•A HPLC–MS/MS method was developed to detect hydrazine and acetylhydrazine in human plasma.•A new derivatization reagent p-tolualdehyde was applied.•Sample pretreatments were simple without additional heating and purification.A high performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) method was developed for simultaneous quantitative analysis of hydrazine and acetylhydrazine in human plasma based on the strategy of p-tolualdehyde derivatization. The derivatization reactions were easily realized by ultrasonic manipulation for 40 min. Good separation of the derivatization products was achieved using a C18 column by gradient elution. The optimized mass transition ion-pairs (m/z) monitored for the two hydrazine derivatives were m/z 237.1 ≫> 119.9 and m/z 176.9 ≫> 117.8, respectively. The limit of detection (LOD) and limit of quantification (LOQ) for hydrazine were 0.002 and 0.005 ng mL−1 separately. And they were 0.03 and 0.05 ng mL−1 for acetylhydrazine, respectively. The linear range was 0.005–50 ng mL−1 for hydrazine and 0.05–500 ng mL−1 for acetylhydrazine with R2 greater than 0.999. The recovery range was determined to be 95.38–108.12% with the relative standard deviation (RSD) in the range of 1.24–14.89%. The method was successfully applied to detect 30 clinical plasma samples of pulmonary tuberculosis patients treated with isoniazid. The concentrations were from 0.04–1.99 ng mL−1 for hydrazine and 0.06–142.43 ng mL−1 for acetylhydrazine. The results indicated that our developed method had the potential for the detection of hydrazine toxicology in complex biological samples. Furthermore, the method has an important significance to clinical treatment with drugs.

•An integrated microfluidic platform is established to recapitulate in vivo blood-brain barrier and glioma microenvironment.•The drug permeability is directly analyzed by ESI MS after μSPE pretreatment.•It is the first report to simultaneously evaluate drug permeability and cytotoxicity of CNS drug candidates.•The device enables the rapid analysis of drug candidates, which may accelerate the drug development.Since most of the central nervous system (CNS) drug candidates show poor permeability across the blood-brain barrier (BBB), development of a reliable platform for permeability assay will greatly accelerate drug discovery. Herein, we constructed a microfluidic BBB model to mimic drug delivery into the brain to induce cytotoxicity at target cells. To reconstitute the in vivo BBB properties, human cerebral microvessel endothelial cells (hCMEC/D3) were dynamically cultured in a membrane-based microchannel. Sunitinib, a model drug, was then delivered into the microchannel and forced to permeate through the BBB model. The permeated amount was directly quantified by an electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-Q-TOF MS) after on-chip SPE (μSPE) pretreatment. Moreover, the permeated drug was incubated with glioma cells (U251) cultured inside agarose gel in the downstream to investigate drug-induced cytotoxicity. The resultant permeability of sunitinib was highly correlated with literature reported value, and it only required 30 min and 5 μL of sample solution for each permeation experiment. Moreover, after 48 h of treatment, the survival rate of U251 cells cultured in 3D scaffolds was nearly 6% higher than that in 2D, which was in accordance with the previously reported results. These results demonstrate that this platform provides a valid tool for drug permeability and cytotoxicity assays which have great value for the research and development of CNS drugs.A microfluidic blood-brain barrier model was developed to evaluate drug permeability using MS detection and assess antitumor activity for central nervous system drug screening.

Recently, MALDI-TOF MS has emerged as a popular analytical tool with the advantages of easy sample preparation and good salt tolerance, as well as fast and high throughput data acquisition ability. However, for the existing matrices, they were limited in detecting low-molecular-weight analytes due to the matrix-related peaks and low reproducibility. Therefore, the development of new matrices is highly desirable. Here, a new matrix N,S-CDs, for the first time, was synthesized and used for small molecule analysis. Comprehensive assessments of N,S-CDs were conducted including structural characterization, comparisons with conventional CHCA, 9-AA and N-CDs matrices in detecting amino acids, peptides, nucleosides and anticancer drugs in different ion modes. The results showed that N,S-CDs could be a sensational matrix which had good signal intensity of the analytes without matrix-related interference, especially in negative ion mode. What's more, the N,S-CDs matrix displayed good salt tolerance and signal reproducibility. In addition, the N,S-CDs matrix was further applied for serum detection, quantitative determination of endogenous glucose, and the classification of different subtypes of breast cancer cell lines. The detection of various endogenous metabolites in serum analysis and significantly distinguishing between two cell lines indicated the ability of our new matrix to be applied in complex biological samples. Furthermore, the ionization mechanism of the N,S-CDs as a matrix was also discussed comparing with N-CDs in the transformation of structure and composition. This work creates a new application branch for N,S-CDs and provides an improved MALDI matrix for small molecule analysis.

Application of matrix-assisted laser-desorption/ionization mass spectrometry (MALDI MS) to analyze small molecules have some limitations, due to the inhomogeneous analyte/matrix co-crystallization and interference of matrix-related peaks in low m/z region. In this work, carbon dots (CDs) were for the first time applied as a binary matrix with 9-Aminoacridine (9AA) in MALDI MS for small molecules analysis. By 9AA/CDs assisted desorption/ionization (D/I) process, a wide range of small molecules, including nucleosides, amino acids, oligosaccharides, peptides, and anticancer drugs with a higher sensitivity were demonstrated in the positive ion mode. A detection limit down to 5 fmol was achieved for cytidine. 9AA/CDs matrix also exhibited excellent reproducibility compared with 9AA matrix. Moreover, by exploring the ionization mechanism of the matrix, the influence factors might be attributed to the four parts: (1) the strong UV absorption of 9AA/CDs due to their π-conjugated network; (2) the carboxyl groups modified on the CDs surface act as protonation sites for proton transfer in positive ion mode; (3) the thin layer crystal of 9AA/CDs could reach a high surface temperature more easily and lower transfer energy for LDI MS; (4) CDs could serve as a matrix additive to suppress 9AA ionization. Furthermore, this matrix was allowed for the analysis of glucose as well as nucleosides in human urine, and the level of cytidine was quantified with a linear range of 0.05–5 mM (R2 > 0.99). Therefore, the 9AA/CDs matrix was proven to be an effective MALDI matrix for the analysis of small molecules with improved sensitivity and reproducibility. This work provides an alternative solution for small molecules detection that can be further used in complex samples analysis.

Single-cell trapping and high-throughput mass spectrometry analysis remain challenging now. Current technologies for single-cell analysis have several limitations, such as throughput, space resolution, and multicomponent analysis. In this study, we demonstrate, for the first time, the combination of microfluidic chip and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for high-throughput and automatic single-cell phospholipids analysis. A microwell-array-based microfluidic chip was designed and fabricated for cell array formation on an indium tin oxide (ITO)-coated glass slide. Mass spectrometry imaging measurement with 25 μm pixel size was performed with a MALDI ion source. Eight phospholipids in a single A549 cell were detected, and their structures were further identified by MS/MS spectra. Selected ion images were generated with a bin width of Δm/z ± 0.005. The selected ion images and optical images of the cell array showed excellent correlation, and mass spectrometry information on phospholipids from 1–3 cells was extracted automatically by selecting pixels with the same fixed interval between microwells on the chip. The measurement and data extraction could be processed in several minutes to achieve a high-throughput analysis. Through the optimization of different microwell sizes and different matrices, this method showed potential for the analysis of other metabolites or metabolic changes at the single-cell level.

•The interaction of HSA with a flavonoid derivative (3d) we designed was investigated.•3d can bind to HSA with high affinity and it can be transported and deposited by HSA.•Molecular modeling showed that 3d can enter into a hydrophobic cavity of HSA at its subdomain IIA.•The conformation of HSA changed due to the formation of 3d–HSA complex.Human serum albumin (HSA) has been developed as a model protein to study drug–protein interaction. In the present work, the interaction between our synthesized flavonoid derivative 3d (possessing potent antitumor activity against HepG2 cells) and HSA was investigated using fluorescence spectroscopy, circular dichroism spectroscopy, UV–vis spectroscopy and molecular modeling approach. Fluorescence spectroscopy showed that the fluorescence of HSA can be quenched remarkably by 3d under physiological condition with a slight shift of maximum fluorescence emission bands from 360 nm to 363 nm. Calculated results from Stern–Volmer equation and modified Stern–Volmer equation indicated that the fluorescence was quenched by static quenching processing with association constant 5.26±0.04×104 L mol−1 at 298 K. After comprehensive consideration of the free energy change ΔG, enthalpy change ΔH and entropy change ΔS, electrostatic interactions were confirmed as the main factor that participate in stabilizing the 3d–HSA complex. Both dichroism spectroscopy and UV–vis spectroscopy indicated conformational change of HSA after binding to 3d. Moreover, the structure of HSA was loosened and the percentage of α-helix decreased with increasing concentration of 3d. Molecular modeling results demonstrated that 3d could bind to HSA well into subdomain IIA, which is related to its capability of deposition and delivery. Three cation–π interactions and three hydrogen bonds occurred between 3d and amino acid residuals ARG218, ARG222 and LYS199. In conclusion, flavonoid derivative 3d can bind to HSA with noncovalent bond in a relatively stable way, so it can be delivered by HSA in a circulatory system.

A sensitive and robust on-line LC/MS method was developed for quantitative determination of linoleic acid, docosahexaenoic acid and docosanoic acid from edible oil samples. The oil samples were dissolved in chloroform-isopropyl alcohol (20:80, v:v) solution and the three fatty acids were separated by HPLC with a C4 column using 10 mmol/L ammonium acetate-isopropyl alcohol-acetonitrile (20:40:40, v:v:v) mobile phase in isocratic elution. Electrospray ionization mass spectrometry with the selected ion recording monitoring was used to detect and quantify the fatty acid. The calibration curves were linear in the range of 10.00–5000 pg/mL for linoleic acid and docosanoic acid, and 1.000–500.0 pg/mL for docosahexaenoic acid. The limit of detection was 2.0 pg/mL for linoleic acid, 3.0 pg/mL for docosanoic acid, and 0.20 pg/mL for docosahexaenoic acid. The results showed that the method described in this paper could be utilized for rapid determination of three fatty acids at picogram levels in edible oils.