Simultaneous determination of lactic acid and pyruvic acid in tissue and cell culture media by gas chromatography after in situ derivatization-ultrasound-assisted emulsification microextraction.

Lactic acid and pyruvic acid are important metabolites in the tricarboxylic acid cycle that can reflect the cytoplasmic redox state and mitochondrial respiratory chain function. The combination of these acids is considered as a screening index for mitochondrial disorders. Due to their biological effects, a derivatization method was developed to simultaneously detect pyruvic acid and lactic acid in tissue and cell culture media using gas chromatography. In this work, the combined derivatization method with methoxyamine hydrochloride and isobutyl chloroformate was first proposed. To improve the efficiency of derivatization, in situ derivatization-ultrasound-assisted emulsification microextraction (USAEME) was used in this study. After optimizing the volume of reagents and reaction times, good linearity values were obtained from 50 to 1000 µmol/L and 1 to 100 µmol/L for lactic acid and pyruvic acid, respectively. The limits of detection (LODs) were 0.12 µmol/L for lactic acid and 0.29 µmol/L for pyruvic acid. The recoveries of the two analytes were between 93.60 and 102.80%, and the precisions were less than 6.20%. This method was successfully applied to quantify pyruvic acid and lactic acid in the animal and cellular hypoxia models which provided an auxiliary means for the diagnosis of mitochondrial diseases. Graphical abstract ᅟ.

Development of Loop-Mediated Isothermal Amplification (LAMP) for Universal Detection of Enteroviruses.

Enteroviruses are found in most environments and cause several diseases in humans. Loop-mediated isothermal amplification (LAMP) was adapted and evaluated for the rapid detection of enteroviruses. Based on the highly conserved 5' untranslated region (5'-UTR) of the human enteroviruses (HEVs), particularly human enterovirus A (HEV-A) and HEV-B, a set of universal primers was designed. The LAMP amplification was carried out under isothermal conditions at 61 °C, depending on the template concentration results were obtained within 45-90 min. The detection limits were found to be 10(1) copies of cloned enterovirus 71 fragments, more sensitive than conventional PCR. Nine water samples collected from drinking water sources during three seasons and 19 stool specimens collected from HFMD patients were analyzed. By using the LAMP assay, the majority of samples was tested positive, 9/9 (100 %) and 18/19 (94.7 %), respectively. LAMP is a practical method for the rapid detection of enteroviruses in environmental and clinical samples.

[Development of a multi-residue detection method for 27 typical pharmaceuticals and personal-care products in plants and analysis of their migration patterns in sprouts].

An analytical method based on ultra-performance liquid chromatography-tandem mass spectrometry was developed for the simultaneous determination of 27 pharmaceutical and personal-care product (PPCP) residues in plants. The enrichment and cleanup of PPCPs in plants were achieved using an HLB extraction column, and the separation was performed on a BEH C18 column (100 mm×2.1 mm, 1.7 µm) with 0.1% formic acid water-acetonitrile as the mobile phase via gradient elution. PPCPs were detected with electrospray ionization mass spectrometry in positive-ion multiple-reaction monitoring (MRM) mode. The limits of detection and quantification of the 27 PPCPs in plants were 0.01-0.30 µg/kg and 0.03-0.98 µg/kg, respectively. Good linearities were observed with coefficients of determination (r2) >0.99. The spiked recoveries were between 80.8% and 122.3% with relative standard deviations (RSDs) between 1.0% and 9.9%. The method was subsequently used to study sprouts grown in different concentrations of PPCPs. A total of 10 PPCPs were detected in sprouts grown in medium with a low concentration PPCPs, 13 PPCPs were detected in sprouts grown in medium with a moderate concentration of PPCPs, and 19 PPCPs were detected in sprouts grown in medium with a high concentration of PPCPs. These results showed that plants grown in water bodies contaminated with PPCPs or irrigated with water contaminated with PPCPs absorbed and accumulated these substances and that the amount and type of PPCPs absorbed by plants were closely related to the levels of PPCPs in the external environment. Analysis of the contents of PPCPs in different plant tissues revealed a general distribution of root>stem>leaf. Haemosibutramine showed a tissue distribution of leaf>stem>root, while glibenclamide showed a distribution of root>leaf>stem; these results revealed differences in the distribution of PPCPs in plants. Calculation of the transfer factor (TF) of the PPCPs in plants demonstrated significant differences in the transferability of different PPCPs, with TF=2.34 for haemosibutramine and TF=1.25 for chlorosibutramine. The results showed that among the drugs that migrated in plants, haemonosibutramine and chlorosibutramine had the strongest migration ability in sprouts, followed by nicardipine and chlorpheniramine maleate, and amantadine, N-monodesmethyl sibutramine, carbamazepine and flumequine had the weakest migration ability. Once absorbed, these compounds were transferred to the stems and/or leaves, where they accumulate and cause potential harm by contaminating other plant organs. Therefore, PPCPs such as homosibutramine and chlorosibutramine, which easily migrate in plants, should be given extra attention in future studies. The method is simple in pre-treatment, sensitive and accurate, and can be widely applied to the detection of PPCP residues in plant samples.

Determination of higenamine in multi-matrix by gas chromatography-mass spectrometry combined with derivatization technology.

Higenamine (HG), a cardioactive component of some foods and medicines, has been listed in the doping category by the International Olympic Committee, which may lead to misuse by athletes. We report the development of a gas chromatography-mass spectrometry (GC-MS) method for determination of HG in various matrix samples (biological samples, different forms of Chinese patent medicine, Chinese herbal medicine) based on acylation derivatization of HG by heptafluorobutyric anhydride. Under optimal conditions, the linearity of HG in the range of 5-200 ng mL-1 was acceptable (R2 > 0.999), and the limit of detection (LOD) and limit of quantitation (LOQ) for HG was 1.52 ng mL-1 and 5 ng mL-1, respectively. Low, medium, and high concentrations (25, 100 and 160 ng mL-1) of HG were added to plasma, urine, oral liquid, capsule, watered bolus, honeyed bolus and Chinese herbal medicine samples, with recovery ranging from 82.70 to 109.80%, intra-day and inter-day precisions were both less than 3.39%. The results indicated that the method had sufficient sensitivity for analysis of biological samples, and Chinese patent and herbal medicine.

Derivatization method for the quantification of lactic acid in cell culture media via gas chromatography and applications in the study of cell glycometabolism.

Lactic acid represents an important metabolite that reflects mitochondria function and may further serve as energy source for cancer cells. In light of this physiological and pathological significance, we developed a novel and sensitive gas chromatography method to detect lactic acid in cell culture media. Here, ethyl chloroformate was selected as derivative reagent and the derivatization process was further optimized in terms of number of reagents and reaction time as well as extraction reagents. Under optimal conditions, good linearity was achieved in the tested calibration range. The limit of detection (LOD) was determined to be 0.67 µmol/L, the recovery rates were 99.6%-106% and the precision rate RSD was <5.49%. Furthermore, this method has been applied to quantify the secretion of lactic acid in cells exposed to mono­2­ethylhexyl phthalate at different doses and in cancer cells over time. Taken in concert, our method proved to be both sensitive and reliable and may be applied for studies on mitochondrial function and cell glycolysis conditions.

Development of a derivatization method for the quantification of hydrogen sulfide and its application in vascular calcification rats.

Hydrogen sulfide (H2S) plays major functional and structural roles in diverse physiological functions and the pathogenesis of a variety of disorders in biological matrices. The significance of H2S has prompted the development of sensitive and selective methods to determine its concentration in biological samples. The fluorescent reagent monobromobimane (MBB) has been widely used to measure various thiol-containing species through alkylation. MBB may prevent the oxidation of sulfide and the reaction of sulfide with several different species (such as superoxide radicals, hydrogen peroxide and peroxynitrite). An isomers of MBB, 3-(bromomethyl)-2, 6, 7-trimethyl-1H, 5H-pyrazolo [1,2-a] pyrazole-1, 5-dione (MMB), is cheaper than MBB and its use in the analysis of H2S has not previously been reported. In the present study, we compared the derivatization reactions of hydrogen sulfide with MMB and MBB and developed a sensitive method to quantify H2S in blood. In our method, H2S was incubated in the dark with excess MMB in 0.1M Tris-HCl buffer (pH 10.1) at 50°C for 120min. 50µL aliquots of the derivatized product were analyzed using HPLC system with gradient elution of 0.1% (v/v) formic acid-acetonitrile. The limit of detection for the derivatized product was 0.03nmol/mL. The derivatization reaction was suitable for detecting low concentrations of H2S. The derivate product is stable over time, permitting batch storage and analysis.

[Development of ELISA-kit of quantitative analysis for Zearalenone].

OBJECTIVE: To develope a rapid, sensitive, quantitative ELISA-kit for Zearalenone and determine zearalenone in cereals. METHODS: On the base of monoclonal antibodies against ZEN, apply indirect ELISA to study the performance parameter of the kit. RESULTS: The limited concentration of detection of the ELISA-kit was 1ng/ml, linear range was 1-200 ng/ml, the linear equation was Y = 0.99 - 0.40 x (R2 = 0.99). The inhibition concentration of 50% against ZEN was 16.3 ng/ml. The average recovery rate of spiked corn and wheat was 96.5% and 95.5%, respectively, the coefficient of variant was 13.2% and 10.9%, respectively. The kit can be stored at 4 degrees C over 6 months. The cross reaction rate with the other mycotoxins was less than 1%, and coefficient of variant within-laboratory and between-laboratory was less than 15% and less than 20%, respectively. Detecting the VICAM sample with ELISA method and HPLC method, the results were within the range of the sample, and there was no statistic difference between the two methods. CONCLUSION: This ELISA-kit was quick, sensitive, stable and specific and can be used to determine ZEN in cereals.

[Determination of trace cadmium in environment water samples by flame atomic absorption spectrometry using flow injection on-line preconcentration with double microcolumns].

A rapid and sensitive methed for the determination of trace cadmium in environment water samples based on flowinjection on-line preconcentration on two-microcolumn system-flame atomic absorption spectrometry has been developed. The cadmium in samples was sequentially retained on two microcolumns with cation exchange resin and was eluted directly in the nebulizer by 1.5 mol x L(-1) HCl solution. The characteristic concentration (preconcentration time 1 min) for cadmium was 0.931 microg x L(-1). The relative standard deviation at the 5 microg x L(-1) level was 2.69% and the corresponding detection limit (3sigma) was 0.808 microg x L(-1). The method has been successfully applied to the determination of cadmium in water reference material GBW08608 and other water samples.

Determination of chromium(III) and total chromium in water by derivative atomic absorption spectrometry using flow injection on-line preconcentration with a double microcolumn.

A rapid and sensitive method has been proposed for the sequential determination of chromium(III) and total chromium in water samples by flame atomic absorption spectrometry combined with a flow injection on-line preconcentration on a double-microcolumn. The chromium(III) and total chromium in samples were retained on a double-microcolumn with a cation exchange resin, respectively, and eluted directly into a nebulizer by 3 mol L(-1) HNO3. The characteristic concentration (gives a derivative absorbance of 0.0044) and the detection limit (3sigma) for chromium were 0.512 microg L(-1) and 0.647 microg L(-1) for a preconcentration time of 1 min, respectively. This is an improvement of 20 and 14-times than those of conventional FI-FAAS. The proposed method allows the determination of chromium in the range of 0-90 microg L(-1) with a relative standard deviation of 3.63% at the 10 microg L(-1) level. The method has been applied for the analysis of chromium in reference water of National Research Center for Certified Reference Materials (GBW08607) and other water samples with satisfactory results.

[Determination of zinc in relish by derivate flame atomic absorption spectrometry with microsampling pulse nebulization].

A simple, rapid, microsampling method for the determination of zinc in relish by derivative FAAS is described. The influences of microsampling volume and other factors are discussed. The detection limit and sensitivity of the proposed method are 0.013 and 0.004 microg x mL(-1), respectively. The samples were centrifugalized and diluled with 1.5% HCl solution, and then analysized directly. The method was applied to the determination of zinc in relish with a recovery of 93.3%-113.3% and relative standard deviation of 3.0%-4.4%.

[Determination of copper, iron and zinc in tapwater by ion exchange microcolumn preconcentration-derivative flame atomic absorption spectrometry].

A new microsample-pulse method is described for the determination of copper, iron and zinc by ion exchange microcolumn preconcentration-derivative flame atomic absorption spectrometry. The optimum conditions concerning the sensitivity were studied. The method was applied to the determination of Cu, Fe and Zn in tapwater with sensitivity being 0.29, 0.59 and 0.06 microgram.L-1, respectively. The recovery range and the relative standard deviation of the proposed method were 91.13%-101.34% and 1.95%-4.28%, respectively. The detection limits were found to be 1.28, 5.85 and 0.68 micrograms.L-1, respectively. The method is sensitive, accurate, precise and rapid.

[Determination of chromium(III) and chromium(VI) in water using flow injection on-line preconcentration and separation with flame absorption spectrometric detection].

A rapid and sensitive method for the sequential determination of Cr(III) and Cr(VI) in water samples based on flow injection on-line preconcentration and separation with two-microcolumn system-flame atomic absorption spectrometry has been developed. The Cr(III) and Cr(VI) in water samples were respectively retained in a microcolum with cation exchange resin and in a microcolumn with anion exchange resin and were eluted directly by 15% HNO3 and 8% NH4NO3, respectively. The characteristic concentrations (pre-concentration time of 1 min) for Cr(III) and Cr(VI) were 1.50 micrograms.L-1 and 1.39 micrograms.L-1, respectively, The relative standard deviations at 10 micrograms.L-1 level were 3.41% and 1.80%, and the corresponding detection limits (3 sigma) were 1.03 micrograms.L-1 and 0.54 microgram.L-1, respectively. The satisfactory recovery of 93.48%-107.5% could be obtained from water samples.