Simultaneous determination of l- and d-lactic acid in plasma by capillary electrophoresis

A novel method for simultaneous determination of d- and l-lactic acids in plasma was presented by capillary electrophoresis with photodiode array detection at 195nm. The separation was performed in an uncoated fused-silica capillary. The parameters influencing the resolution and the migration time of lactic acids were optimized. When 150mM phosphate-Tris buffer (pH 7.0) consisting of 220mM 2-hydroxypropyl-beta-cyclodextrin and 0.2mM tetradecyltrimethylammonium bromide was utilized as the running buffer, highly effective chiral separation of d- and l-lactic acids was achieved at about 42min at an effective voltage of -25kV. The resolution of lactic acid enantiomers was >/=1.25. The limits of detection of d- and l-lactic acids in standard solution without any pretreatment were 80 and 50muM (S/N=3), respectively. Sample pretreatment was preceded by protein-removal procedure with acetonitrile. With a pre-concentration procedure by 10 times, the limits of detection of d- and l-lactic acids were 20 and 15muM (S/N=10), respectively. The satisfactory analytical performance of the proposed method was validated.     

Simultaneous determination of clopidogrel and its carboxylic acid metabolite by capillary electrophoresis

A capillary electrophoresis method was developed and validated for the first time for the analysis of clopidogrel and its carboxylic acid metabolite. Prior to method optimization, the pH dependence of effective mobility of both compounds was determined in order to define the initial pH of the running buffer. The optimized method demonstrated to be selective, and linear in the concentration range of 2–100 μM for both compounds. The method limits of detection and quantification were, respectively, 1.2 and 3.7 μM for clopidogrel and 1.1 and 3.2 μM for the carboxylic acid metabolite. Moreover, method validation demonstrated acceptable results for method repeatability (RSD < 7%), intermediate precision (RSD < 7%) and accuracy (85–96%) and is suitable for the quantitative analysis of clopidogrel and its metabolite in serum samples. The validated method was also applied to the determination of the kinetic parameters of the enzymatic hydrolysis of clopidogrel. An apparent K_m of 145 ± 30 μM and V_max of 0.4, 1.5 and 3.4 μM/min, respectively for the enzyme concentrations 1.0, 2.0 and 4.0 U/ml, were obtained.     

白花蛇舌草的化学成分研究

从白花蛇舌草(Hedyotis diffusaWilld)的全草中分离得到12个化合物,应用波谱学方法鉴定为p-香豆酸(p-coumaric acid,1)、阿魏酸(ferulic acid,2)、齐墩果酸(oleanolic acid,3)、熊果酸(ursolic acid,4)、2-甲基-3-羟基-蒽醌(2-methyl-3-hydroxyanthraquinone,5)、2-甲基-3-甲氧基蒽醌(2-methyl-3-methoxyanthraqui-none,6)、东莨菪内酯(scopolin,7)、槲皮素(quercetin,8)、山奈酚(kaeperferol,9)、胡萝卜苷(daucosterol,10)、豆甾醇(stigmasterol,11)和β-谷甾醇(-βsitosterol,12)。化合物2、7为首次从该植物中分得。     

Simultaneous electrochemiluminescence determination of sulpiride and tiapride by capillary electrophoresis with cyclodextrin additives

Sulpiride and tiapride are often used in the treatment of depression, schizophrenia and psychopathology of senescence, gastric or duodenal ulcers and are also partly excreted by kidney. This work developed a simple and sensitive method for their simultaneous monitoring in human urine based on capillary electrophoresis coupled with electrochemiluminescence detection by end-column mode. beta-Cyclodextrin (beta-CD) was used as an additive to the running buffer to obtain the absolute separation of sulpiride and tiapride. Under optimized conditions the proposed method displayed a linear range from 1.0 x 10(-7) to 1.0 x 10(-4) M for both sulpiride and tiapride with the correlation coefficients more than 0.995 (n = 6). Their limits of detection were 1.0 x 10(-8) M (45 amol) and 1.5 x 10(-8) M (68 amol) at a signal to noise ratio of 3, respectively. The relative standard deviations for six determinations of 2.0 microM sulpiride and 3.0 microM tiapride were 1.8 and 2.5%, respectively. For practical application an extract step with ethyl acetate at pH 11 was performed to eliminate the influence of ionic strength in sample. The recoveries of sulpiride and tiapride at different levels in human urine were between 84 and 95%, which showed that the method was valuable in clinical and biochemical laboratories for monitoring sulpiride and tiapride for various purposes.     

Simultaneous quantification of twelve bioactive components in San-huang-xie-xin-tang by HPLC

A simple and reliable high-performance liquid chromatographic method with ultraviolet detection (280 nm) has been developed for the simultaneous analysis of 12 bioactive components in San-huang-xie-xin-tang (SHXXT), a traditional Chinese medicine containing Rhei rhizome, Coptidis rhizome and Scutellariae radix. A relatively simple extraction procedure was employed and optimised, and separation of the components was obtained within 1 h using a reversed-phase column under gradient elution with acetonitrile and a buffer containing 0.01 M sodium 1-pentanesulphonate (pH 3). The lower limit of detection for the analytes ranged from 25 to 75 ng/mL. The correlation coefficients associated with each calibration curve were greater than 0.99. The precision and accuracy of the method ranged from 1.0 to 10.5% at low concentration levels, 0.8 to 8.7% at medium levels and 1.2 to 5.8% at high levels. In commercial products of SHXXT, baicalin and berberine were present in the highest amounts with levels up to 4.0 and 3.3%, respectively, in one sample. The HPLC method was able rapidly and efficiently to analyse constituents in crude herb and traditional Chinese medicinal preparations containing Rhei rhizome, Coptidis rhizome and Scutellariae radix.     

Simultaneous determination of aminomethylbenzoic acid,cefminox sodium and etamsylate in human urine by capillary electrophoresis

A sensitive and selective capillary electrophoresis method is developed, for the first time, for effective separation and simultaneous determination of aminomethylbezoic acid (PAMBA), cefminox sodium (CMNX) and etamsylate (ETM). The electrophoresis conditions were investigated and optimized. A 25 mM phosphate solution (pH 8.5) was used as a buffer and the peak area was determined with UV detection at 216 nm wavelength under 18 kV separation voltage. Under optimal conditions, the three drugs can be separated effectively. Good linearity was achieved in 3.13–150 μg/mL for PAMBA, 6.25–150 μg/mL for CMNX and 3.13–150 μg/mL for ETM, with the correlation coefficients of >0.999. The limit of detection (LOD) for PAMBA, CMNX and ETM was 1.04, 2.08 and 1.04 μg/mL, respectively. Their recoveries in human urine were in the range from 90.2% to 101% with the RSD (n = 5) of 0.7–3.1%. The proposed method is simple, rapid and accurate, and provides the sensitivity and linearity necessary for analysis of the test drugs in human urine at clinically relevant concentrations.     

Simultaneous determination of iodate and periodate by capillary zone electrophoresis: application to carbohydrate analysis

Iodate and periodate were rapidly (in 11 min) separated from each other with high column efficiency by capillary zone electrophoresis, using a fused silica tube (50 microns i.d., 80 cm) and 100 mM acetate buffer, pH 4.5, as carrier. On-column uv detection at 222 nm allowed sensitive detection down to the picomole level, and measurement of relative peak area to that of pyromellitic acid (internal standard) enabled reproducible determination of these ions. This method was proved useful for periodate oxidation analysis of various carbohydrates.     

Simultaneous determination of phenylethanoid glycosides and aglycones by capillary zone electrophoresis with running buffer modifier

Although the separation efficiency of capillary electrophoresis (CE) is much higher than that of other chromatographic methods, it is sometimes difficult to adequately separate the complex ingredients in biological samples. This article describes how one effective and simple way to develop the separation efficiency in CE is to add some modifiers to the running buffer. The suitable running buffer modifier β-cyclodextrin (β-CD) was explored to fast and completely separate four phenylethanoid glycosides and aglycones (homovanillyl alcohol, hydroxytyrosol, 3,4-dimethoxycinnamic acid, and caffeic acid) in Lamiophlomis rotata (Lr) and Cistanche by capillary zone electrophoresis with ultraviolet (UV) detection. It was found that when β-CD was used as running buffer modifier, a baseline separation of the four analytes could be accomplished in less than 20 min and the detection limits were as low as 10⁻³ mg L⁻¹. Other factors affecting the CE separation, such as working potential, pH value and ionic strength of running buffer, separation voltage, and sample injection time, were investigated extensively. Under the optimal conditions, a successful practical application on the determination of Lr and Cistanche samples confirmed the validity and practicability of this method.     

On-line chemiluminescence determination of mitoxantrone by capillary electrophoresis

A novel capillary electrophoresis (CE) with chemiluminescence (CL) detection method for the determination of mitoxantrone (MTX) has been developed, which based on the CL reaction of potassium ferricyanide with luminol in sodium hydroxide medium sensitized by MTX. Under optimum analytical conditions, MTX is determined over the range of 7.0 × 10⁻⁸–1.0 × 10⁻⁶ M with a detection limit of 1.0 × 10⁻⁸ M. The relative standard deviation (RSD) was 3.7%, 2.6% and 3.0% for 7.0 × 10⁻⁸, 5.0 × 10⁻⁷ and 1.0 × 10⁻⁶ M MTX (n = 11), respectively. In laboratory-built CE–CL apparatus, the proposed method has been applied to determination of MTX in commercial drug and spiked in human urine and plasma with satisfactory results.     

Simultaneous determination of 19 intracellular nucleotides and nucleotide sugars in Chinese Hamster ovary cells by capillary electrophoresis

Twelve nucleotides and seven nucleotide sugars in Chinese Hamster ovary (CHO) cells were determined by capillary electrophoresis (CE). The CE operating conditions of buffer pH value, ion strength, capillary temperature, polymer additive and cell extraction method were investigated. Optimum separation was achieved with 40 mM sodium tetraborate buffer (pH 9.5) containing 1% (w/v) polyethylene glycol (PEG) at a capillary temperature of 22 degrees C. Acetonitrile and chloroform were used for intracellular extraction. This method can be used to monitor intracellular carbohydrate metabolism.     

白花蛇舌草的组织培养和植株再生

1 植物名称白花蛇舌草(Hedyotis diffusa). 2 材料类别带顶芽或腋芽的茎段,取自本校花圃. 3 培养条件诱导培养基:(1)MS+NAA 0.2 mg*L-1(单位下同)+6-BA 2.0;(2)MS+NAA 0.5+6-BA 2.0;(3)MS+NAA 1.0+6-BA 2.0;(4)MS+NAA 2.0+6-BA 2.0;(5)MS+NAA 2.0+6-BA 1.0;(6)MS+NAA 2.0+6-BA 0.5;(7)MS+NAA 2.0+6-BA 0.2.丛生芽增殖培养基:(8)MS+6-BA 3.0+NAA 0.1.壮苗培养基:(9)MS+6-BA 2.0+IBA 0.5.生根培养基(10)1/2MS+NAA 0.5.以上培养基均附加蔗糖30 g*L-1,琼脂7 g *L-1,pH 5.8,培养温度(25±2)℃,光照12 h*d-1,光照度1 500～2 000 lx.     

Simultaneous quantification of sialyloligosaccharides from human milk by capillary electrophoresis

The acidic oligosaccharides of human milk are predominantly sialyloligosaccharides. Pathogens that bind sialic acid-containing glycans on their host mucosal surfaces may be inhibited by human milk sialyloligosaccharides, but testing this hypothesis requires their reliable quantification in milk. Sialyloligosaccharides have been quantified by anion exchange high-performance liquid chromatography (HPLC), reverse- or normal-phase HPLC, and capillary electrophoresis (CE) of fluorescent derivatives; in milk, these oligosaccharides have been analyzed by high pH anion exchange chromatography with pulsed amperometric detection and, in our laboratory, by CE with detection at 205nm. The novel method described here uses a running buffer of aqueous 200mM NaH2PO4 (pH 7.05) containing 100mM sodium dodecyl sulfate (SDS) mixed with 45% (v/v) methanol to baseline resolve 5 oligosaccharides and separate all 12. This allows automated simultaneous quantification of the 12 major sialyloligosaccharides of human milk in a single 35-min run. This method revealed differences in sialyloligosaccharide concentrations between less and more mature milk from the same donors. Individual donors also varied in expression of sialyloligosaccharides in their milk. Thus, the facile quantification of sialyloligosaccharides by this method is suitable for measuring variation in expression of specific sialyloligosaccharides in milk and their relationship to decreased risk of specific diseases in infants.     

Stereoselective determination of clenbuterol in human urine by capillary electrophoresis

The effectiveness of capillary electrophoresis (CE) in the field of stereoselective determination of drugs in biological matrices is demonstrated by analyzing clenbuterol in human urine. Due to the very low therapeutical doses of 20–40 μg per day the total concentrations in urine are 1–10 ng/ml. The sample was extracted with hexane–tert.-butyl methyl ether (99.5:0.5). The reconstituted sample was injected electrokinetically (50 s, 10 kV). Using phosphate buffer, pH 3.3 and hydroxyethyl-β-cyclodextrin as chiral selector the total analysis time was below 15 min. The limit of determination was estimated as 0.5 ng/ml per enantiomer. S-(−)-Bupranolol was used as internal standard. Both precision and accuracy of the method were within the limits for biological samples. The application to human urine from patients having received therapeutical doses showed a slightly predominant excretion of the (+)-enantiomer to the (−)-enantiomer.     

Simultaneous determination of vigabatrin and amino acid neurotransmitters in brain microdialysates by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD) coupled to in vivo microdialysis sampling was used in order to monitor simultaneously a drug and several neurotransmitters in the brain extracellular fluid. Determination of the antiepileptic drug vigabatrin and the amino acid neurotransmitters glutamate (Glu), l-aspartate (l-Asp) and gamma-aminobutyric acid (GABA) was performed on low-concentration samples which were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) and separated using a pH 9.2 75 mM sodium borate running buffer containing 60 mM sodium dodecyl sulfate (SDS) and 5mM hydroxypropyl-beta-cyclodextrin (HP-beta-CD). Glu, l-Asp and vigabatrin derivatized at a concentration of 1.0 x 10(-9) M, and GABA derivatized at a concentration of 5.0 x 10(-9) M, produced peaks with signal-to-noise ratios of 8:1, 8:1, 4:1 and 5:1, respectively. The nature of the neurotransmitter peaks found in rat brain microdialysates was confirmed by both electrophoretic and pharmacological validations. This method was used for monitoring vigabatrin and amino acid neurotransmitters in microdialysates from the rat striatum during intracerebral infusion of the drug and revealed rapid vigabatrin-induced changes in GABA and Glu levels. This original application of CE-LIFD coupled to microdialysis represents a powerful tool for pharmacokinetic/pharmacodynamic investigations.     

Chemiluminescence determination of pharmacologically active compounds by capillary electrophoresis.

A simple and rapid capillary electrophoresis with direct chemiluminescence method has been developed for the determination of five natural pharmacologically active compounds including rutin, protocatechuic aldehyde, chlorogenic acid, luteolin and protocatechuic acid. The luminol as a component of the separation electrolyte buffer was introduced at the head of the separation capillary. The separation of five compounds was carried out in a fused-silica capillary with 15.0 mmol/L tetraborate, 1.0 mmol/L SDS and 0.42 mmol/L luminol (pH 8.5). The analytes was determined by enhancing the chemiluminescence of luminol with 0.13 mmol/L K3Fe(CN)6 in 0.05 mol/L NaOH, which was introduced at the post-column stage. The voltage applied was 16 kV. Under the optimum conditions, the analytes were separated within 10 min. The excellent linearity was obtained over two to three orders of magnitude with a detection limit (signal:noise = 3) of 0.012-0.055 micromol/L for all five analytes. The method was successfully used in the analysis of pharmaceutical and biological samples, and the assay results were satisfactory.     

Simultaneous determination of NO and NO in fisheries wastewaters high in chloride concentration by capillary ion electrophoresis with direct UV detection

The simultaneous determination by capillary ion electrophoresis of nitrate and nitrite in wastewaters with chloride concentrations of 15 to 23g/l is described. Chloride concentrations over 200mg/l hampered the determination; thus, lead, mercuric and silver acetate were used to precipitate chloride. Silver acetate added in 1.5 times the stoichiometric amount for AgCl formation gave the best results in terms of nitrate and nitrite peak resolution.     

Rapid simultaneous determination of glucagon and insulin by capillary electrophoresis immunoassays

A rapid capillary electrophoresis (CE) with laser-induced fluorescence (LIF) competitive immunoassay has been developed for the determination of glucagon in biological mixtures. In the assay, fluorescein-conjugated glucagon is mixed with the sample followed by addition of anti-glucagon. Free and antibody-bound, tagged glucagon could be separated in 3 s using CE to obtain quantitative determination of glucagon with a concentration detection limit of 760 pM. The assay was combined with a previously developed competitive immunoassay for insulin to produce a simultaneous immunoassay for both peptides. The method was used to determine glucagon content of islets of Langerhans.     

Indirect determination of pyruvic acid by capillary electrophoresis with amperometric detection

A method of indirectly measuring pyruvic acid (PA) by capillary electrophoresis with amperometric detection is proposed for the first time. It is based on the oximation reaction between PA and hydroxylamine (NH(2)OH), and the quantification of PA was performed by direct and sensitive amperometric detection of excessive NH(2)OH after the oximation reaction. This method displayed a good sensitivity, and the detection limits of NH(2)OH and PA are 1.76 x 10(-7) and 3.88 x 10(-7)mol/L, respectively at S/N=3. The linear relationship between the peak current and PA concentration is exhibited over the range from 4 x 10(-6) to 1 x 10(-4)mol/L. This method has been applied to determine PA in rat plasma with satisfactory results.     

Simultaneous determination of flavones and phenolic acids in the leaves of Ricinus communis Linn. by capillary electrophoresis with amperometric detection

Capillary electrophoresis (CE) with amperometric detection (AD) has been developed for the separation and determination of disaccharide glycoside rutin, gentistic acid, quercetin, and gallic acid in the leaves of Ricinus communis Linn. for the first time. The effects of the acidity and the concentration of the running buffer, separation voltage, injection time, and detection potential were investigated to acquire the optimum conditions for the determination of the four analytes. The detection electrode was a 300microm diameter carbon disc electrode at a detection potential of +0.90V (versus saturated calomel electrode (SCE)). The four analytes could be well separated within 10min in a 40cm length fused silica capillary at a separation voltage of 15kV in a 50mM borate buffer (pH 9.0). The relation between peak current and analyte concentration was linear over about 3 orders of magnitude with detection limits (S/N=3) ranging from 0.8 to 2.9microM for all the analytes. The proposed method has been successfully applied to monitor flavones and phenolic acids in the real plant samples with satisfactory assay results.     

Simultaneous detection of reduced and oxidized glutathione in tissues and mitochondria by capillary electrophoresis

We have developed a rapid and precise method for glutathione quantitation by capillary electrophoresis, that allows a low amount of both redox forms to be measured. Small fragments of rat heart or liver tissues (20 mg wet weight) and the corresponding mitochondria (1 mg protein) were homogenized in 1% perchloric acid and the acid-soluble phase ultrafiltered by centrifugation with a microconcentrator (M_r cut-off 3000 Da). The analysis was performed at a constant temperature (28°C) using a Beckman P/ACE System 2100, equipped with a UV absorbance detector set to 200 nm. The limit of quantitation in heart tissue was 1.8 μM for GSH and 1.2 μM for GSSG. Myocardial concentrations of GSH and GSSG were 8.1±2.6 and 0.45±0.15 (nmol/mg protein±S.D.), respectively. The ratio of GSH to GSSG was 17.8±1.3 for heart tissue, whereas it was much higher (>100) in the mitochondria. An oxidative stress decreased the myocardial tissue GSH/GSSG ratio, indicating that the CE analysis of both glutathione forms is also a useful method to study biological redox modification.