Determination of β-(3,4-dihydroxyphenyl) lactic acid, protocatechuic acid and protocatechuic aldehyde in radixSalviae Miltiorrhizae by capillary electrophoresis

Summary The separation and determination of β-(3,4-dihydroxyphenyl) lactic acid, protocatechuic acid and protocatechuic aldehyde in RadixSlviae Miltiorrhizae injection were investigated by capillary zone electrophoresis. With boric acid (200 mmol L⁻¹) adjusted to pH 8.11 as a background electrolyte, 28 kV applied voltage and 30°C thermostating temperature, complete separation of β-(3,4-dihydroxyphenyl)lactic acid, protocatechuic acid and protocatechuic aldehyde was readily achieved within 9 min. Results indicate that capillary electrophoresis promises to be applicable to quality control of radixSalviae Miltiorrhiza injection.     

Reproducibility of sample separation using liquid or forced air convection thermostatted high performance capillary electrophoresis systems

Run-to-run sample separation reproducibility has been compared on two commercial high performance capillary electrophoresis units which differ in the mode by which the capillary temperature is thermostatted. Three standard analytes, differing dramatically in molecular character and size, were used for the analysis: benzoic acid, a 14 amino acid peptide from human chorionic gonadotropin, and ribonuclease A represent, respectively, small stable organic molecules, small peptides with little or no secondary structure, and proteins with secondary structure. These standards were evaluated with regard to reproducibility of migration time, peak area, and peak height. The analyses, performed in buffers of optimum pH for the separations, demonstrated that the liquid and forced air convection thermostatted systems both performed extremely well. The reproducibility, as judged by the percent coefficient of variance (% CV) of replicate analyses, was generally found to be less than 1 % (migration time); the reproducibility decreased in the order migration time > peak height > peak area. Whereas the absolute % CV values for MT_rel (migration relative to a standard) observed with the liquid thermostatted system were 2- to 4-fold lower than those observed with the forced air convection thermostatted system, there was little statistically significant difference between the two. As expected, the data indicated a reduction in reproducibility as the complexity of the analyte increased, perhaps as the result of an increased potential for wall interactions. Comparing separations in which low (≈?1 watt/meter [W/m] of capillary) and high (>5 W/m) Joule heat was generated by altering the sodium chloride content of the buffer revealed few statistically significant differences in the reproducibility obtained from the two systems. With these particular standard analytes and their respective buffer systems, there appears to be little difference between forced air convection and liquid thermostatting of the capillary.     

Separation and Determination of Magnolol and Honokiol in Crude and Burn-Fried Magnolia officinalis

Abstract

The application of capillary electrophoresis (CE) to the separation and determination of the two active ingredients, magnolol and honokiol, in Magnolia officinalis and its processed product was described. Optimum separation was achieved with a fused-silica capillary tube(60.5cm × 75μm I.D.) and a Na₂B₄O₇-NaH₂PO₄ buffer(20 mmol/L) at pH=9.0 containing 20% of methanol. The applied voltage was 20 kV and the capillary thermostating temperature was kept constant at 25 deg;C.     

Detection of point mutations by capillary electrophoresis with temporal temperature gradients.

By combining the advantages of capillary electrophoresis and temperature gradient gel electrophoresis, a method was developed to detect point mutations in polymerase chain reaction (PCR) fragments. Increasing and decreasing temporal temperature gradients were established by means of a computer-controlled Peltier module. Native and denaturing conditions were achieved by cooling to 25 degrees C and heating to 70 degrees C, respectively, a thermostating liquid surrounding the capillary. To separate nucleic acid fragments, a sieving media, containing 4% linear polyacrylamide, 1 x Tris borate EDTA buffer (TBE) and 6 M urea, was found appropriate. Renewal of the sieving matrix before each run significantly improved the reproducibility of fragment separation. The ability of this capillary electrophoresis system to detect point mutations is demonstrated with the human prion-protein gene.     

Statistical Optimization Applied to Simultaneous Determination of Maprotiline, Desipramine, and Moclobemide by Capillary Zone Electrophoresis

Summary. A method of Capillary Zone Electrophoresis (CZE) for separation of three most-frequently prescribed antidepressants in the market: maprotiline, desipramine, and moclobemide was developed. The proposed method is fully validated for a ternary laboratory mixture but it is also suitable for individual determination of the investigated components in pharmaceutical dosage forms. Since the preliminary investigations did not show complete separation because of close migration time of desipramine and maprotiline, a complete set, 2³ experimental design was applied. All the factors that affect separation as well as their mutual interactions were investigated. Voltage, temperature of capillary and the pH of phosphate buffer were independent variables or factors to be investigated in two levels. Applying response surface methodology, from experimental points the appropriate graphs were constructed and optimal chromatographic conditions for the separation were defined. The optimum conditions were: running voltage of 20 kV, phosphate buffer (pH = 2.35), temperature 25°C, and UV detection at 200 nm. An uncoated (fused silica) capillary (50 μm i.d.) with a total length of 50 cm and a distance of 47 cm between the injection and detection points was used.     

Analysis of oxygen heterocyclic compounds in citrus essential oils by capillary electrochromatography and comparison with HPLC

Summary A new method for the analysis of oxygen heterocyclic compounds in bergamot, mandarin and sweet organge oils by capillary electrochromatography (CEC) is reported. The fused silica capillaries employed were slurry-packed in house with 3 μm ODS1 stationary phase. The repeatability of the technique has been investigated with two neutral standard mixtures. Conditions for the analysis have been optimised by varying several parameters such as applied voltage, temperature, composition of the buffer and percentage of organic solvent. The best separations were obtained using the buffer acetonitrile/TRIS 10 mM at pH 7.8 in the ratio 80∶20. The results were compared with those obtained by high performance liquid chromatography (HPLC) in terms of time of analysis, resolution and efficiency. A study on the behaviour of oxygen heterocyclic compounds analysed with a “non-aqueous” mobile phase has also been carried out.     

Enantiomer Separation of the Four Stereoisomers of 1-(4-Hydroxy-3-Methoxy)-Phenyl-2-[4-(1,2,3-Trihydroxy-Propyl)-2-Methoxy]-Phenoxy-1,3-Propandiol from Hydnocarpus annamensis by Capillary Zone Electrophoresis with HP-β-CD as Chiral Selector

A capillary zone electrophoresis method with HP-β-CD as chiral selector was established for the chiral separation of four stereoisomers of 1-(4-hydroxy-3-methoxy)-phenyl-2-[4-(1,2,3-trihydroxy-propyl)-2-methoxy]-phenoxy-1,3-propandiol for the first time, which were isolated from Hydnocarpus annamensis. The effects of chiral selector type and concentration, buffer composition, pH and concentration, and cartridge temperature on the enantioseparation were investigated. A baseline separation of the four stereoisomers was achieved in less than 18 min under the optimized conditions: 40 mmol L⁻¹ Borax–NaOH buffer (pH 10.02) in the presence of 100 mmol L⁻¹ HP-β-CD at 15°C and 30 kV. The experimental results showed that the method by capillary zone electrophoresis for the separation of four stereoisomers is powerful, sensitive and fast, requires less amounts of reagents, and can be employed as a reliable alternative to other methods.     

Analysis of flavonols ofSedum telephium L. leaves by capillary electrophoresis and HPLC-mass spectrometry

Summary Flavonol glycosides ofSedum telephium L. were analysed by MEKC. Baseline separation was achieved within 14 min using a fused silica capillary and a borate/phosphate buffer solution (35 mM, pH 5.8) containing 35 mM SDS and 4% MeOH. The applied voltage was 25 kV and the thermostating temperature was kept constant at 40°C. Injection was performed via the pressure mode for 3 s, the detection wavelength was 205 nm. The optimized MEKC method was used for the quantitative determination of flavonol glycosides in extracts ofS. telephium leaves. Analysis was also performed by HPLC-MS using an electrospray ionization (ESI) interface. The good agreement between the quantitative CE results and those obtained by LC clearly demonstrated the applicability of the methods presented.     

Tudy on drug displacement interactions by capillary electrophoresis-frontal analysis

The interaction between 18-methyl norethindrone and ketoprofen, including the displacement of ketoprofen from human serum albumin binding sites, was investigated by the capillary electrophoresis-frontal analysis method (CE-FA) at room temperature. A very large sample plug was introduced hydrostatically into the capillary (65 cm × 50 μm i.d.; effective length of 35 cm) over 80 s at a height difference of 11 cm. The working conditions for CE-FA separation are as follows: operating voltage, 10 kV; running buffer, 67 mmol·L⁻¹ phosphate, pH 7.4. The unbound ketoprofen concentration was directly measured from the height of the frontal peak. When the concentration of 18-methyl norethindrone was increased from 0 to 200 μmol/L, the unbound ketoprofen concentration was found to increase from 22.4 to 26.4 μmol/L at 100 μmol/L total ketoprofen concentration and from 82.1 to 106.2 μmol/L at 200 μmol/L total ketoprofen concentration. From these data, it may be deduced that the administration of high concentration of 18-methyl norethindrone can displace ketoprofen from its secondary binding site. __________ Translated from Chinese Journal of Chromatography, 2005, 23(2)(in Chinese)     

In-line solid-phase extraction–capillary electrophoresis coupled with mass spectrometry for determination of drugs of abuse in human urine

In-line solid-phase extraction–capillary electrophoresis coupled with mass spectrometric detection (SPE–CE–MS) has been used for determination of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), codeine (COD), hydrocodeine (HCOD), and 6-acetylmorphine (6AM) in urine. The preconcentration system consists of a small capillary filled with Oasis HLB sorbent and inserted into the inlet section of the electrophoresis capillary. The SPE–CE–MS experimental conditions were optimized as follows: the sample (adjusted to pH 6.0) was loaded at 930 mbar for 60 min, elution was performed with methanol at 50 mbar for 35 s, 60 mmol L⁻¹ ammonium acetate at pH 3.8 was used as running buffer, the separation voltage was 30 kV, and the sheath liquid at a flow rate of 5.0 μL min⁻¹ was isopropanol–water 50:50 (v/v) containing 0.5% acetic acid. Analysis of urine samples spiked with the four drugs and diluted 1:1 (v/v) was studied in the linear range 0.08–10 ng mL⁻¹. Detection limits (LODs) (S/N = 3) were between 0.013 and 0.210 ng mL⁻¹. Repeatability (expressed as relative standard deviation) was below 7.2%. The method developed enables simple and effective determination of these drugs of abuse in urine samples at the levels encountered in toxicology and doping.     

Chiral separation of newly synthesized arylpropionic acids by capillary electrophoresis using cyclodextrins or a glycopeptide antibiotic as chiral selectors

Summary The chiral separation of two newly synthesized arylpropionic acids of pharmaceutical interest, namely 2-[(5′-benzoil-2′-hydroxy)phenyl]-propionic acid (DF-1738y) and 2-[(4′-benzoiloxy-2′-hydroxy)phenyl]-propionic acid (DF-1770y), was performed by Capillary Zone Electrophoresis (CZE) using either cyclodextrins or antibiotics as chiral selectors in coated capillary. In order to optimize the separation, the effect on the migration time and resolution of type and concentration of the chiral selector, the buffer pH and the capillary temperature were studied. Several cyclodextrins, namely the charged 6^A-monomethylamino-β-cyclodextrin (MeNH-β-CD) and the neutral methyl-β-cyclodextrins (M-β-CD) and heptakis-2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CD), were tested for the enantiomeric separation of aryl propionic acids (APAs) compounds. Of these TM-β-CD provided the highest enantiomeric resolution at pH 5, however only DF-1738y optical isomers were baseline resolved. Using background electrolytes (BGEs) at higher pHs (pH=6–7) supported with the above listed CDs, an enantioresolution increase was recognized only for compound DF-1738y. In contrast DF-1770y exhibited the highest resolution at the lowest pH value studied (pH 4). The macrocyclic antibiotic vancomycin was therefore added to the BGE and tested as chiral selector using the partial filling counter current mode in order to obtain a sensitive analysis, high resolution and reduced antibiotic adsorption on the capillary wall. 5 mM vancomycin dissolved in the BGE at pH 5 and 25°C provided relatively high enantiomeric resolution (R _DF-1738y=3.4,R _DF-1770y=2.22) of both compounds.     

Determination of Ephedrine, Pseudo-Ephedrine and Caffeine in a Dietary Product by Capillary Electrophoresis

This paper reports the determination of caffeine, ephedrine and pseudo-ephedrine in a dietary product by two rapid and simple methods utilising capillary electrophoresis (CE). The solutes were extracted from the product using 0.2 M HCl and determined by CE with background electrolytes containing 7.5% highly sulfated-β-cyclodextrin (7–11 sulfate groups per β-CD molecule) at pH 2.5 and pH 7.6. Determination of ephedrine and pseudo-ephedrine was accomplished at pH 2.5 with the anode at the detection side of the capillary whereas caffeine was quantified at pH 7.6 with a normal electrophoresis polarity mode. Triethanolamine was added to the running buffer at pH 2.5 in order to reverse the electroosmotic flow (EOF) and thereby speed up the separation of ephedrine and pseudo-ephedrine. Revised: 18 November 2005 and 2 January 2006     

Capillary-Channeled Polymer (C-CP) Films as Processing Platforms for Protein Analysis by Matrix-Assisted Laser/Desorption Ionization Mass Spectrometry (MALDI-MS)

Polypropylene (PP) capillary-channeled polymer (C-CP) films have parallel, μm-sized channels that induce solution wicking via capillary action. Efficient mass transport from the solution phase to the channel surface leads to adsorption of hydrophobic protein solutes. The basic premise by which C-CP films can be used as media to manipulate analyte solutions (e.g., proteins in buffer), for the purpose of desalting or chromatographic separation prior to MALDI-MS analysis is presented here. Cytochrome c and myoglobin prepared in a Tris-HCl buffer, and ribonuclease A, lysozyme, and transferrin prepared in phosphate buffered saline (PBS), are used as the test solutions to demonstrate the desalting concept. Protein analysis is performed after deposition on a C-CP film with and without a water washing step, followed by spray deposition of a typical sinapinic acid matrix. Extracted MALDI mass spectra exhibit much improved signal-to-noise characteristics after water washing. A mixture of cytochrome c and myoglobin (2 μL of 2.5 μM each in Tris-HCl buffer) was applied, washed with water and spatially separated via simple capillary action (wicking) using a reversed-phase solvent composition of 0.1% trifluoroacetic acid (TFA) in 50:50 acetonitrile (ACN):H₂O. Subsequent application of sinapinic acid followed by imaging of the film using MALDI-MS reveals that as the protein solution is wicked down the film, separation occurs.     

Buffer Standards for the Physiological pH of the Zwitterionic Compound,DIPSO, from 5 to 55 °C

The values of the second dissociation constant, pK ₂, and related thermodynamic quantities of 3-[N,N-bis (2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO) have already been reported over the temperature range 5 to 55 °C including 37 °C. This paper reports the pH values of four NaCl-free buffer solutions and four buffer composition containing NaCl salt at I=0.16 mol⋅kg⁻¹. Conventional pa _H values are reported for all eight buffer solutions. The operational pH values have been calculated for four buffer solutions recommended as pH standards, at 25 and 37 °C after correcting the liquid junction potentials with the flowing junction cell.     

Microextraction,capillary electrophoresis,and mass spectrometry for forensic analysis of azo and methine basic dyes from acrylic fibers

Designed experiments based on a simplex mixture design were employed to explore the effects of three solvent components (water, formic acid, and aqueous acetic acid), extraction time, and extraction temperature for the automated microextraction of basic (cationic) dyes from acrylic fibers. Extractions were conducted by an automated liquid handling system, and dye extraction was evaluated using a UV/visible microplate reader. Highest extraction efficiency for two subclasses of basic dyes (methine and azo) from acrylic fibers was achieved with an extraction solvent containing 88% formic acid/12% water. Cationic dyes were analyzed by capillary electrophoresis using a 45 mM ammonium acetate buffer in acetonitrile–water at pH 4.7. The utility of microextraction combined with capillary electrophoresis–mass spectrometry for analysis of extracts from trace fibers was demonstrated by the detection and characterization of three basic dyes extracted from a 2-mm length of single acrylic fiber.     

Purity control of oxytetracycline by capillary electrophoresis

The applicability of capillary electrophoresis for the purity control of oxytetracycline (OTC) was investigated. OTC is a broad-spectrum antibiotic belonging to the group of the tetracyclines. Several related substances can be present due to fermentation or degradation, such as 4-epioxytetracycline, -apooxytetracycline, β-apooxytetracycline, anhydrooxytet racycline, 2-acetyl-2-decarboxamidooxytetracycline, tetracycline and 4-epitetracycline. Using fused-silica capillaries, the influence of buffer type, buffer pH and buffer concentration were investigated. In all cases 1 mM EDTA was added to prevent metal-ion complexation. The influence of the buffer counter-ion type was examined. Consequently, some instrumental parameters were changed such as capillary length and diameter as well as capillary temperature and applied voltage. The following method is finally proposed: fused-silica capillary, l (effective length) = 38 cm, L (total length) = 44 cm, 50 μm I.D.; buffer, sodium carbonate 20 mM-EDTA 1 mM, pH 11.25; voltage, 10 kV; temperature, 10°C. Linearity, limit of detection and limit of quantitation were determined as well as the relative standard deviations for all the analytes involved. This method is less selective then existing liquid chromatographic methods but it may be used as a complementary tool in purity control and stability studies.     

Effects of the operation parameters on Hydrophilic Interaction Liquid Chromatography separation of phenolic acids on zwitterionic monolithic capillary columns

Strongly polar phenolic acids are weakly retained and often poorly separated in reversed-phase (RP) liquid chromatography. We prepared zwitterionic polymethacrylate monolithic columns for micro-HPLC by in situ co-polymerization in fused-silica capillaries. The capillary monolithic columns prepared under optimized polymerization conditions show some similarities with the conventional particulate commercial ZIC-HILIC silica-based columns, however have higher retention and better separation selectivity under reversed-phase conditions, so that they can be employed for dual-mode HILIC-RP separations of phenolic acids on a single column. The capillary polymethacrylate monolithic sulfobetaine columns show excellent thermal stability and improved performance at temperatures 60–80 °C. The effects of the operation conditions on separation were investigated, including the type and the concentration of the organic solvent in the aqueous-organic mobile phase (acetonitrile and methanol), the ionic strength of the acetate buffer and temperature. While the retention in the RP mode decreases at higher temperatures in mobile phases with relatively low concentrations of acetonitrile, it is almost independent of temperature at HILIC conditions in highly organic mobile phases. The best separation efficiency can be achieved using relatively high acetate buffer ionic strength (20–30 mmol L⁻¹) and gradient elution with alternately increasing (HILIC mode) and decreasing (RP mode) concentration of aqueous buffer in aqueous acetonitrile. Applications of the monolithic sulfobetaine capillary columns in alternating HILIC-RP modes are demonstrated on the analysis of phenolic acids in a beer sample.     

Analysis of chlortetracycline and related substances by capillary zone electrophoresis: Development and validation

Summary A capillary zone electrophoresis method has been developed and validated for the analysis of chlortetracycline and related substances. The influence of the type of buffer, pH and concentration of the buffer were investigated. In all cases 1 mM EDTA was added to prevent metal ion complexation. Instrumental parameters such as capillary temperature and applied voltage were optimised. The following methods is proposed: capillary: fused silica, 44 cm (36 cm effective length), 50 μm i.d.; buffer: 120 mM sodium tetraborate including 1mM EDTA at pH 8.5; voltage: 10 kV; temperature: 25°C; detection wavelength: 280 nm. The robustness of the method has been examined by means of a full-fraction factorial design. The parameters for validation namely relative standard deviation, linearity, precision, limit of detection and limit of quantitation are also reported.     

Non-ideal properties of the TRIS—TRIS·HCl−NaCl−H2O buffer system in the 0–40 °C temperature interval. Application of the pitzer equations

The buffer solution TRIS—TRIS·HCl−NaCl−H₂O was studied in the 0–40 °C temperature region and ionic strength interval of (0.1–4)m (m is molality) by the e.m.f. method using two types of cells without liquid junction composed of platinum-hydrogen, silverchloride, and sodium-glass electrodes. For temperatures of 5 and 15 °C and the (1–4)m concentration region, the osmotic coefficients of the TRIS·HCl−H₂O solutions were measured by the isopiestic method. The results were processed in the framework of the Pitzer method, and the parameters of interaction of the components of the buffer system were calculated. The associative character of the interactions in the TRIS·HCl−H₂O solution was shown. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 670–675, April, 2000.     

Anion exchange silica monolith for capillary liquid chromatography

An anion exchange monolithic silica capillary column was prepared by surface modification of a hybrid monolithic silica capillary column prepared from a mixture of tetramethoxysilane (TMOS) and methyltrimethoxysilane (MTMS). The surface modification was carried out by on-column copolymerization of N-[3-(dimethylamino)propyl]acrylamide methyl chloride-quaternary salt (DMAPAA-Q) with 3-methacryloxypropyl moieties bonded as an anchor to the silica surface to form a strong anion exchange stationary phase. The columns were examined for their performance in liquid chromatography (LC) and capillary electrochromatography (CEC) separations of common anions. The ions were separated using 50 mM phosphate buffer at pH 6.6. Evaluation by LC produced an average of 30,000 theoretical plates (33 cm column length) for the inorganic anions and nucleotides. Evaluation by CEC, using the same buffer, produced enhanced chromatographic performance of up to ca. 90,000 theoretical plates and a theoretical plate height of ca. 4 μm. Although reduced efficiency was observed for inorganic anions that were retained a long time, the results of this study highlight the potential utility of the DMAPAA-Q stationary phase for anion separations. Figure Micro-LC performance evaluation of a strong anion exchange silica monolith column, 100H-MOP-DMAPAA-Q, 33 cm in length, with a mobile phase of 50 mM phosphate buffer, pH 2.8; linear velocity: u = 1.8 mm/s; UV-Vis detection at 254 nm. Sample solution (5 mg/mL of each component, 4 mL) was injected in split flow injection mode at a split ratio of ca. 1:1900 with a pump flow rate of 1.5 mL/min