Determination of Phenolic Glucosides in Gentiana piasezkii by Capillary Zone Electrophoresis

A capillary zone electrophoretic method has been developed for the quantitative analysis of five phenolic glucosides, 6′-O-vanilloylarbutin (VA), 7-O-feruloylorientin (FE), lutonarin (LN), isoorient (IO) and luteolin (LL), in Gentiana piasezkii with UV detection at 270 nm. 7-O-β-D-glucosyl-coumarin was selected as the internal standard. The applied voltage was 15 kV and the capillary temperature was kept constant at 25 °C. The effect of pH, the concentration of methanol and boric acid on migration were studied systematically. Optimum separation was achieved with 200 mM boric acid buffer at pH 9.50 containing 10% (v/v) methanol. Regression equations revealed good linear relationship between the peak area ratio of each compound and internal standard and its concentration. The correlation coefficients were 0.9975, 0.9997, 0.9998, 0.9998 and 0.9988 for VA, FE, LN, IO and LL, respectively. The relative standard deviations of migration time and the peak area ratio of each analyte and internal standard were <1.78% and 4.93%, respectively. The contents of the five compounds in Gentiana piasezkii were successfully determined with satisfactory repeatability and recovery.     

Determination of Flavonoids from Paulownia tomentosa (Thunb) Steud by Micellar Electrokinetic Capillary Electrophoresis

A micellar electrokinetic capillary electrophoresis (MEKC) method was developed for the determination of three flavonoids: diplacone (DI), mimulone (MI) and apigenin (AP) in the flowers of Paulownia tomentosa (Thunb) Steud. A buffer solution (pH 10.00) of 20 mM sodium borate, 10 mM sodium dodecyl sulfate (SDS) and 5% methanol was found to be the most suitable electrolyte for this separation. Regression equations revealed linear relationships (correlation coefficients: 0.9997 and 0.9998) between the peak area of each compound (DI, MI and AP) and its concentration. The relative standard deviations of migration times and peak areas were < 1.93 and 6.84%, respectively. The effects of pH, surfactant (SDS) concentration and organic modifier (methanol) on migration were also studied. The contents of three flavonoids in the extracts from the flowers of P. tomentosa applying different solvents were successfully determined within 12 min.     

CE Determination of Droperidol in Tablets and Human Serum

A capillary electrophoretic method was described for the determination of droperidol in pharmaceutical tablets and human serum. Droperidol and internal standard, lansoprazole, were separated on an uncoated fused-silica capillary using a run buffer containing borate (10 mM; pH 9.3) and aqueous methanol (20%, υ/υ). The signals were detected at 210 nm. The migration times for droperidol and internal standard were 2.1 and 2.9 min, respectively. The method has been validated in the range of 2.2 × 10^?5 ? 7.8 × 10^?5 M and applied to both tablets and human serum with good repeatability and no interference.     

Simultaneous Determination of the Active Ingredients in Abelmoschus manihot (L.) Medicus by CZE

A capillary zone electrophoretic method has been developed for the quantitative analysis of four active compounds, quercetin-3-O-robinobioside (LXY3), hyperin (LXY4), isoquercetin (LXY5) and myricetin (LXY7) in A. manihot (L.) Medicus with UV detection at 254 nm. The capillary temperature was kept constant at 25 °C. The effects of buffer composition, pH, and concentration and running voltage on migration were studied systematically. Optimum separation was achieved with 20 mmol L^?1 Borax–NaOH buffer (pH 9.50) at 25 °C and 25 kV. Regression equations revealed good linear relationship between the peak area of each compound and its concentration. All the correlation coefficients were higher than 0.9990. The relative standard deviations of migration time and the peak area were <0.53% and 5.34% (inter-day), and <0.56% and 3.81% (intra-day). The contents of the four compounds in A. manihot (L.) Medicus were successfully determined with satisfactory repeatability and recovery.     

Rapid Determination of Water-Soluble and Fat-Soluble Vitamins in Commercial Formulations by MEEKC

Microemulsion electrokinetic capillary chromatography has been successfully applied to the separation and determination of water-soluble vitamins (thiamine hydrochloride, riboflavin, niacin, pyridoxine hydrochloride, folic acid, cobalamin, ascorbic acid) and a fat-soluble vitamin (α-tocopherol acetate). The optimal microemulsion buffer contained sodium dodecylsulfate (SDS) as surfactant, butan-1-ol as the co-surfactant, ethyl acetate as the oil and pH 9.2 tetraborate buffer, modified with 15% (v/v) 2-propanol. UV detection at 214 nm gave adequate sensitivity without interference from sample excipients. Under the optimized conditions, the vitamins were baseline separated in less than 7 min. Analytical curves of peak area versus concentration presented coefficients of determination (R ² ) > 0.99, acceptable limits of quantification between 8.40 and 16.23 μg mL^?1 were obtained. Vitamin levels in liquid formulation were quantified with intra-day precision better than 0.99% RSD for migration time and 1.19% RSD for peak area ratio. Recoveries ranged between 98.7 and 101.7%. The method was considered appropriate for rapid and routine analysis.     

Direct Determination of Barbiturates in Urine by Capillary Electrophoresis Using a Capillary Coated Dynamically with Polycationic Polymers

A new, simple and rapid capillary electrophoresis method, using hexadimethrine bromide (HDB) as electro-osmotic flow modifier, has been developed for the identification and determination of nine barbiturates, barbital acid, barbital, phenobarbital, pentobarbital, amobarbital, thiobarbituric acid, butobarbital, N-methyl-5-phenyl-ethyl barbital acid and 5-cyclohexenyl-5-ethyl barbital acid in urine with UV detection at 200 nm. The applied voltage was ?25 kV and the capillary temperature was kept constant at 25 °C. The effects of buffer pH, the concentration of HDB and the concentration of α-cyclodextrin were studied systematically. Optimum separation was achieved with 20 mM borate buffer at pH 10.00 containing 0.04% (w/v) HDB and 2.06 mM α-cyclodextrin. Regression equations revealed good linear relationship between the peak area of each compound and its concentration. The correlation coefficients were from 0.9990 to 0.9997. The relative standard deviations of migration times and peak areas were <3.84 and 5.45% (intra-day). The nine barbiturates in urine were successfully determined within 7 min, without a prior preparation step and the method is useful for the investigation of intoxication.     

Analysis of Atorvastatin and Related Substances by MEKC

A new micellar electrokinetic capillary chromatographic (MEKC) method has been developed for simultaneous quantitation of atorvastatin (AT) and its related substances. The separation was carried out in an extended light path capillary at applied voltage of 30 kV using a background electrolyte consisting of 10 mM sodium tetraborate buffer pH 9.5, 50 mM sodium dodecyl sulphate and 20% (v/v) methanol. The addition of methanol to the running buffer resulted in a very effective choice to achieve resolution between the peaks of charged substances adjacent to AT as well as the peaks of neutral drug-related substances. Linear calibration curves were established over the concentration range 100–1,200 μg mL^?1 for AT and 1.0–12.5 μg mL^?1 for related substances. The proposed MEKC procedure has been validated with respect to selectivity, precision, linearity, limits of detection, and quantitation, accuracy and robustness. The method has been successfully applied to the determination of AT and purity evaluation of bulk drug and formulated products.     

Optimisation of Batch Culture Conditions for Cell-Envelope-Associated Proteinase Production from Lactobacillus delbrueckii subsp. lactis ATCC? 7830?

Using a combination of conventional sequential techniques, the batch growth conditions for the production of cell-envelope-associated proteinases have for the first time been studied and optimised in Lactobacillus delbrueckii subsp. lactis 313 (ATCC 7830; LDL 313). Concentrations of inoculum (0.1?<?X?<?10?% vol/vol), agitation speed (0?<?S?<?200?rpm), varying incubation temperature (30?<?T?<?50?°C), starting pH (4.5?<?pH?<?7) and carbon/nitrogen ratio of production medium (0.2?<?r?<?5) had an individual effect on proteinase yield (p?<?0.01). Optimal conditions for proteinase production included an initial pH of 6.0, 45?°C incubation temperature, 2?% (v/v) inoculum size of OD₅₆₀?=?1, 150?rpm agitation speed, and growth medium carbon/nitrogen ratio of 1.0. Maximum proteinase activity obtained for whole cells was 0.99 U/ml after 8?h of incubation. The variables studied are very relevant due to their significance in improving the productivity of proteinase synthesis from LDL 313, under process and, likely, economic optimum conditions.     

Qualitative and Quantitative Determination of Oligonucleotides by Non-Gel Capillary Electrophoresis

A novel simple non-gel capillary electrophoresis (NGCE) method was developed for the qualitative and quantitative determination of oligonucleotides (ODNs). ODNs were synthesized in a DNA synthesizer and separated by NGCE under optimized conditions as follows: 25 mmol L^?1 Tris?Cboric?CEDTA buffer (pH 8.0) with 7 mmol L^?1 urea in the presence of 20% (w/v) polyethylene glycol (PEG) 35000 at 30 °C and ?20 kV. Single-base differences, in which there are differences in the base quantity and the single-base structure with the same base quantity, can be identified by the proposed method, and the quantitative determination was carried out. The obtained regression equation revealed a good linear relationship between the peak area ratio and the ODNs concentration. The correlation coefficient was 0.9986 and the relative standard deviation (RSD) of the precision was <3.6% (intro-day) and <6.2% (inter-day), respectively. The recoveries of three concentrations (high, middle, and low) were between 99.6% and 107.6%. The established method is sensitive, simple, economical, and can be used in gene mutation detection, pharmacokinetics study of nucleic acid drugs, and so on.     

A Convenient Approach to Simultaneous Analysis of a Pharmaceutical Drug and Its Counter-Ion by CE Using Dual-Opposite End Injection and Contactless Conductivity Detection

Capillary electrophoresis (CE) coupled to a capacitively coupled contactless conductivity detector (C⁴D) was used for the determination in a single analysis of a pharmaceutical drug and its counter-ion. Dual-opposite end injection (DOI) was used to introduce hydrodynamically the analytes at each end of the capillary. No modification of the commercial apparatus is required. After applying the voltage, the cations and anions migrate from each end of the capillary in opposite directions toward the detector placed near the cathode outlet. The electrophoretic conditions were initially developed with three test drugs (chlorpheniramine maleate, metoprolol tartrate, clomiphene citrate) and then applied to two Vinca alkaloids (catharanthine sulfate, vinorelbine ditartrate). The 10 mM histidine–50 mM acetic acid buffer (pH 4.1)–methanol 90:10 (v/v) electrolyte was suitable for the analysis of these high or medium mobile anions by CE–C⁴D due to its low conductivity background and high buffer capacity. Finally, the CE procedure developed was successfully validated for catharanthine sulfate. The method developed herein is fast (<10 min) and accurate (repeatability on migration time < 0.6% and peak areas < 1.3%, n = 6).

     

Use of molybdate as novel complex-forming selector in the analysis of polyhydric phenols by capillary zone electrophoresis

Molybdate was examined as a complex-forming additive to the CE background electrolytes (BGE) to affect the selectivity of separation of polyhydric phenols such as flavonoids (apigenin, hyperoside, luteolin, quercetin and rutin) and hydroxyphenylcarboxylic acids (ferulic, caffeic, p-coumaric and chlorogenic acid). Effects of the buffer concentrations and pH and the influence of molybdate concentration on the migration times of the analytes were investigated. In contrast to borate (which is a buffering and complex-forming agent generally used in CE at pH ≥9) molybdate forms more stable complexes with aromatic o-dihydroxy compounds and hence the complex-formation effect is observed at considerably lower pH. Model mixtures of cinnamic acid, ferulic acid, caffeic acid and 3-hydroxycinnamic acid were separated with 25 mM morpholinoethanesulfonic acid of pH 5.4 (adjusted with Tris) containing 0.15 mM sodium molybdate as the BGE (25 kV, silica capillary effective length 45 cm × 0.1 mm I.D., UV-vis detection at 280 nm). With 25 mM 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulphonic acid/Tris of pH^* 7.4 containing 2 mM sodium molybdate in aqueous 25% (v/v) methanol as the BGE mixtures of all the above mentioned flavonoids, p-coumaric acid and chlorogenic acid could be separated (the same capillary as above, UV-vis detection at 263 nm). The calibration curves (analyte peak area versus concentration) were rectilinear (r > 0.998) for ≈8-35 μg/ml of an analyte (with 1-nitroso-2-naphthol as internal standard). The limit of quantification values ranged between 1.1 mg l⁻¹ for p-coumaric acid and 2.8 mg l⁻¹ for quercetin. The CE method was employed for the assay of flavonoids in medicinal plant extracts. The R.S.D. values ranged between 0.9 and 4.7% (n = 3) when determining luteolin (0.08%) and apigenin (0.92%) in dry Matricaria recutita flowers and rutin (1.03%) and hyperoside (0.82%) in dry Hypericum perforatum haulm. The recoveries were >96%.     

Simultaneous Determination of the Endogenous Free ��-Lipoic Acid and Dihydrolipoic Acid in Human Plasma and Erythrocytes by RP-HPLC with Electrochemical Detection

A highly sensitive, precise, and accurate reversed-phase high-performance liquid-chromatography/electrochemical detection method for simultaneous determination of the endogenous free ??-lipoic acid and dihydrolipoic acid in biological matrices was developed and validated. The two analytes were extracted from the samples with acetonitrile/10% metaphosphoric acid solution_(aqueous) (50/50 v/v). To determine the total lipoic acid, samples were treated with tris(2-carboxyethyl)phosphine solution in phosphate buffer, pH 2.5 with 85% orthophosphoric acid prior to deproteination. The two analytes were separated on a C₁₈ (150 × 4.6 mm, 5 ??m) analytical column using acetonitrile-50 mM phosphate buffer, pH 2.5 with 85% orthophosphoric acid (35/65 v/v) as the isocratic mobile phase pumped at a flow rate of 2.0 mL min^?1 at the column oven temperature of 35 °C. The column eluents were monitored at a potential of 0.9 V. These analytes were efficiently resolved in <7 min. The present method was sufficiently robust and specific for simultaneous determination of the two analytes and demonstrated acceptable values for linearity (r ² = 0.999 in the range of 0.1?C500 and 0.25?C1,000 ng mL^?1 for ??-lipoic acid and dihydrolipoic acid, respectively), recovery (>97%), precision (RSD% <2), and sensitivity (on column limit of detection, 150 and 375 fg for ??-lipoic acid and dihydrolipoic acid, respectively and limit of quantification: 0.5 and 1.25 pg for ??-lipoic acid and dihydrolipoic acid, respectively), indicating that the proposed method was more sensitive, precise, economical, and versatile, and has higher throughput than the previously reported methods for simultaneous determination of the two analytes.     

Determination of Folic Acid by CE in Various Cultivated Variety of Lentils

A simple extraction and determination method for folic acid (FA) in lentil samples was developed employing capillary electrophoresis. The analysis was performed in a 75 μm ID fused silica capillary using a running buffer of 10 mM sodium borate (10%, v/v, methanol, pH 9) at +18 kV and a detection wavelength at 200 nm. Methylparaben was used as the internal standard. FA signal response was linear in the range between 1.2 × 10^?5 and 4.8 × 10^?5 M. Limit of detection (inter-day) was determined at 6.12 × 10^?7 M (3.3 σs^?1). The amount of FA found in green, red and mignon lentils was found to range between 0.408 and 0.742 mg g^?1.     

Optimization of Micellar LC Conditions for the Flavonoid Separation

The chromatographic conditions for the separation of a complex set of flavonoids (aglycones and glycosides) by micellar liquid chromatography with spectrophotometric detection were optimized. A good separation for all analytes was obtained and satisfactory peak shapes were achieved by isocratic elution with Ultrasphere ODS column (250 mm × 4.6 mm, 5 μm). The optimal mobile phase range for flavonoids separation is: SDS concentration between 0.014 and 0.018 mol L^?1 and 1-propanol volume fraction between 2.2 and 4.5% (v/v) in a diluted (1:5) phosphate buffer solution pH 6.86. The flavonoids (robinin, rutin, hyperoside, quercitrin, liquroside, luteolin-7O-glucoside, apigenin-7O-glucoside, isosalipurposide, myricetin, fisetin, luteolin, apigenin, quercetin and caempferol) were successfully separated within 40 min with isocratic elution. The developed method is an alternative to reversed-phase LC in the assay of flavonoids in plants, plant extracts and plant extract containing drugs.     

Multiobjective Optimization Approach in Evaluation of Chromatographic Behaviour of Zolpidem Tartrate and Its Degradation Products

Multiresponse optimization methodology in combination with experimental design was employed as a powerful technique for simultaneous optimization of input variables significant for evaluation of chromatographic behaviour of zolpidem tartrate, zolpacid, oxozolpidem, zolpyridine and zolpaldehyde towards various responses. In the first stage of the investigation fractional factorial design was used to decrease the number of variables that should be studied in detail. Among examined variables, pH of the mobile phase, percentage of organic modifier and buffer concentration showed to be statistically important and were consequently optimized with central composite design and Derringer??s desirability function. Four responses were considered, the retention factors of zolpacid and zolpaldehyde (the first and last peak) and the resolutions between critical peaks. Optimal conditions included Luna C₁₈(2) analytical column (250?mm?x?4.6?mm, 5???m particle size), mobile phase consisted of methanol?C10?mM ammonium acetate (68.4:31.6, v/v, pH 5.4) and column temperature of 35???C. The flow rate of the mobile phase was 1?mL?min^?1 and the detection was performed at 254?nm. At the end, the method was successfully validated in accordance with ICH guideline and subsequently applied to the analysis of commercially available zolpidem tartrate tablets.     

RP-LC Determination of Residual Monomers in Polycarboxylate Superplasticizers

A procedure was developed for the determination of residual monomers in polycarboxylate superplasticizer by reversed-phase high performance liquid chromatography. Seven kinds of residual monomers were quantitatively determined on a SinoChrom ODS-BP (C18) column and UV detector at 205 nm. The mobile phases which were used to determine micromolecular monomers were composed of acetonitrile and phosphate buffer solution (0.05 mol L^?1, pH = 3) in the ratio of 8:92 (v/v). While the mobile phases for long side-chain monomers testing were composed of acetonitrile and phosphate buffer solution (0.05 mol L^?1, pH = 6.5) in the ratio of 40:60 (v/v). The linear response ranged from 4.0 × 10^?6?C2.0 × 10^?3 mol L^?1. The detection limit was 0.12 × 10^?5?C0.8 × 10^?5 mol L^?1. Determination of real samples showed that relative standard deviation of high conversion rate samples was 3.1?C8.7% and standard addition recovery ratio was 91.5?C102.8%. While the relative standard deviation of low conversion rate samples was less than or close to 1% and the standard addition recovery ratio was 96.3?C103.1%.     

Determination of Ibandronate and its Degradation Products by Ion-Pair RP LC with Evaporative Light-Scattering Detection

A simple method has been developed for analysis of ibandronate and related substances by ion-pair reversed-phase high-performance liquid chromatography (RPIC) with evaporative light-scattering detection (ELSD). After optimization of the chromatographic conditions satisfactory separation of the compounds was achieved on an Intersil C₈ column with an isocratic mobile phase—8:4:88 (v/v) acetonitrile–methanol–12 mM ammonium acetate buffer containing 35 mM n-amylamine (pH 7.0). The mobile phase flow rate was 1.0 mL min^?1. The calibration plot was linear in the range 352 to 1760 µg mL^?1 for ibandronate. The precision and reproducibility were 0.3% and 0.5%, respectively. The average recovery of ibandronate was 100.4% and RSD was 0.6%. The method was validated and shown to be precise, accurate, and specific for assay of ibandronate in bulk material and dosage forms. The proposed liquid chromatographic method can be satisfactorily used for quality control of ibandronate.     

Determination of Florfenicol in Freshwater, Sediments and Bryophyte Fontinalis antipyretica by HPLC with Fluorescence Detection

Novel procedures for the determination of florfenicol in freshwater, sediments and bryophyte Fontinalis antipyretica, using reversed-phase high-performance liquid chromatography are described. Liquid chromatography was performed on a 5 µm PuroSpher RP-18E^® column using methanol and 0.05 M phosphate buffer (18/82 v/v, pH 7.3) as mobile phase (0.8 ml min^?1) and fluorescence detection (excitation wavelength 265 nm and emission wavelength 295 nm). Florfenicol was determined in centrifuged freshwater samples. Florfenicol was extracted from sediments and bryophytes samples by using a solid-liquid extraction step followed by a solid phase extraction step. Linearity was confirmed over the concentration range 25–1000 ng mL^?1 water and 50-1000 ng g^?1 sediment or bryophyte. Limits of detection and quantitation were 8 and 25 ng mL^?1 water and 17 and 50 ng g^?1 sediment or bryophyte respectively. Mean extraction recoveries of florfenicol from sediments and bryophyte were from 85.9 to 109.1%.     

Separation and Determination of Six Active Components in Two Myricaria Plants by Capillary Chromatography

A capillary zone electrophoretic (CZE) method for determination of six active components of two Myricaria plants has been developed for the first time. The analytes were completely separated within 15 min. The electrophoresis buffer was 25 mmol L^?1 sodium borate containing 15% (v/v) acetonitrile (pH 10.20). The correlation coefficients of the calibration curves for the six analytes were 0.9998 or 0.9999 over the concentration ranges examined. Recoveries of the six constituents ranged from 96.3 to 106.8%. The method, combined with a relatively simple extraction procedure, was successfully used for analysis of two Myricaria plants and the assay results were satisfactory.     

Electrophoretic stacking for sensitive determination of antibiotic ceftazidime in human blood and microdialysates from diabetic foot

An electrophoretic stacking method has been developed for monitoring the therapeutic level of the antibiotic ceftazidime in blood plasma and microdialysates taken from peripheral soft tissues of the lower limbs of patients with diabetic foot syndrome. The biological samples are treated by addition of acetonitrile in an amount of 75% v/v and injected into a capillary in a large volume; after turning on the separation voltage, the residual acetonitrile is forced out of the capillary by the application of hydrodynamic pressure. The clinical samples were separated in an optimised background electrolyte composed of 50 mM chloroacetic acid +20% v/v methanol +0.5% v/v INST coating solution. The attained LOD for ceftazidime equalled 0.42 μg mL⁻¹ (0.8 μM) and the migration time equalled 3.75 min when using a 25 μm capillary with minimum length of 31.5 cm. The separation was controlled by a maximum voltage of +30 kV and the movement of the analyte was accelerated by a pressure of 50 mbar. The RSD values for intra-day repeatability of the migration time and peak area are 0.14% and 3.8%, respectively; the inter-day values equalled 0.25% for the migration time and 7.3% for peak area, respectively. Pharmacological studies revealed that ceftazidime passes from the blood circulation to the peripheral tissues of the lower limbs with an efficiency of 20%. The introduction of CE control of ceftazidime level in diabetic foot represents a very important improvement in achieving the targeted therapeutic effect.