胶束电动色谱-激光诱导荧光检测法测定肌松弛药巴氯芬

以4-氯-7-硝基苯并-2-氧杂-1,3-二唑(NBD-Cl)为柱前衍生试剂,建立了胶束电动色谱-激光诱导荧光检测法测定肌松弛药巴氯芬(BAL)的新方法。经过实验条件的优化,采用15 mmol/L硼砂、20 mmol/L十二烷基硫酸钠、10%(v/v)乙腈、pH 9.75的缓冲体系,在分离电压为17.5 kV、柱温为25 ℃的条件下,压力进样3.45 kPa(0.5 psi)×3 s,巴氯芬及其内标物的衍生产物在7 min内实现较好的基线分离,线性范围为0.025～25 mg/L,相关系数为0.9999,检出限(S/N=3)和定量限(S/N=10)分别为0.90 μg/L和6.25 μg/L。该方法被应用于巴氯芬制剂及加入巴氯芬对照品的尿液样品分析,回收率范围分别为101.6%～107.9%和107.0%～109.6%。该方法有望应用于巴氯芬药物制剂的质量监控以及为巴氯芬药物代谢的研究提供辅助手段。     

Mass distribution in a dynamic sample zone inside a flow injection manifold: modelling integrated conductimetric profiles

A mathematical model for fitting the experimental ICM (integrated conductimetric method) curves developed by the authors in a previous work, is presented for the first time in this study. The proposed model fits the experimental curves with great precision and allows to predict physical dispersion for single-line flow injection system. The correlation of the model’s parameters with typical reactionless FIA peak parameters is also assessed. The IDQ coefficient—a novel dispersion estimator previously reported by the authors—can also be predicted when operational FIA variables are changed. Experimental and modelled profiles are compared as a function of the system’s variables, showing an excellent agreement.     

Detection of N-nitroso-bile acids at 285 nm in reverse-phase HPLC

In the present study, we developed a novel, simple, and specific detection method using an RP-HPLC at UV 285 nm for the separation and quantification of N-nitroso-bile acids. First, we found that N-nitroso-bile acids have a specific spectrophotometric absorbance at 285 nm. Using this 285 nm detection system, we could especially measure N-nitroso-bile acids, even in co-existence of non-N-nitroso-bile acids. Next, we observed the decomposition of N-nitroso-glychocholate under alkaline, acidic, and neutral conditions. N-nitroso-glychocholate rapidly decomposed under alkaline conditions (pH 9) (t(1/2) = 0.96 h), but remained fairly stable under acidic (pH 2) (t(1/2) = 12.8 h) and neutral (pH 7) (t(1/2) = 7.8 h) conditions. This study is the first report, which simply and specifically analyzes N-nitroso-bile acids using an RP-HPLC system.     

Chromatographic Analysis of Trans Resveratrol in Italian Wines: Comparisons between FL, UV and MS Detection

Resveratrol (3,5,4′-trihydroxystilbene) is a phytoalexin that belongs to the group of stilbenes, known to occur in grapes and consequently in grape products. Its presence in wine is an important qualitative parameter because of the several beneficial effects on human health. The aim of this work is the development of a high-performance liquid chromatographic (HPLC) method for the determination of trans resveratrol in wines, and comparisons between the results obtained by different detection techniques: UV-vis spectroscopy, fluorescence spectroscopy and mass spectrometry. Resveratrol is analysed on a C-18 column using gradient elution. The method permits direct injection of sample, revealing to be time-saving, overcoming the need of sample pre-treatment steps. Detection limits were 154.8 ng mL⁻¹ by HPLC-UV, 118.0 ng mL⁻¹ by HPLC-FL and 48.0 ng mL⁻¹ by HPLC-MS. Trans resveratrol has been then quantified in a group of 52 wines derived from different Italian regions, cultivars and winemaking technologies by HPLC-UV.     

Single cell manipulation, analytics, and label-free protein detection in microfluidic devices for systems nanobiology

Single cell analytics for proteomic analysis is considered a key method in the framework of systems nanobiology which allows a novel proteomics without being subjected to ensemble-averaging, cell-cycle, or cell-population effects. We are currently developing a single cell analytical method for protein fingerprinting combining a structured microfluidic device with latest optical laser technology for single cell manipulation (trapping and steering), free-solution electrophoretical protein separation, and (label-free) protein detection. In this paper we report on first results of this novel analytical device focusing on three main issues. First, single biological cells were trapped, injected, steered, and deposited by means of optical tweezers in a poly(dimethylsiloxane) microfluidic device and consecutively lysed with SDS at a predefined position. Second, separation and detection of fluorescent dyes, amino acids, and proteins were achieved with LIF detection in the visible (VIS) (488 nm) as well as in the deep UV (266 nm) spectral range for label-free, native protein detection. Minute concentrations of 100 fM injected fluorescein could be detected in the VIS and a first protein separation and label-free detection could be achieved in the UV spectral range. Third, first analytical experiments with single Sf9 insect cells (Spodoptera frugiperda) in a tailored microfluidic device exhibiting distinct electropherograms of a green fluorescent protein-construct proved the validity of the concept. Thus, the presented microfluidic concept allows novel and fascinating single cell experiments for systems nanobiology in the future.     

Electroanalysis of sulfonamides by flow injection system/high-performance liquid chromatography coupled with amperometric detection using boron-doped diamond electrode

Sulfonamides (SAs) were electrochemically investigated using cyclic voltammetry at a boron-doped diamond (BDD) electrode. Comparison experiments were carried out using a glassy carbon electrode. The BDD electrode provided well-resolved oxidation, irreversible cyclic voltammograms and higher current signals when compared to the glassy carbon electrode. Results obtained from using the BDD electrode in a flow injection system coupled with amperometric detection were illustrated. The optimum potential from a hydrodynamic voltammogram was found to be 1100 mV versus Ag/AgCl, which was chosen for the HPLC-amperometric system. Excellent results of linear range and detection limit were obtained. This method was also used for determination of sulfonamides in egg samples. The standard solutions of 5, 10, and 15 ppm were spiked in a real sample, and percentage of recoveries was found to be between 90.0 and 107.7.     

Determination of tobramycin in human serum by capillary electrophoresis with contactless conductivity detection

A study on the determination of the antibiotic tobramycin by CE with capacitively coupled contactless conductivity detection is presented. This method enabled the direct quantification of the non-UV-absorbing species without incurring the disadvantages of the indirect approaches which would be needed for optical detection. The separation of tobramycin from inorganic cations present in serum samples was achieved by optimizing the composition of the acetic acid buffer. Field-amplified sample stacking was employed to enhance the sensitivity of the method and a detection limit of 50 microg/L (S/N = 3) was reached. The RSDs obtained for migration time and peak area using kanamycin B as internal standard were typically 0.12 and 4%, respectively. The newly developed method was validated by measuring the concentration of tobramycin in serum standards containing typical therapeutic concentrations of 2 and 10 mg/L. The recoveries were 96 and 97% for the two concentrations, respectively.     

Determination of thiol by high-performance liquid chromatography and fluorescence detection with 5-methyl-(2-(<Emphasis Type="Italic">m</Emphasis>-iodoacetylaminophenyl)benzoxazole

A new high-performance liquid chromatographic (HPLC) method for measuring low molecular weight (LMW) thiol-containing compounds, including cysteine (CysH), glutathione (GSH), N-acetylcysteine (Nac), penicillamine (PA), and 2-mercaptoethanol (2-ME), has been developed by using 5-methyl-(2-(m-iodoacetylaminophenyl)benzoxazole (MIPBO) as fluorescence-labeling reagent. The derivatization and separation conditions have been investigated in detail. Detection limits ranging from 3.5 to 15.0 fmol were achieved for the thiols investigated in a 16 min separation with detection wavelengths 310 and 375 nm for the excitation and emission, respectively. The utility of the proposed method has been validated by measuring CysH in human urine samples.     

离子色谱-积分脉冲安培检测法测定大蒜多糖的单糖组成

采用氨基酸分离柱(Amino PAC PA10,2×250 mm),用10.0 mmol/L NaOH溶液作为流动相,以Au为工作电极,Ag/AgCl为参比电极的脉冲积分安培离子色谱法,分离检测了大蒜多糖水解产生的单糖成分及相对含量。实验结果表明,大蒜多糖中含有半乳糖、葡萄糖、甘露醇和果糖,检出限在0.3~6μg/L范围,样品加标回收率为95%~112%。该法具有灵敏度高、精密度好、分离效果好和样品不需要衍生处理的优点,适用于各种植物多糖的单糖组成分析。     

Determination of coenzyme Q10 in human breast milk by high-performance liquid chromatography

An isocratic HPLC method was developed for the determination of coenzyme Q(10) (CoQ(10)) in human breast milk. After a single-step liquid-liquid extraction, the milk extract was injected directly into the HPLC system. The analytical method is based on pre-column inline treatment of CoQ(10). Chromatographic separation of CoQ(10) and coenzyme Q(9) (CoQ(9)) internal standard was achieved using a reversed-phase Microsorb-MV C(18) analytical column. CoQ(10) and CoQ(9) were monitored by an electrochemical detector (ECD). An excellent linearity (r = 0.999) was observed for CoQ(10) in the concentration range 0.06-2.5 micromol L(-1) in breast milk. The limit of quantitation (LOQ) was 60 nmol L(-1). Coefficients of variations (CVs) for intra-day and inter-day assay precisions were less than 5%. A total of 194 breast milk samples were analyzed for the CoQ(10) concentration; the mean value was 0.32 +/- 0.21 micromol L(-1).