approaches.

Serum metabolite profiling of cutaneous T-cell lymphoma based on a multiplatform approach

Cutaneous T-cell lymphoma (CTCL) is a class of non-Hodgkin lymphoma with a difficult early diagnosis. The overall annual age-adjusted incidence of CTCL had consistently increased to around 10.2 cases per million persons. However, our knowledge regarding its mechanism of disease origin and progression remains unclear. In this study, serum samples from 31 CTCL patients and 31 matched healthy volunteers were analyzed in depth to screen metabolites capable of differentiating CTCL from controls. To obtain a higher coverage of metabolome with various hydrophilicity, a multiplatform approach with GC-MS and UHPLC-QTOF-MS has been employed. Data were analyzed by multivariate statistical analysis and CTCL group was separated from control group successfully using supervised OPLS-DA model. A total of 51 CTCL-regulated metabolites were identified, among which 15 differential metabolites have an AUC > 0.9 in receiver operating characteristic (ROC) curve analysis. Glycerophospholipid metabolism, tryptophan metabolism and purine metabolism were highlighted as 3 major altered pathways in CTCL serum. These alterations revealed impacts to membrane stability and weakened immune as well as ATP depletion associated with CTCL. Overall, these results aid in improving understanding of the mechanism related to CTCL, and demonstrate this multiplatform approach is suitable for serum metabolomics researches.

Sorting nexin 10 controls mTOR activation through regulating amino-acid metabolism in colorectal cancer

Amino-acid metabolism plays a vital role in mammalian target of rapamycin (mTOR) signaling, which is the pivot in colorectal cancer (CRC). Upregulated chaperone-mediated autophagy (CMA) activity contributes to the regulation of metabolism in cancer cells. Previously, we found that sorting nexin 10 (SNX10) is a critical regulator in CMA activation. Here we investigated the role of SNX10 in regulating amino-acid metabolism and mTOR signaling pathway activation, as well as the impact on the tumor progression of mouse CRC. Our results showed that SNX10 deficiency promoted colorectal tumorigenesis in male FVB mice and CRC cell proliferation and survival. Metabolic pathway analysis of gas chromatography–mass spectrometry (GC-MS) data revealed unique changes of amino-acid metabolism by SNX10 deficiency. In HCT116 cells, SNX10 knockout resulted in the increase of CMA and mTOR activation, which could be abolished by chloroquine treatment or reversed by SNX10 overexpression. By small RNA interference (siRNA), we found that the activation of mTOR was dependent on lysosomal-associated membrane protein type-2A (LAMP-2A), which is a limiting factor of CMA. Similar results were also found in Caco-2 and SW480 cells. Ultra-high-performance liquid chromatography–quadrupole time of flight (UHPLC-QTOF) and GC-MS-based untargeted metabolomics revealed that 10 amino-acid metabolism in SNX10-deficient cells were significantly upregulated, which could be restored by LAMP-2A siRNA. All of these amino acids were previously reported to be involved in mTOR activation. In conclusion, this work revealed that SNX10 controls mTOR activation through regulating CMA-dependent amino-acid metabolism, which provides potential target and strategy for treating CRC.