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Core-shell structured magnetic covalent-organic frameworks (Fe

Lysophosphatidylcholine (LPC) was commonly known as a class of significant differential metabolites of high relevance with many diseases including psoriasis, of which the accurate determination is of great importance to diagnosis or prediction to many diseases. However, it is challenging and complicated because of the enormous biological sample complexity and impurities interference. In this study, we synthesized a magnetic nanocomposite MG@PD@TiO2 and took advantage of the interactions of Lewis acid-base between the phosphate groups in LPCs and Ti ions on MG@PD@TiO2 nanomaterials for selective separation and enrichment of LPCs from complex biological matrix. The solid-phase extraction sample pretreatment process by means of MG@PD@TiO2 nanomaterials coupled with LC-MS/MS method was then applied to actual determination of six typical LPCs (LPC 10:0, 14:0, 16:0, 18:0, 18:1, 22:0) in human plasma. The extraction conditions were scientifically optimized by single-factor test (adsorbent amount, adsorption and desorption time, elution solvent type, eluant volume). Under the optimal conditions, the detection limits (LOD, S/N = 3) and quantification limits (LOQ, S/N = 10) were 1 and 5 ng/mL for LPC 10:0 and LPC 14:0, 0.02 and 0.1 ng/mL for LPC 16:0 and LPC 18:1, 0.05 and 0.2 ng/mL LPC 18:0 and LPC 22:0, respectively. The intra- and inter-day precisions were 3.82-12.60 % (n = 6) and 3.29-13.50 % (n = 6) respectively, the recoveries were in the range of 91.92-113.69 % and the stability of the analytes in the matrix performed well with RSDs≤15.51 %. Finally, the developed method was successfully applied to the accurate determination of six LPCs biomarkers of plasma in patients with psoriasis (n = 10) and control groups (n = 10).

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.

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.

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.

An in vivo study of Strychnos alkaloids metabolites in rats by ultra high performance liquid chromatography with linear ion trap Orbitrap MS is reported for the first time. Two major Strychnos alkaloids compounds including strychnine and brucine were investigated. To obtain optimal extraction efficiency, samples were pretreated by using an SPE plate. The structures of metabolites and their fragment ions were characterized based on the accurate mass and MSn data. Forty-seven metabolites were identified in rat urine, of which 25 were reported for the first time. Four new metabolism pathways were proposed on the basis of the identified metabolites. This study provides a practical approach for rapidly identifying complicated metabolites, a methodology that could be widely applied not only in forensic and clinically toxicological relevant cases, but also for the structural characterization of metabolites of other compounds.

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.

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.

For the first time a sensitive, specific and rapid LC–MS–MS assay is presented for the simultaneous determination of levodopa (L-DP), 3-O-methyldopa (3-OMD) and benserazide (BSZ) in human serum. The three compounds were extracted from human serum by protein precipitation followed by dilution of the supernatant with aqueous formic acid. In serum, linearity was observed between 50 and 1,000 ng mL−1 of L-DP, 3-OMD and BSZ, respectively. Intra-day and inter-day RSD values were below 10.56 and 6.22% at concentrations of 120, 360 and 720 ng mL−1. The presented method showed excellent specificity and sensitivity compared with other methods reported. It was applied to a pharmacokinetic study and demonstrated its applicability to pre-clinical and clinical pharmacological research.

A reliable and validated LC–MS method was established for a hexokinase inhibitor study based on adenosine 5′-triphosphate (ATP) determination. By adding 5 mM ammonium acetate in the aqueous phase, this method enabled the determination of ATP by LC–MS and greatly increased the MS signal of ATP. This method was used in the study of the anticancer mechanism of Momordica cochinchinensis, an exact ingredient which had exhibited certain hexokinase inhibitor activity. This might reveal the anticancer mechanism of Momordica cochinchinensis.

A sensitive and effort-saving method was established and validated for the quantitative determination of recombinant Arg-Gly-Asp-hirudin (rRGD-hirudin) in human urine samples. The assay was performed on a uncoated fused silica capillary of 70 cm x 50 microm I.D. and a positive voltage of 30 kV was applied. The sample was injected under pressure of 50 mbar for 300 s and the temperature of capillary was kept 25 degrees C. Sheath liquid consisting of 30% methanol and 70% of 0.1% formic acid aqueous solution flowing at 7 microL/min was supplied to the CE-electrospray interface. Utilizing the dynamic pH junction technique, a lower limit of quantitation of approximately 35 nM was achieved (concentration coefficiency was about 100-fold) without complex sample preprocessing procedure. CE-MS conditions and parameters were also optimized to obtain better performance. The method has been successfully applied in clinical research of rRGD-hirudin.

Rhodamine 123 has been frequently used to evaluate the functional activity of P-glycoprotein, assess the related drug interactions, analyze mitochondrial distribution and function, sort functionally distinct cell subpopulations and measure mitochondrial or cellular membrane potential. We developed a sensitive and rapid liquid chromatographic method with fluorescence detection after simple sample preparation procedure for the determination of Rhodamine 123 in P-glycoprotein efflux studies. The mobile phase consisted of methanol and 15 mM dibasic sodium phosphate buffer (pH 6.0) (80:20,v/v), delivered at a rate of 1.0 mL min−1. 15 μL of the samples were injected into a reversed-phase C18 column with a fixed excitation wavelength at 505 nm and altered emission wavelengths. The whole LC analysis was accomplished within 6 min. The established linearity range from 1 to 100 ng mL−1, with the inter-day and intra-day RSD below 7.57 and 4.89% at concentrations of 4.4, 44 and 88 ng mL−1. All the calibration standards and quality controls were prepared in cell lysate and were stable for three freeze-thaw cycles, for 12 h at 4 °C and for 6 h at room temperature. This rapid LC method has been applied to the quantification of Rhodamine 123 in cell lysate obtained from P-glycoprotein efflux study conducted in rat brain capillary endothelial cells.

We report the development and validation of a high-performance liquid-chromatographic–tandem mass spectrometric method for determination of artemether (ARM) and its active metabolite dihydroartemisinin (DHA) in human plasma; artemisinin was used as internal standard (IS). Chromatographic separation was performed on a 150 mm × 4.6 mm i.d., 5 μm particle, C18 column coupled with a 4.0 mm × 3.0 mm i.d., 5 μm particle, C18 guard column. The mobile phase was acetonitrile–0.1% formic acid solution, 80:20 (v/v), at a flow-rate of 1 mL min−1. An atmospheric-pressure chemical-ionization (APCI) interface was used to produce sample ions, and positive ions were quantified by using the MS detector in selected-reaction-monitoring mode, using the reaction m/z 221 to 163 for determination of ARM and DHA and the reaction m/z 283 to 219 for determination of the IS. Plasma samples were prepared by extraction with methyl t-butyl ether, evaporation of the extract to dryness, and reconstitution of the residue with mobile phase. Extraction recovery for ARM and DHA ranged from 74.74 to 99.39%. High specificity and a limit of quantification of 5 ng mL−1 were achieved for ARM and DHA. Linearity was confirmed over the concentration range 5–500 ng mL−1; the correlation coefficients (R) were >0.99. The relative standard deviation for intra-day and inter-day assay of both compounds was

An LC-ESI-MS method was developed and validated for the assay of apomorphine in canine plasma using one-step liquid-liquid extraction. The analytes were separated on a Phenomenex Gemini C18 (150 mm x 2.0 mm id 3 microm) column and determined by MS in the positive ion mode. The linear range was 0.4-40 ng/mL with an LOD of 0.2 ng/mL for apomorphine in plasma. The intraday and interday precision and accuracy of quality control samples were5.9% RSD and7.5% bias for apomorphine. Extraction recoveries were80%. The validated method was successfully applied to analyze canine plasma samples in a pharmacokinetic study of apomorphine in dogs and detailed pharmacokinetic parameters were calculated.

An HPLC method was established for preparation of scutellarein from scutellarin using an enzymolysis reaction. The aglycone was identified as scutellarein with its chromatogram, UV, LC‐MS, and 1H‐NMR spectra. Acid hydrolysis was tried in preliminary experiments, but enzymolysis was adopted after optimization of reaction conditions. High purity scutellarein (content 98.04%∼98.36%) was prepared in this way for the first time, which could be used as a working reference substance.

A reliable and validated reversed-phase high-performance liquid chromatography (HPLC) method using fluorescence detection is reported for the simultaneous quantitation of mycophenolic acid (MPA) and valproic acid (VPA) in human plasma. The method is based on the pre-column derivatization of valproic acid with 4-bromomethyl-6, 7-dimethoxycoumarin (BrMMC) and online solvatochromism of MPA by pH adjustment. The linear calibration range was 0.50-30 microg/mL for MPA and 5.00-150 microg/mL for VPA. The relative standard deviations of the method of intra- and inter-day analyses (n = 6) were below 6.5 and 6.7% for MPA, and 5.8 and 6.3% for VPA, respectively. Dichloromethane was used for the simultaneous extraction of MPA and VPA from acidified plasma. This reliable method can be applied in the analysis of MPA and VPA in human plasma using only a small volume (100 microL).

Two simple high-performance liquid chromatographic (HPLC) methods have been established for simultaneous determination of mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) in human urine, and of their total and unbound forms in human plasma. For total MPA and MPAG analysis sample preparation entailed precipitation of protein with acetonitrile and isolation of the free analytes from the plasma by ultrafiltration. For urine samples, fivefold dilution with water was used. MPAG was determined by UV detection whereas MPA was quantified by fluorescence detection after post-column derivatization with 0.2 M sodium hydroxide solution. For plasma, response was found to be linearly dependent on concentration over the ranges 0.1–40 µg mL-1 and 0.01–1 µg mL-1 for total and free MPA, respectively, and 10–200 µg mL-1 and 2.5–100 µg mL-1 for total and free MPAG, respectively. For urine, linearity was observed from 0.1 to 50 µg mL-1 for MPA and 10 to 500 µg mL-1 MPAG in the urine before dilution. The methods reported were found to be accurate and reproducible for quantifying the level of MPA and MPAG and can thus be used for clinical pharmacokinetic studies and for therapeutic drug monitoring.

A simple, specific and sensitive high performance liquid chromatography-mass spectrometry (LC-MS) method for the determination of risperidone and its active metabolite 9-hydroxyrisperidone in human plasma has been developed and validated. The analytes were prepared through a single-step liquid-liquid extraction (LLE) procedure with the solvent methyl tert-butyl ether and quantitated by MS detection in the positive mode using selected ion monitoring (SIM). Each analytical run was completed within 9 min. Results showed that the LC-MS method enabled to detection of both compounds down to 0.1 ng.mL−1 (S/N > 3) and the linear range was 0.2–24 ng.mL−1, with the correlation coefficients above 0.99. At the concentration of 0.2, 0.5, 10 and 20 ng.mL−1, the inter-day and intra-day RSD were both below 15%. The method has been successfully used to support the routine therapeutic drug monitoring (TDM) and the pharmacokinetics study of risperidone.

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.

We employed CE to identify mixtures of the toxic alkaloids lappaconitine, bullatine A, atropine sulfate, atropine methobromide, scopolamine hydrobromide, anisodamine hydrobromide, brucine, strychnine, quinine sulfate, and chloroquine in human blood and urine, using procaine hydrochloride as an internal standard. The separation employed a fused-silica capillary of 75 microm id x 60 cm length (effective length: 50.2 cm) and a buffer containing 100 mM phosphate and 5% ACN (pH 4.0). The sample was injected in a pressure mode and the separation was performed at a voltage of 16 kV and a temperature of 25 degrees C. The compounds were detected by UV absorbance at wavelengths of 195 and 235 nm. All the ten alkaloids were separated within 16 min. The method was validated with regard to precision (RSD), accuracy, sensitivity, linear range, LOD, and LOQ. In blood and urine samples, the detection limits were 5-40 ng/mL and linear calibration curves were obtained over the range of 0.02-10 microg/mL. The precision of intra- and interday measurements was less than 15%. Electrophoretic peaks could be identified either by the relative migration time or by their UV spectrum.

In the current paper, we report the development of a new capillary electrophoresis method using pre-column derivatization and laser-induced fluorescence detection for the determination of ephedrine and amphetamine drugs. Our new method allows for the identification and quantification of six commonly used illicit drugs namely pseudoephedrine, ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, and 3,4-methylenedioxymethylamphetamine, respectively, as well as propafenone (internal standard). Following derivatization with fluorescein isothiocyanate, a total of six amphetamine drugs and the internal standard could readily be separated using a fused-silica 75 μm ID × 60 cm length (effective length: 50.2 cm) capillary column. The mobile phase consisted of buffer containing 20 mM borate (pH 12, adjusted with sodium hydroxide). Samples were injected in pressure mode with the capillary being operated at 25 kV/25 °C, and the detection of the derivatized compounds was sought using a laser-induced fluorescence (LIF) detector ( λ ex = 488 nm and λ em = 520 nm), with a run-time of 20 min. The current method was validated with regard to precision (relative standard deviation, RSD), accuracy, sensitivity, linear range, limit of detection (LOD) and limit of quantification (LOQ). In human blood and urine samples, detection limits were 0.2 ng mL −1 , and the linear range of the calibration curves was 0.5–100 ng mL −1 . The intra-day and inter-day precisions were both less than 13.22%.

A specific, precise and accurate high-performance liquid chromatography (HPLC) method for the determination of aglycone conjugated metabolites of scutellarin in plasma after enzymolysis to scutellarein (the aglycone of scutellarin) was developed and validated. The chromatographic separation was performed on a Lunar C18(2) reversed-phase column at a column temperature of 40 degrees C. The mobile phase, delivered at 1.0 ml/min, consisted of acetonitrile-KH2PO4 buffer (40 mM, pH 2.5) (33:67, v/v). The detection wavelength was set at 335 nm. Scutellarein and I.S. (quercetin) were isolated by a liquid-liquid extraction after incubating the plasma samples with beta-glucuronidase/sulfatase. The method was validated using scutellarin spiked plasma as standards. Linearity was confirmed in the concentration range of 0.2165-4.329 nmol/ml, R.S.D.s were within 8.32%, and the recoveries of scutellarein ranged from 101.2 to 108.6%. The method is applicable to the pharmacokinetic study of aglycone conjugated metabolites of scutellarin in rats after oral administration of scutellarin.

A simple high-performance liquid chromatographic (HPLC) method was developed for the assay of total and free mycophenolic acid (MPA) in human plasma. Prior to analysis, total mycophenolic acid was extracted by protein precipitation and free drug was isolated from plasma samples using ultrafiltration. The extracts were injected onto a Kromasil C8 column at 30 degrees C with excitation and emission wavelengths set at 342 and 425 nm, respectively. The mobile phase was consisted of acetonitrile-32 mM glycine buffer, pH 9.2 (20:80, v/v), at a flow rate of 1.0 ml/min. The method was found to be linear over the concentration range investigated, 0.05-40 mg/l for total mycophenolic acid (r>0.999) and 5-1000 microg/l (r>0.99) for free drug. The percentage error of the analytical method was below 10.9%. The intra- and inter-day reproducibility was adequate with the coefficients of variation of 8.28% or below. The run time were 4 and 6 min for free and total MPA, respectively. The method thus can be effectively applied to measure mycophenolic acid concentrations in clinical samples.