Determination of Plant Thiols by Liquid Chromatography Coupled with Coulometric and Amperometric Detection in Lettuce Treated by Lead(II) Ions

The main aim of this paper is to utilize high performance liquid chromatography with electrochemical detection for determination of thiols content in plants tissues of lettuce treated with lead(II) ions (0, 0.5 and 1 mM). We used two HPLC‐ED instruments: HPLC coupled with one channel amperometric detector and HPLC coupled with twelve channel coulometric detector to detect simultaneously twelve thiols. The detection limits of thiols measured by CoulArray detector were about two magnitudes lower in comparison to those measured by Coulochem III detector and were from tens to hundreds pM. Under the optimal conditions, we utilized HPLC‐CoulArray detector for analysis of tissues from lettuce plants. In addition, distribution and accumulation of lead ions with high spatial resolution was monitored using laser induced breakdown spectroscopy.     

RP-HPLC analysis of phenolic compounds and flavonoids in beverages and plant extracts using a CoulArray detector

Methods were developed for the analysis of natural antioxidants including phenolic compounds and flavonoids in beverages and plant extracts using gradient HPLC with multi-channel electrochemical coulometric detection. Suitability of various reversed-phase columns for this purpose was compared; pH and mobile phase gradients were optimized with respect to the separation selectivity and sensitivity of detection. Because of different target compounds in various sample types, the overlapping resolution maps and the normalized resolution product approaches described earlier were used to select optimum columns and gradients to suit the analysis of the individual sample types. The methods were applied to the analysis of phenolic compounds and flavonoids in beer, wine, tea, and yacon extracts. 32 phenolic compounds were identified and determined, including derivatives of benzoic and cinnamic acids, flavones, and a few related glycosides. Eight-channel CoulArray detection offers high selectivity and sensitivity with limits of detection in the low microg L(-1) range, at least an order of magnitude lower than single-channel coulometric detection using the Coulochem detector. No special sample pretreatment is necessary and, because of the compatibility of the CoulArray detector with gradient elution, phenolic antioxidants of different polarities can be determined in a single run. In addition to the retention times, the ratios of the areas of the pre-dominant and post-dominant peaks to the area of the dominant peak can be used for improved identification of natural antioxidants.     

Optimisation of gradient HPLC analysis of phenolic compounds and flavonoids in beer using a coularray detector

A method was developed for simultaneous analysis of natural antioxidants in beer using multichannel electrochemical detection with a CoulArray detector, which enables selective and sensitive antioxidant detection in gradient HPLC and facilitates the identification of analytes based on the ratios of signals recorded at different potentials applied to the detection cells arranged in series. The separation conditions were optimised for 27 phenolic compounds including derivatives of benzoic and cinnamic acids, flavones, and a few related glycosides identified in beer samples. Separation selectivities of 11 columns with different stationary phase chemistries were compared, and the pH and gradient programs were optimised for the individual columns to provide best resolution and high number of resolved peaks, using the window-diagram approach. The effects of pH on the sensitivity of electrochemical coulometric detection were considered in the optimisation approach. The optimised conditions were applied to the analysis of real beer samples.     

Application of an electrochemical detector to the determination of procarbazine hydrochloride by high-performace liquid chromtography

An amperometric flow-through detector with a carbon paste working electrode was utilized as a high-performance liquid chromatographic (HPLC) detector to determine procarbazine hydrochloride, an antineoplastic agent, in both buffer solution and biological fluids. The HPLC system included an amino-cyano stationary phase and an aqueous (pH 7)-methanolic mobile phase which enabled the separation of procarbazine from its only electroactive degradation product, N-isoprophyl-a- (2-methylhydrazono)-p-toluamide. The electrochemical detector, with an approximate limit of detection of 2 ng procarbazine injected, was 20 times more sensitive to procarbazine than a typical UV detector. The low dead volume (I μl) and superior selectivity of the electrochemical detector enabled the HPLC determination of procarbazine in untreated human urine and plasma.     

色谱指纹图谱在苹果酒质量评价中的应用

采用反相高效液相色谱-电化学检测法研究了14种苹果酒样品的指纹图谱。以标准品绿原酸进行定位,通过对图谱分析和相对保留时间计算,确定了8个共有峰。根据共有峰的峰面积用相关系数法和向量夹角余弦法计算相似度,两种方法的计算结果一致。实验结果表明同一厂家生产的苹果酒相似度较好。该法为苹果酒的产品分析提供了有效的微观信息,为苹果酒的质量控制、新产品的研发以及苹果酒行业标准的制定提供一种可行思路。     

Reversed‐Phase High‐Performance Liquid Chromatography with Electrochemical Detection of Anthocyanins

Abstract

Anthocyanins, flavonoid compounds present in grapes and wines, were determined by reverse‐phase high‐performance liquid chromatography (RP‐HPLC) with electrochemical detection (RP‐HPLC‐ED). The method developed consists of RP‐HPLC gradient elution with voltammetric detection using a glassy carbon electrode after separation in an Inertsil ODS‐3V analytical column. Good peak resolution was obtained following direct injection of a 50 µL sample of anthocyanins in a mobile phase of pH 2.20. The results show that six different anthocyanins: cyanidin‐3‐O‐glucoside chloride (kuromanin chloride), cyanidin‐3,5‐di‐O‐glucoside chloride (cyanin chloride), malvidin‐3‐O‐glucoside chloride (oenin chloride), malvidin‐3,5‐di‐O‐glucoside chloride (malvin chloride), delphinidin‐3‐O‐glucoside chloride (myrtillin chloride), and peonidine‐3‐O‐glucoside chloride, all with antioxidant properties, can be separated in a single run by direct injection of solution. The limit of detection (LOD) for these compounds was lower than 0.3 µM. The method can also be applied to the analysis of these compounds in red wines and in skins and pulp extracts of red grapes, since all these antioxidants are electroactive.     

Quantitative and pharmacokinetic analysis of naloxone in plasma using high-performance liquid chromatography with electrochemical detection and solid-phase extraction

In this study we present a method for measuring naloxone in plasma after intravenous and oral administration of naloxone to humans, in order to study its pharmacokinetic profile. The method consists of a solid-phase extraction step followed by detection on a high-performance liquid chromatographic (HPLC) system equipped with an electrochemical dual-electrode detector. The extraction step employs cyanopropyl columns optimized for naloxone extraction to allow for elution of naloxone by the HPLC mobile phase; this eluate is then directly injected in the HPLC instrument. The HPLC system employs a radial compression phenyl column with a mobile phase containing 18% (v/v) acetonitrile and pentanesulfonic acid as ion-pairing agent; this system shows extraordinary high plate counts for naloxone. The detection limit is 3 ng (signal-to-noise ratio = 3) free naloxone per ml plasma. Following intravenous injection of 30 mg naloxone hydrochloride in two subjects, it was possible to determine the free naloxone concentration in the plasma for 8 h, more than four times the half-life of naloxone in plasma in humans.     

Determination of tetracyclines in ovine milk by high-performance liquid chromatography with a coulometric electrode array system

A method has been developed to analyze residual tetracyclines (TCs) (oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), methacycline (MTC), doxycycline (DC)) in ovine milk, using high-performance liquid chromatography (HPLC) with a coulometric electrode array system. The samples were pretreated, using liquid-liquid extraction based on hexane. The chromatography was performed, using a C18 column (150 mm x 4 mm i.d. and 5 microm) with a mobile phase: sodium phosphate monobasic dihydrate (pH 2.2, 0.05 M)-acetonitrile (78:22, v/v). The flow rate of mobile phase was kept constantly at 1ml/min. The residues were monitored by an ESA electrochemical detector. Potentials of four electrodes in series were set at 400, 660, 680 and 700 mV, respectively. The first electrode was set to remove those interfering substances that may co-elute with TCs and the other three electrodes were used for quantification. The maximal potential of our detection was 700 mV. Calibration curve showed good linearity and the detection limit of TCs was 12.5, 20, 25, 10 and 25 ng/ml, respectively. Optimization of the pH of the mobile phase, the proportion of acetonitrile and the pH of the pretreatment were also performed. Recoveries of TCs from spiked samples were more than 88% and the relative standard deviations were less than 4.3%. This method was reliable, sensitive, economical and suited for routine monitoring of TC residues in ovine dairy milk.     

Validated HPLC determination of the two fixed dose combinations (chlordiazepoxide hydrochloride and mebeverine hydrochloride; carvedilol and hydrochlorothiazide) in their tablets

Simple, rapid, and selective RP-HPLC methods with UV detection were developed for simultaneous determination of chlordiazepoxide hydrochloride and mebeverine hydrochloride (Mixture I) and carvedilol and hydrochlorothiazide (Mixture II). The chromatographic separation in both mixtures was achieved by using an RP-C8 (octylsilyl) analytical column. For Mixture I, a mobile phase composed of acetonitrile-0.05 M disodium hydrogen phosphate-triethylamine (50 + 50 + 0.2, v/v/v), pH 2.5, was used; the detector wavelength was 247 nm. For Mixture II, the mobile phase consisted of acetonitrile-0.05 M disodium hydrogen phosphate (50 + 50, v/v), pH 4.0, and the detector was set at 220 nm. Quantification of the analytes was based on measuring their peak areas. Both mixtures were resolved in less than 6 min. The reliability and analytical performance of the proposed HPLC procedures were statistically validated with respect to linearity, range, precision, accuracy, selectivity, robustness, LOD, and LOQ. The linear dynamic ranges were 2.5-150 and 2.5-500 microg/mL for chlordiazepoxide HCI and mebeverine HCI, respectively, and 0.25-200 and 0.25-150 microg/mL for carvedilol and hydrochlorothiazide, respectively. The validated HPLC methods were successfully applied to the analysis of their commercial tablet dosage forms, for which no interfering peaks were encountered from common pharmaceutical adjuvants.     

Rapid separation of three glucosylated resveratrol analogues from the invasive plant Polygonum cuspidatum by high‐speed countercurrent chromatography

Three glucosylated resveratrol analogues (piceid, piceatannol glucoside, resveratroloside) were successfully isolated from the crude MeOH extract of the invasive plant species Polygonum cuspidatum by semi‐preparative high‐speed countercurrent chromatography with a two‐phase solvent system composed of cyclohexane‐ethyl acetate‐methanol‐water (1:5:1:5, v/v/v/v). Piceid (23 mg), resveratroloside (17 mg), piceatannol glucoside (15 mg) of purities over 80% were isolated from 500 mg crude MeOH extract in one step. Subsequent passage over a SPE column was used to quickly bring their purities to over 90%. The purities were determined by HPLC analysis and their structures were elucidated by proton nuclear magnetic resonance (¹H‐NMR), HMBC, ESI‐MS and HR‐MS.     

High performance liquid chromatography with an electrochemical detector in the cathodic mode as a tool for the determination of p-nitrophenol and assay of acid phosphatase in urine samples

Utilizing a commercially available helium-purging device and PEEK tubes for all tubing, especially for connection between the mobile phase and pump, high performance liquid chromatography with an electrochemical detector (ECD/HPLC) at the cathodic mode is a simple and precise method for the determination of p-nitrophenol (NP). Studies with cyclic and hydrodynamic voltammetry indicated that 25% aqueous MeOH containing 0.1% (v/v) CF(3)CO(2)H and -0.8 V vs. Ag/AgCl are the best mobile phase and detection potential for cathodic ECD/HPLC. With the present system, the limits of detection and determination were 0.2 and 0.25 microM, respectively, and up to 50 microM, a linear calibration curve was afforded. Within-day precisions for the analysis of 5 and 50 microM NP were 0.8 and 0.7% (n=6), respectively, and between-day precisions (n=6) for these samples were 3.5 and 2.2%, respectively. Compared with the commonly used Bessey-Lowry-Brock method, cathodic ECD/HPLC was useful for the assay of acid phosphatase in urine samples with p-nitrophenyl phosphate disodium salt as a substrate.     

Determination of Belotecan in the Plasma,Bile, and Urine of Rats by High-Performance Liquid Chromatography with Fluorescence Detection and Its Application to a Pharmacokinetic Study

Abstract

A simple and reliable high-performance liquid chromatographic (HPLC) method was developed for the determination of belotecan in the plasma, urine, and bile samples of rats. Belotecan was analyzed with HPLC using a C18 column with fluorescence detector. A mixture of acetonitrile–0.1 M potassium phosphate buffer at pH 2.4 (25:75, v/v) and 0.2% trifluoroacetic acid was used as the mobile phase. The lower limits of quantitation (LOQ) were 5 ng mL^?1 for the plasma and 5 µg mL^?1 for the urine and bile samples. The method has been readily applied for the routine pharmacokinetic study of belotecan in small laboratory animals.     

Isolation and determination of estrogens in water samples by solid‐phase extraction using molecularly imprinted polymers and HPLC

Advanced SPE with molecularly imprinted polymers (MIP) was used in this study. A noncovalent imprinting approach was applied to separate 17β‐estradiol, estriol, and estrone from water samples. Polymer material was prepared by bulk polymerization with methacrylic acid as a functional monomer, divinylbenzene and ethyleneglycol dimethacrylate as crosslinkers, and acetonitrile, acetonitrile/toluene (3:1, v/v) or isooctane/toluene (1:99, v/v) as a porogen. We also prepared an MIP film on a silica gel surface with methacrylic acid and ethyleneglycol dimethacrylate as monomers and acetonitrile as a solvent. Qualitative and quantitative hormone analyses were carried out by HPLC with various detection techniques, including UV/visible spectroscopic detection (diode array detection) and electrochemical detection (CoulArray). The results of the study indicate that MIP technology is an excellent method for the quality control of estrogens in environmental analyses with a low quantification limit for 17β‐estradiol of around 26 (diode array detection) and 0.25 ng/mL (electrochemical detection). The proposed method was found to be suitable for routine determinations of the analyzed compound in environmental laboratories.     

Separation of B6 vitamers with micellar liquid chromatography using UV and electrochemical detection

Separation of six vitamers of vitamin B6 was performed by RP-HPLC using micellar mobile phase, UV and electrochemical detection. Effect of temperature, type and amount of organic modifier in mobile phase on efficiency and asymmetry factor showed that, the appropriate conditions were temperature of 35 degrees C and 3.0-5.0% (v/v) 1-butanol in mobile phase. Variations of selectivity factor versus 1-butanol concentration, pH of mobile phase, and SDS concentration was investigated and the following optimized conditions were selected for the separation: 3.0% (v/v) 1-butanol, pH=5.5 and 65 mM SDS in mobile phase. Electrochemical behavior of vitamers in optimized mobile phase was investigated using cyclic voltammetry, and potential of +1.2 V versus Ag/AgCl(Sat.) was chose as working potential. Finally, separation of B6 vitamers using UV detection at 254 nm and electrochemical detection at +1.2 V was compared.     

Determination and comparison of flavonoids and anthocyanins in Chinese sugarcane tips,stems, roots and leaves

The flavonoids and anthocyanins in Chinese sugarcane (Saccharum sinensis Roxb.) tips, stems, roots and leaves were separately analyzed by HPLC‐UV diode array detector, respectively. The results indicated that the content of flavonoids in sugarcane leaves was considerably high in comparison with previous reports in Brazil sugarcane (S. officinarum L.) leaves. Moreover, the content of flavonoids in sugarcane tips and roots was also high in comparison with sugarcane stems. For another, the content of anthocyanins in sugarcane roots was higher than that in other parts of the sugarcane, such as leaves, tips and stems. In addition, two anthocyanins, named petunidin 3‐O‐(6″‐succinyl)‐rhamnoside and cyanidin‐3‐O‐glucoside, were first identified from S. sinensis by HPLC‐UV diode array detector and HPLC‐MS/MS.     

Determination and validation of duloxetine hydrochloride in capsules by HPLC with pre-column derivatization and fluorescence detection

A high-performance liquid chromatographic (HPLC) method is described for the determination of duloxetine hydrochloride in capsules. The method was based on pre-column derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole using the fluorimetric detection technique. Duloxetine hydrochloride was analyzed by HPLC using an Inertsil C18 column (5 μm, 150 × 4.6 mm) and mobile phase consisted of methanol and water (65:35, v/v). The fluorescence detector was adjusted at excitation and emission wavelengths of 461 and 521 nm, respectively. The linearity of the method was in the range of 10-600 ng/mL. Limits of detection and quantification were 0.51 and 1.53 ng/mL, respectively. The proposed method was successfully applied for determination of duloxetine hydrochloride in its pharmaceutical preparation. The results were in good agreement with those obtained using a reference method.     

A universal peroxyoxalate-chemiluminescence detection system for mobile phases of differing pH.

A universal peroxyoxalate-chemiluminescence detection system for high performance liquid chromatography, available for a variety of mobile phases, has been developed. The system consisted of a dual-head short-stroke pump and a chemiluminescence detector. The standard conditions using bis(2,4,6-trichlorophenyl) oxalate (TCPO) as aryl oxalate were as follows. The first postcolumn solution was the mixture of 0.5 M imidazole-nitric acid (pH 7.5) and acetonitrile (1:4, v/v). The second was acetonitrile containing TCPO-hydrogen peroxide. These two solutions were delivered by the two pump-heads. After the pH of the column eluate was adjusted to the optimum range (6.5-7.5) by the first postcolumn solution, the solution was mixed with the second postcolumn solution. After flowing through a reaction coil, the chemiluminescence of the mixture was monitored. Using this system, a high sensitivity (fmol level) was obtained for perylene as an analyte with mobile phases having different pH values (2.0-8.0). Polycyclic aromatic hydrocarbons became detectable to a high sensitivity even after the column separation using an acidic mobile phase. The detection sensitivity of nitrated pyrenes after on-line electrochemical reduction using an acidic mobile phase was also increased. This system might be available for other aryl oxalates by some modifications of the postcolumn solutions.     

A Simple Isocratic High-Performance Liquid Chromatographic (HPLC) Determination of Naproxen in Human Plasma using a Microbore Column Technique

Abstract

A simple and sensitive HPLC method was developed for the determination of naproxen in human plasma. The assay employs a microbore column packed with a C18 reversed-phase material (5 μm ODS Hypersil) with an isocratic mixture of acetonitrile and 10 mM phosphate buffer, pH 2.5 (40:60, v/v) as the mobile phase. The mobile phase was pumped at a flow rate of 0.5 ml/min. For sample analysis 200 μl of acetonitrile containing internal standard (flurbiprofen) was added to 100 μl of plasma. After centrifugation 10 mM phosphate buffer, pH 7.4 (200 μl) was added to the tube, then vortexed and centrifuged. The supernatant (20 μl) was injected onto the HPLC column. The chromatographic separation was monitored by a fluorescence detector at an emission wavelength of 350 nm with an excitation wavelength of 225 nm. The direct precipitation of plasma protein using acetonitrile gave a good recovery for both naproxen and the internal standard. The detection limit was 0.1 μg/ml for naproxen. The intra- and inter-assay coefficients of variation at different concentrations evaluated were less than 10%.     

Determination of memantine in plasma and vitreous humour by HPLC with precolumn derivatization and fluorescence detection

A new HPLC procedure with precolumn derivatization and rimantadine as the internal standard for determining memantine, a candidate agent for the treatment of glaucoma in plasma and vitreous humour, has been developed and validated. Precolumn derivatization was performed with 9-fluorenylmethyl-chloroformate-chloride (FMOC-Cl) as the derivatization reagent and followed by a liquid-liquid extraction with n-hexane. Optimal conditions for derivatization were an FMOC-Cl concentration of 1.5 mM, a reaction time of 20 min, the temperature at 30°C, the borate buffer pH 8.5, and a borate buffer-acetonitrile ratio of 1:1. The derivatives were analyzed by isocratic HPLC with the fluorescence detector λex 260 nm λem 315 nm on a Novapack C(18) reversed-phase column with a mobile phase of acetonitrile-water (73:27, v/v), 40°C, and a flow rate of 1.2 mL/min. The linear range was 10-1000 ng/mL with a quantification limit of ～ 10 ng/mL for both types of samples. This analytical method may be suitable for using in ocular availability studies.     

Chiral separation of isoxanthohumol and 8‐prenylnaringenin in beer,hop pellets and hops by HPLC with chiral columns

Xanthohumol, isoxanthohumol, and 8‐prenylnaringenin in beer, hop and hop pellet samples were analyzed by HPLC using an InertSustain phenyl column and the mobile phase containing 40% methanol and 12% 2‐propanol. Fractions of isoxanthohumol and 8‐prenylnaringenin obtained by the above HPLC were separately collected. Isoxanthohumol and 8‐prenylnaringenin were enantioseparated by HPLC using a Chiralcel OD‐H column with a mobile phase composed of hexane–ethanol (90:10, v/v) and a Chiralpak AD‐RH column with a mobile phase composed of methanol–2‐propanol–water (40:20:40, v/v/v), respectively. Isoxanthohumol and 8‐prenylnaringenin from beer, hop and hop pellet samples were found to be present in a racemic mixture. This can be explained by the fact that the two analytes were produced by a nonenzymatic process. The effects of boiling conditions on the conversion of xanthohumol into isoxanthohumol were also studied. A higher concentration of ethanol in heating solvent resulted in a decrease in the conversion ratio and the conversion was stopped by addition of ethanol at >50% (v/v). The isomerization was significantly affected pH (2−10) and the boiling medium at pH 5 was minimum for the conversion. Therefore, it was suggested that xanthohumol was relatively difficult to convert to isoxanthohumol in wort (pH 5−5.5) during boiling.