High-performance Capillary Electrophoresis Method Research Articles

Selection of Optimal Conditions for the Separation of Curcuminoids by High-performance Capillary Electrophoresis (HPCE) Using Micellar Electrokinetic Chromatography (MEKC)

Introduction. Curcuminoids are natural polyphenolic colorants with a wide spectrum of pharmacological activity. Modern hybrid and classical methods are used for the determination of curcuminoids. However, the problem of the analysis of curcuminoids in the composition of plant and medicinal objects remains relevant. In this work, capillary electrophoresis (HPCE) in the variant of micellar electrokinetic chromatography with diode array detection (MEKC/DAD) was used as such a method. Aim. The purpose of this analysis is the choice of optimal conditions for the separation of curcuminoids using capillary electrophoresis in the form of micellar electrokinetic chromatography. Materials and methods. Separation of curcuminoids was carried out on an Agilent 7100 CE capillary electrophoresis instrument with a diode array detector and an Agilent Chem Station monitoring and data acquisition system. Borate buffer (20 mM, pH 9.3) with the addition of sodium dodecyl sulfate – SDS (30 mM) in ratios of 1 : 1 was used like the electrolyte. The sample was injected hydrodynamically – 50 mbar/3 sec, electrode voltage is +25 kV, quartz capillary – total capillary length/effective capillary length = 30/40 cm, ID = 50 µm, capillary temperature +20 ° С. The output of curcuminoids was monitored at the wavelength of the diode-matrix detector – λ max = 425 nm/4 nm, the refereed wavelength is 360 nm/100 nm. Results and discussion. Curcuminoids in a borate buffer solution with the addition of SDS are practically not separated, which is associated with a high activity of the electroosmotic flow, to suppress which 95 % ethyl alcohol was added. In the course of research, it was found that the addition of ethyl alcohol in an amount of 20 % relative to the buffer solution makes it possible to separate the mixture of curcuminoids. Conclusion. Thus, the conditions for the separation of the total curcuminoids by the HPCE method in the variant of micellar electrokinetic chromatography (MEKC) were selected. It has been established that for the separation of curcuminoids by this method, it is necessary to use an electrolyte consisting of a mixture of equal volumes of borate buffer (20 mM) and sodium dodecisulfate (30 mM) and ethyl alcohol in an amount of 20 % of the electrolyte volume. A further increase in the concentration of ethyl alcohol in the electrolyte is unreasonable, since it can adversely affect the stability of micelles.

Compositional Monosaccharide Analysis of Morus nigra Linn by HPLC and HPCE Quantitative Determination and Comparison of Polysaccharide from Morus nigra Linn by HPCE and HPLC.

Background: Morus nigra Linn is not only treated as health food but also medicine in Chinese history.Objective: Here, we have extracted and separated the heteroglycan to monosaccharides of the dry fruits.Method: After heteroglycan being hydrolysised, then were we derivatived the monosaccharides with 1-phenyl-3-methyl-5-pyrazolone (PMP), and then subsequently HPCE and HPLC were used to separate. HPCE, which held an uncoated capillary (d=75μm) and detected by PDA at 245 nm with borate buffer, the voltage was set at 15 kV and capillary temperature was 25°C. There is Yilite column (Hypersil BDS C18 5μm, 4.6mm×250mm) with HPLC and detected by UV at 245 nm and capillary temperature 25°C.The High Performance capillary electrophoresis method and High Performance Liquid Chromatog-raphy method were compared on monosaccharide composition and quantitative determination of polysac-charide from the black mulberry.Results: Through several indicators including standard curve, precision, reproducibility, stability and recovery rate, the result of the two methods is basically consistent while they have complementary ad-vantages.Conclusion: Both methods are suitable for the determination of the black mulberry polysaccharide, but each has its own merits.

A study of ofloxacin and levofloxacin photostability in aqueous solutions

Introduction. The photostability is one of the most important properties of drugs. A comprehensive study of ofloxacin (OFX) and levofloxacin (LVX) photostability in aqueous solutions was performed. Ofloxacin is a chemotherapeutic agent belonging to the second generation fluoroquinolones and is a racemate of (R)-(+)-ofloxacin and (S)-(-)-ofloxacin (LVX).Material and Methods. Samples of OFX and LVX were subjected to stress conditions of UV irradiation using a mercury‑vapor lamp. The study involved development of enantioselective high‑performance liquid chromatography (HPLC) and high‑performance capillary electrophoresis (HPCE) methods for separation of OFX enantiomers and their degradation products. These methods were used to monitor the degradation process of OFX and LVX under irradiation and to determine the kinetics of degradation of these antibacterial agents. Moreover, the identification of photoproducts was also attempted. The structure of the main photoproducts was examined by mass spectrometry (MS).Results and Conclusions. Using HPLC method it was possible to observe two products of OFX degradation and only one for LVX, while using HPCE method eight products of OFX degradation and six of LVX were observed. Some of the photoproducts retain character of optically active compounds. The trend of the photodegradation of both tested compounds was described by autocatalytic reaction proceeding according to the Prout‑Tompkins model. Some of the products of the decomposition catalyze this reaction. The rate of degradation was similar for both enantiomers but t0.5 was slightly longer for LVX than OFX. Based on MS experiments the photodegradation products of the studied fluoroquinolones and possible pathways of UV induced decay were identified.

A study of ofloxacin and levofloxacin photostability in aqueous solutions

Introduction. The photostability is one of the most important properties of drugs. A comprehensive study of ofloxacin (OFX) and levofloxacin (LVX) photostability in aqueous solutions was performed. Ofloxacin is a chemotherapeutic agent belonging to the second generation fluoroquinolones and is a racemate of (R)-(+)-ofloxacin and (S)-(-)-ofloxacin (LVX).Material and Methods. Samples of OFX and LVX were subjected to stress conditions of UV irradiation using a mercury-vapor lamp. The study involved development of enantioselective high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE) methods for separation of OFX enantiomers and their degradation products. These methods were used to monitor the degradation process of OFX and LVX under irradiation and to determine the kinetics of degradation of these antibacterial agents. Moreover, the identification of photoproducts was also attempted. The structure of the main photoproducts was examined by mass spectrometry (MS).Results and Conclusions. Using HPLC method it was possible to observe two products of OFX degradation and only one for LVX, while using HPCE method eight products of OFX degradation and six of LVX were observed. Some of the photoproducts retain character of optically active compounds. The trend of the photodegradation of both tested compounds was described by autocatalytic reaction proceeding according to the Prout-Tompkins model. Some of the products of the decomposition catalyze this reaction. The rate of degradation was similar for both enantiomers but t0.5 was slightly longer for LVX than OFX. Based on MS experiments the photodegradation products of the studied fluoroquinolones and possible pathways of UV induced decay were identified.

Selective Determination of Bisphenol A Based on Molecularly Imprinted Poly(ortho-phenylenediamine) /Multi- walled Carbon Nanotubes Composite Film

Background: Considering that BPA has emerged as a major public health issue, there is an urgent need to establish a simple, effective, and highly sensitive method for monitoring the levels of BPA. The aim of this work was to construct an electrochemical sensor for the detection of BPA by the combination of molecularly imprinted technique and MWCNTs. The optimization of test conditions and the performance of the imprinted sensor were discussed. The application of the sensor in real water samples was evaluated. Methods: The sensor was prepared by electropolymerizing o-phenylenediamine with bisphenol A after depositing carboxylfunctionalized multi-walled carbon nanotubes onto the surface of a glassy carbon electrode. The template can be quickly removed in methanol–acetic acid (1:1, v/v) solution. The formation of PoPD film was monitored by FT-IR spectra. The morphological structures of the modified electrodes were tested by SEM. K3[Fe(CN)6] served as an electrochemical probe in this work. Cyclic voltammetry and linear sweep voltammetry were employed for the electrochemical characterization of the sensor. Results: The molecularly imprinted polymer based sensor displayed an excellent recognition capacity toward BPA compared with other structurally similar molecules, and the existence of some inorganic ions and organic compounds has no obvious effect on the determination of the analyte. Additionally, the decrease of the peak current of K3Fe(CN)6 was proportional to the BPA concentration in the range of 4.0 x 10-7 to 8 .0 x 10-5 mol L-1 with a limit of detection of 6.0 x 10-8 mol L-1. The prepared sensor also showed stable repeatability. The results in real samples obtained by the proposed method agreed with those obtained from high performance capillary electrophoresis method. Conclusion: An electrochemical sensor for BPA based on electropolymerization of the oPD in the presence of BPA on the MGCE has been successfully fabricated. When the resulting sensor was applied to the determination of BPA, it presented high sensitivity, stability and selectivity. In addition, the fabrication procedure was quite simple. The use of MWCNTs enhanced the sensitivity of detection and resulted in a better LOD; in combination with the specific selectivity of the molecularly imprinting technique, the proposed sensor provided a bright potential for the determination of BPA in practical samples. Keywords: Bisphenol A, electropolymerization, molecularly imprinted polymer, multi-walled carbon nanotubes, poly-o-phenylenediamine.

Study on Determination of Synephrine and Artemisinin in <em>Phellinus vaninii</em> by High Performance Capillary Electrophoresis

This study established high performance capillary electrophoresis method to determine synephrine and Artemisinin in Phellinus vaninii. In 30 mmol/L Na2B4O7 buffer solution (pH = 10.00), effective separation of components to be tested was achieved, which provided a new approach of quality control of Chinese medicine Phellinus vaninii.

Multiwalled carbon nanotube-dispersive solid-phase extraction followed by high performance capillary electrophoresis for simultaneous determination of six adulterants in antihypertensive functional foods

A novel, simple and accurate high performance capillary electrophoresis method after multiwalled carbon nanotube-dispersive solid-phase extraction was developed for simultaneous determination of hydrochlorothiazide (HCT), chlortalidone (CTD), indapamide (IDP), reserpine (RSP), nifedipine (NDP) and valsartan (VST) in antihypertensive functional foods.

Determination of Synephrine and Naringenin in Aifunuangong Pills by High Performance Capillary Electrophoresis

This paper established high performance capillary electrophoresis method to determine synephrine and naringenin in aifunuangong pills. In 30mmol/L Na2B4O7 buffer solution (pH=10.00), effective separation of components to be tested was achieved, which provided a new approach of quality control of Chinese medicine aifunuangong pills.

Analysis of phenolic pollutants in human samples by high performance capillary electrophoresis based on pretreatment of ultrasound-assisted emulsification microextraction and solidification of floating organic droplet

A novel, simple and rapid method, termed ultrasound-assisted emulsification microextraction and solidification of floating organic droplet (UAEM–SFO) coupled to high performance capillary electrophoresis (HPCE), was developed for preconcentration and analysis of five phenolic compounds (PCs) in human urine and blood samples. The proposed method is based on microextraction of target analytes in less-toxic organic solvent under assistance of ultrasound. A micro-droplet of less-toxic organic solvent floating on the surface of liquid samples in a sealed vial can be dispersed into sample solutions under the ultrasound frequency, solidified under ice bath, collected with a medicine spatula, molten at ambient temperature, and finally subjected to HPCE analysis. The parameters of UAEM–SFO procedure including type and volume of extraction solvent, extraction temperature, time of ultrasound-assisted extraction and centrifugation, sample pH, and ionic strength were optimized. The influence parameters on HPCE resolution such as pH, concentration of running buffer, applied voltage and injection time were also investigated. This method requires only 40μL of 2-dodecanol extraction solvent and 8min of pretreatment time. The enrichment factors of analytes were in the range of 114–172 and extraction recoveries (69–86%) were obtained. Good linearity was achieved for five analytes in the range of 0.05–100μgL−1 and the correlation coefficients ranged from 0.9934 to 0.9999. The limits of detection were 0.01μgL−1for triclosan (TCS) and biophenol A (BPA), and 0.02μgL−1 for pentachlorophenol (PCP), 2,4-dichlorophenol (2,4-DCP) and 4-nonylphenol (4-NP) in urine samples, and 0.02μgL−1 for TCS and BPA, 0.04μgL−1 for PCP, 2,4-DCP and 4-NP in blood samples. The developed UAEM–SFO–HPCE method has a great potential in routine residual analysis of trace PCs in biological samples.

Simultaneous Determination of Seven Adulterants in Functional Foods by High-Performance Capillary Electrophoresis

Abstract A high-performance capillary electrophoresis method has been established for the simultaneous determination of seven functional food adulterants, namely, fenfluramine, pseudoephedrine, norpseudoephedrine, amfepramone, sibutramine, sildenafil, and strychnine. A mixture of 20 mM sodium tetraborate-10 mM sodium dodecyl sulfate-5% acetonitrile was used as a running buffer (pH 9.0), and analysis was carried out at a detection wavelength of 195 nm under a separation voltage of 17 kV at 25 °C. The upper limit of the linear ranges was 100 mg L −1 , with the linear correlation coefficients being greater than 0.998. The relative standard deviations ranged between 4.5% and 7.9% ( n = 7). The average recoveries were in the range of 79.6%–112.0%. The limits of detection (three times the signal-to-noise ratio, S/N = 3 ) were between 0.16 and 0.65 mg L −1 . The method is simple, fast, sensitive, accurate and cheap. It has been successfully applied for the determination of the seven target chemicals in functional foods

Simultaneous determination of six flavonoids in Hyperici Japonici Herba by HPCE-DAD

To establish a high performance capillary electrophoresis method with diode array detection (HPCE-DAD) for simultaneous determination of rutin, isoquercitrin, hyperoside, quercitrin, kaempferol and quercetin in Hyperici Japonici Herba. Based on the mode of capillary zone electrophoresis, 40 mmol x L(-1) borax was used as buffer solution (pH 8.62), uncoated fused silica capillary (56 cm x 64.5 cm x 75 microm) was used, separation voltage was 25 kV, detection wavelength was at 206 nm, column temperature was maintained at 25 degrees C, and sample was injected at 50 mbar, 8 s. Six flavonoids showed good linearity (r > 0.9953) in the range of the tested concentration, the average recoveries of the method were between 98.8%-102.9%. The method is simple, accurate and reproducible, and can be used for quality control of Hyperici Japonici Herba.

Analysis of Five Pharmacologically Active Compounds from the Tibetan Medicine Elsholtzia with Micellar Electrokinetic Capillary Chromatography

A high performance capillary electrophoresis method with diode array detector detection for the determination of five bioactive ingredients in Tibetan medicine Elsholtzia, namely quercetin, rutin, saussurenoside, kaempferol, and oleanolic acid, has been developed. The effects of several factors, such as the acidity, concentration of running buffer, separation voltage, temperature, and SDS concentration were investigated. The optimal conditions were 44 mmol/L boric acid running buffer (pH 8.5), 45 mmol/L SDS, 16 KV voltage, 20°C, and 10.0% (V/V) of acetonitrile. Under the optimum conditions, five components could be separated with a good baseline resolution within 17 min. The calibration curves showed good linear relationship over the concentration range of 5×10−4∼0.1 mg/mL for quercetin, rutin, saussurenoside, kaempferol, and 1×10−3∼0.1 mg/mL for oleanolic acid. The average recoveries of the method and RSD were (99.2%, 3.2%) for quercetin, (102.1%, 2.1%) for rutin, (99.4%, 1.5%) for saussurenoside, (98.9%, 1.8%) for kaempferol, and (99.0%, 2.9%) for oleanolic acid, respectively. The detection limits (S/N=3) were 1.1×10−4 mg/mL for quercetin, 2.6×10−4 mg/mL for rutin, 1.8×10−4 mg/mL for saussurenoside, 2.9×10−4 mg/mL for kaempferol, and 6.3×10−4 mg/mL for oleanolic acid, respectively. The method was simple, rapid, and reproducible and could be applied for the determination of quercetin, rutin, saussurenoside, kaempferol, and oleanolic acid in Tibetan medicine Elsholtzia, and the assay results were satisfactory.

Development and validation of a procedure for the determination of minoxidil in hair-regrowth formulations using two variants of capillary zone electrophoresis

A high performance capillary electrophoresis method was developed and validated for purity assessment of minoxidil bulk drug and for determination of minoxidil in Rogaine ®. The principal use of the method was in analyzing illicit minoxidil-containing hair-regrowth samples. Although validated for Rogaine, the procedure proved equally viable on illicit minoxidil-containing preparations. The developed method fulfilled the goal of providing an orthogonal technique to HPLC for confirmation of the presence of minoxidil in these imitations. The method was validated on two instruments, one utilizing EK injection, the other gravity injection. It is selective for minoxidil, which is separated from known process impurities and the single degradation impurity. Validation figures of merit for linearity/recovery (accuracy) and precision were in accordance with current expectations for method validation.

A high performance capillary electrophoresis method for determination of methotrexate in serum

目的 建立快速检测人血清中氨甲蝶呤药物浓度的方法.方法用高效毛细管电泳技术,选用直径75 μm,总长60 cm,有效长度50.5 cm熔融石英毛细管,以75 mmol/L,pH 7.4的磷酸盐为电泳缓冲液,检测波长为306 nm,在25 kV电压下的快速分离检测氨甲蝶呤,并对此方法进行方法学评价.结果氨甲蝶呤的出峰时间在10 min内,线性范围在1.1～1 100.0 μmol/L间,最低检出浓度为0.55 μmol/L,回收率为88.2%～98.2%,批间精密度为5.4%,批内精密度为4.2%.结论高效毛细管电泳方法具有快速、准确且成本低的特点,为临床和科研中测定血清氨甲蝶呤提供了一种有效的手段。

Determination of quinolizidine alkaloids inSophora tonkinensis by HPCE

A simple, rapid and reliable high-performance capillary electrophoresis method has been developed to determine quantitatively the alkaloid content of Sophora tonkinensis, a Chinese herb commonly known as shan-dou-gen. A total of seven quinolizidine alkaloids (cytisine, sophocarpine, matrine, lehmannine, sophoranol, oxymatrine and oxysophocarpine) could be readily separated within 15 min. The running buffer was 50 mM phosphate buffer (pH 2.5) containing 1% hydroxypropyl-beta-cyclodextrin and 3.3% isopropanol in water. The applied voltage was 25 kV, the capillary temperature was 25 degrees C, the detection wavelength was 200 nm and scopolamine butylbromide was used as internal standard. The method was used to analyse the chemical constituents of two commercial alternatives to shan-dou-gen. The alkaloid constituents of authentic shan-dou-gen gave a specific HPCE electropherogram that could be used to distinguish the drug from potential substitutes. Furthermore, the content of oxymatrine and the total content of the seven quinolizidine alkaloids could be used as quantitative markers in order to assess the quality of S. tonkinensis.

High performance capillary electrophoresis method for determination of ibuprofen enantiomers in human serum and urine

A direct and stereospecific capillary zone electrophoresis (CZE) method for quantification ibuprofen enantiomers in biological matrices: human serum and urine, has been developed. Chiral separation of the enantiomers of ibuprofen and (+)- S-indobufen [(+)- S-INDB, internal standard, IS] was obtained in an uncoated silica capillary filled with a background electrolyte (BGE), consisted of heptakis 2,3,6-tri- O-methyl-β-cyclodextrin (TM-β-CD) in buffer of pH 5.0. The complete enantioselective analysis of ibuprofen and its 1-hydroxy metabolite confirmed appropriate specificity of the method. The electrophoretic parameters: electroosmotic ( μ EOF) and electrophoretic ( μ ep) mobility and resolution factor ( R s) were determined. Extraction procedures with organic solvent and solid phase extraction (SPE) with C 18 stationary phase for isolation of enantiomers from biological fluids were compared. SPE method for further studies was chosen. Stereoselective extraction of IBP enantiomers from serum at basic pH has been discovered. Validation of the method was carried out. Calibration curves of ibuprofen enantiomers were linear in the range of 0.1–25.0 μg/ml in serum and of 0.5–250.0 μg/ml in urine. Recovery of both enantiomers from serum and urine amounted 74–86 and 90–98%, respectively. Intra- and inter-day measurement precision and accuracy were below 15%. Limits of detection for IBP enantiomers amounted 0.05 and 0.25 μg/ml in samples of serum and urine, respectively. Limit of quantitation was also estimated. IBP enantiomers proved to be stable following three freeze and thaw cycles and during storage in autosampler at ambient temperature. The validated methods enable pharmacokinetic studies of enantiomers in both media. The elaborated HPCE method can be alternative to HPLC.

High-performance capillary electrophoretic method for the quantification of 5-methyl 2'-deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues.

A new approach to the evaluation of the relative degree of genomic DNA methylation through the quantification of 2'-deoxynucleosides is proposed. Detection and quantification of 5-methyl 2'-deoxycytidine in genomic DNA has been performed using micellar high-performance capillary electrophoresis (HPCE) with UV-Vis detection. This approach has been demonstrated to be more sensitive and specific than other HPCE methods for the quantification of DNA methylation degree and also to be faster than other HPLC-based methods. The detection and quantification of nucleosides through enzymatic hydrolyses notably increases the specificity of the technique and allows its exploitation in the analysis of poorly purified and/or concentrated DNA samples such as those obtained from meristematic plant regions and paraffin-embedded tissues.

Ultrarapid capillary electrophoresis method for the determination of reduced and oxidized glutathione in red blood cells.

We describe a very rapid high-performance capillary electrophoresis method for the separation and quantification of reduced (GSH) and oxidized (GSSG) glutathione in red blood cells. Two procedures for sample preparation have been compared, Microcon-10 membrane filtration and acid precipitation. The separation is obtained in an uncoated capillary using a high ionic strength borate buffer at pH 7.8. The intra-assay coefficients of variation (CVs%) are 1.53 and 1.66 for GSH and GSSG, respectively. The run is shorter than 90 s and the migration time is highly reproducible both for GSH (CV% 0.22) and GSSG (CV% 0.17). When the filtration step is used only GSH is found, whereas both GSH and GSSG are detectable after acid precipitation, suggesting that GSSG revealed after acid treatment may be an artefact due to GSH oxidation. Because of its good analytical performance this method could be used for routine red blood cell glutathione measurement in healthy or pathological conditions.

Determination of fenticonazole and its impurities by capillary electrophoresis and high performance liquid chromatography

Fenticonazole, a topical antifungal agent containing a stereogenic centre, is used in therapy as a racemic mixture. Five related compounds can be found as impurities in the drug. We propose a HPLC method using as stationary phase a RP-8 column eluted with different gradients of acetonitrile/phosphate buffer (pH 6) for simultaneous determination of the drug and these impurities. We also studied a high performance capillary electrophoresis (HPCE) method for quality control of fenticonazole. The separation of fenticonazole from all its impurities by HPCE was obtained in a relative short capillary (40 cm, effective length 34 cm, 50 μm ID) with a running buffer of 30 mM phosphate (pH 3) containing 8 mM trimethyl-β-cyclodextrin. Under these experimental conditions very good separation of fenticonazole from each individual impurity is obtained in less than 20 minutes. The choice of cyclodextrin added to the running buffer was dictated by the chiral nature of fenticonazole; trimethyl-β-cyclodextrin is mainly used as chiral selector. The optimised HPLC and HPCE methods are compared.

Determination of histidine and urocanic acid isomers in the human skin by high-performance capillary electrophoresis

Histidine was baseline separated from histamine, 1-methylhistamine and cis- and trans-urocanic acid using high-performance capillary electrophoresis (HPCE) on a fused-silica column (50 cm×75 μm) with 0.05 M NaH 2PO 4 buffer, pH 5.0, and 12 kV. The detection limit of histidine, trans- and cis-urocanic acid was 10 −6 M at a wavelength of 214 nm. The detection limit of the urocanic acid isomers was slightly enhanced to 5·10 −7 M at 267 nm. The transformation of the trans-urocanic acid standard in vitro into the cis-isomer was dependent on the time of exposure and the energy of the light source. UVB light induced a significantly faster conversion than UVA light. The HPCE method was used for the characterization and measurement of histidine and urocanic acid in human skin eluates. The concentrations of histidine, trans- or cis-urocanic acid in ethanol washes from the skin of healthy, non-allergic volunteers were 2.22±0.40·10 −5, 0.96±0.26·10 −5 and 1.04±0.30·10 −5 M, respectively, (mean±SEM, n=8). The results obtained by HPCE correlated well with data obtained by HPLC. Correlation coefficients of r 2=0.981, r 2=0.814 and r 2=0.956 were found for histidine, trans- and cis-urocanic acid, respectively.