Chiral capillary electrophoretic determination of the enantiomeric purity of homocamptothecin derivatives, promising antitumor topoisomerase I inhibitors, using highly sulfated CDs and fluorescence detection

EKC methods for the enantiomeric resolution of homocamptothecin derivatives, potent anticancer agents targeting DNA topoisomerase I selected for clinical trials, were developed using highly sulfated beta-CD as chiral selectors at acidic pH. Optimal electrophoretic conditions, with migration times under 15 min, were as follows: for the neutral homocamptothecin analog 1, a BGE of 75 mM phosphate buffer pH 2.5 (H(3)PO(4) + triethanolamine)/ACN - 95/5 v/v, with 7.5% w/v highly S-beta-CD, an applied field of 0.2 kV/cm and a fused capillary temperature control of 30 +/- 0.1 degrees C (typical current approximately 175 microA); for the cationic homocamptothecin 2, a BGE of 25 mM phosphate buffer pH 2.5 (H(3)PO(4) + TEA)/ACN - 90/10 v/v, with 2.5% w/v highly S-beta-CD, an applied field of 0.15 kV/cm and a fused capillary temperature control of 25 +/- 0.1 degrees C (typical current approximately 45 muA), and both are validated. The best results in terms of LOQ were obtained by EC with fluorescence detection: 10 ng/mL and 20 ng/mL for 1 and 2, respectively (LOQ divided by 150 for 1 and 5 for 2 with respect to UV), thus making this method particularly convenient for enantiomeric purity determination of galenic forms. UV detection appears to be an alternative to fluorescence for the analysis of the main component either for the control of galenic forms or for therapeutic adaptation. Moreover, this method exhibits better performances than HPLC.     

HPLC method for the simultaneous determination of atenolol and chlorthalidone in human breast milk

A high-performance liquid chromatographic method was optimized and validated for the determination of atenolol and chlorthalidone (CT) in human breast milk. The milk samples were extracted and purified using ACN and phosphoric acid for precipitation of proteins followed by removal of ACN and milk fats by extraction with methylene chloride. The samples were applied, after an extraction procedure, to a cyanide column using a mobile phase consisting of ACN/water (35:65 v/v) and buffered at pH 4.0 with flow rate of 1.0 mL/min. Quantitation was achieved with UV detection at 225 nm using guaifenesin as the internal standard. The effectiveness of protein precipitation and clean up procedure were investigated. The method was validated over the range of 0.3-20 microg/mL for atenolol and 0.25-5 microg/mL for CT.     

Measurement of tobramycin by reversed-phase high-performance liquid chromatography with mass spectrometry detection

Analysis of tobramycin faces challenges owing to its significant basicity, hydrophilicity and lack of a UV absorbing chromophore. Chromatographic methods, coupled with derivatization to introduce chromophores for tobramycin analysis, were extensively studied. A direct reversed-phase HPLC method for tobramycin analysis has not been reported. Here, we would like to report a simple LC/MS method for quantitative analysis of tobramycin in pharmaceutical formulations. Reversed-phase HPLC analysis of tobramycin was achieved using a pH stable C18 column with basic (pH 11) aqueous mobile phase (ammonium hydroxide buffer), while direct detection was carried out employing a single quadruple mass detector in negative mode via electrospray ionization. This unique separation-detection combination provided simple and specific determination of tobramycin. This method was found to be linear at a tobramycin concentration range of 0.2-0.8 mg/mL with a correlation coefficient value of 0.999. The quantitation limit and detection limit were calculated as 0.210 and 0.063 μg/mL, respectively, with 99.994% confidence. This method was successfully applied to measure tobramycin content in matrices containing tobramycin and other pharmaceutical formulation ingredients. Recoveries of 101.8, 97.8 and 106.7% were obtained for tobramycin spiked in the pharmaceutical formulation at concentrations of 1.68, 1.0 and 0.35 mg/mL, respectively. The relative standard deviations for six injections of spiked samples ranged from 0.2 to 3.2%, indicating good method repeatability.     

Novel nonaqueous capillary electrophoresis separation and determination of bioactive flavone derivatives in Chinese herbs

Nonaqueous CE (NACE) coupled to UV detection is described for the separation and determination of bioactive flavone derivatives in Chinese herbs extraction. After optimization of electrophoresis parameters, including the electrolyte nature and the organic solvent composition, a reliable separation of the analytes in an ACN/methanol (60:40, v/v) mixture containing 80 mM Tris and 10 mM sodium cholate was performed. The detection was performed at 254 nm. Method performances, including migration time and peak area reproducibility, linearity, sensitivity, and accuracy, were evaluated. The method was applied to determine bioactive flavone derivatives in seven Chinese herbs.     

Development of a capillary electrophoresis method for the determination of soybean proteins in soybean-rice gluten-free dietary products

CE has been applied for the first time to the simultaneous separation of soybean and rice proteins. Treated and untreated capillaries with different effective lengths as well as separation media at different pHs were tested. For that purpose, samples and standard solutions were prepared in 25:75 ACN-water media containing 0.3% v/v acetic acid. The use of an untreated capillary of 50 cm effective length together with an 80 mM borate buffer (pH 8.5) modified with 20% v/v ACN and UV detection at 254 nm were the conditions working the best. These conditions enabled the determination of soybean proteins in gluten-free dietary commercial products elaborated with soybean protein and/or soybean flour and rice flour using the standard additions calibration method. The method was linear up to 26 mg/mL of soybean proteins, the precision (expressed as RSD) was always better than 6%, and recoveries obtained for soybean proteins when spiking commercial products were very close to 100%.     

Development and validation of a reversed-phase liquid chromatographic method for analysis of demeclocycline and related impurities

A simple, robust, and rapid reversedphase high-performance liquid chromatographic method for the analysis of demeclocycline and its impurities is described. Chromatographic separations were achieved on a Symmetry Shield RP8 (75 mm × 4.6 mm, 3.5 μm) column kept at 40°C. The mobile phase was a gradient mixture of acetonitrile, 0.06 M sodium edetate (pH 7.5), 0.06 M tetrapropylammonium hydrogen sulphate (pH 7.5) and water, A (2:35:35:28 v/v/v/v) and B (30:35:35:0 v/v/v/v) pumped at a flow rate of 1 mL/min. UV detection was performed at 280 nm. The developed method was validated according to the ICH guidelines for specificity, limit of detection, limit of quantification, linearity, precision, and robustness. An experimental design was applied for robustness study. Results show that the peak shape, chromatographic resolution between the impurities, and the total analysis time are satisfactory and better than previous methods. The method has been applied for the analysis of commercial demeclocycline bulk samples available on the market.     

Separation and determination of four active anthraquinones in Chinese herbal preparations by flow injection-capillary electrophoresis

A simple, rapid, and accurate method for the separation and determination of physcion, chrysophanol, aloe-emodin, and emodin in Rhubarb, Juemingzi, and Chinese herbal preparations was developed by combination of flow injection-capillary zone electrophoresis for the first time. The analysis was carried out using an unmodified fused-silica capillary (75 mm x 50 microm ID x 375 microm OD, effective separation length of 48 mm) and direct ultraviolet detection at 254 nm. By a series of optimization, the sample solvent consisted of NaOH (100 mmol/L) and ACN (1:1 v/v), and a running buffer composed of 15 mmol/L sodium borate - 12.5 mmol/L sodium dihydrogen phosphate - 42% v/v ACN (pH 10.1) was applied for the separation of the four anthraquinones. The separation was rapid and highly reproducible, with complete resolution of all four compounds within 6 min. The sample throughput rate could reach up to 12 per h. The repeatability (defined as relative standard deviation) was 4.45, 4.44, 4.34, 0.61% with peak height evaluation and 1.62, 0.89, 2.49, 2.19% with peak area evaluation for physcion, chrysophanol, aloe-emodin, and emodin, respectively.     

Simultaneous determination of diethylene glycol and propylene glycol in pharmaceutical products by HPLC after precolumn derivatization with p-toluenesulfonyl isocyanate

A simple and reliable HPLC method was developed for the simultaneous quantitative analysis of diethylene glycol (DEG) and propylene glycol (PG) in pharmaceutical products by precolumn derivatization. The derivatization reagent p-toluenesulfonyl isocyanate (TSIC, 10 microL, 20% in ACN v/v) was added to 100 microL of the sample, and then 10 muL of water was added. The resulting derivatives were separated using a C(18)analytical column and a mobile phase composed of 0.01 M KH(2)PO(4)buffer (adjusted to pH 2.5 with phosphoric acid) and ACN (47:53 v/v) at 1 mL/min and 25 degrees C. For detection, UV light at 227 nm was used. The derivatization conditions including reaction time, temperature, and concentration of TSIC were optimized. The calibration curves were linear from 0.062 to 18.6 microg/mL (r(2)= 0.9999) and from 0.071 to 21.3 microg/mL (r(2) = 0.9999) for DEG and PG, respectively. The RSD values of intra- and interday assays were all below 4% for DEG and PG. The proposed method was then successfully applied to analyze two Armillarisin A injection samples and two spiked syrup samples.     

Determination of peimine and peiminine in Bulbus Fritillariae Thunbergii by capillary electrophoresis by indirect UV detection using N-(1-naphthyl)ethylenediamine dihydrochloride as probe

A simple and inexpensive CE method was developed for the determination of peimine and peiminine. Because of the lack of an UV chromophore of peimine and peiminine, the detection method chosen was indirect UV detection, with N‐(1‐naphthyl)ethylenediamine dihydrochloride (NED) as the UV absorbing probe. It was thought that NED, a chromophoric ion, may form hydrogen bonding pairs with the analytes to cause significant changes in separation selectivity. Additionally, the hydrophobic interactions between analytes and the probe also play a crucial role in achieving a resolution between the two analytes. The analyses were carried out with a background electrolyte composed of 66% MeOH–ACN (1:1, v/v), 34% aqueous buffer containing 15 mM NaH₂PO₄, 2.5 mM NED, 4 mM H₃PO₄. MeOH–ACN mixtures used as organic modifiers can not only reduce the adsorption of NED to the capillary wall, but also decrease the baseline noise and drift. The method provided a linear response ranging from 5 to 200 μg/mL. The limits of detection (LODs) for peimine and peiminine were 3.9 and 4.1 μg/mL, respectively. The repeatabilities (n = 3) reached relative standard deviation values (RSDs) of 3.4 and 4.1% for the peak areas, 4.0 and 4.4% for the peak heights, and 0.29 and 0.30% for the migration time of peimine and peiminine, respectively. Regression equations revealed linear relationships (r = 0.9995–0.9996) between the peak area of each analyte and the concentration. The method developed was successfully applied to quantify peimine and peiminine in chloroform extracts of the ground Bulbus Fritillariae Thunbergii.     

Factorial design of electrolyte systems for the separation of fatty acids by capillary electrophoresis

In this work, a capillary zone electrophoretic methodology using UV indirect detection (224 nm) for the analysis of fatty acids (FAs) in saponified oils is proposed. The electrolyte consisted of a 5 mmol l(-1) phosphate buffer, pH 7. containing 4 mmol l(-1) sodium dodecylbenzenesulfonate (SDBS) as chromophore, 4 mmol l(-1) dimethyl-beta-cyclodextrin and 45% acetonitrile (ACN). The composition of the electrolyte was optimized by a 2(3) factorial design with triplicate at the central point. The design established practical concentration boundaries for SDBS and ACN. In a defined concentration range of 2-4 l(-1), SDBS can certainly be used as a chromophore for indirect detection without imparting excessive baseline noise. For ACN, a suitable interval of 45-55% was found to enhance FAs solubilization without overflowing the system with bubble formation and current interruption. Additionally, the design revealed the importance of dimethyl-beta-cyclodextrin in the resolution of difficult pairs and its function as a solubilizing agent for long chain FAs. At the optimized conditions, nine FAs from C10 to C20, including mono- di- and tri-unsaturated C18 fatty acids were baseline separated in less than 10 min. The proposed method was applied to the separation of FAs in edible oils and polyunsaturated fatty acid enriched margarine. Additionally, spectral monitoring at 206 nm was used to confirm peak identity in the samples.     

Analyses of alkaloids in different products by NACE-MS

A simple method for the separation and characterization of five nicotine-related alkaloids by NACE employing UV and MS detections is described here for the first time. Several factors, including NACE parameters (compositions of running solution) and MS parameters (such as nature and flow rate of sheath liquid, pressure of nebulization gas, and flow rate of dry gas), were optimized in order to obtain both an adequate CE separation and high MS signals for the alkaloid compounds used in this study. A reliable CE separation of five alkaloids was achieved in 50 mM ammonium formate that was dissolved in an ACN/methanol mixture (50:50, v/v) of pH* 4.0 (apparent pH 4.0). The optimal electrospray MS measurement was carried out in the positive ionization mode using a coaxial sheath liquid composed of isopropyl alcohol and water in the ratio of 80:20 v/v at a flow rate of 180 microL/h. In addition, the proposed NACE method was also applied in the analyses of alkaloids in several products including chewing gums, beverages, and tobaccos. This NACE-MS method was found to provide a better detection ability and separation resolution for the analysis of nicotine alkaloids when compared to other aqueous CE-MS reports.     

Method development and validation for the simultaneous determination of four coumarins in Saussurea superba by capillary zone electrophoresis

A capillary zone electrophoretic method has been developed for the determination of four coumarins--skimmin, scopolin, scopoletin, and umbelliferone-in Saussurea superba with UV detection at 254 nm. The capillary temperature was kept constant at 25 degrees C. Effects of buffer pH, electrolyte concentration, organic modifier, and applied voltage on migration behavior were studied systematically. The optimum conditions for separation were achieved by using 30 mM borate buffer at pH 9.02 containing 15% (v/v) methanol as the electrolyte and 25 kV as the applied voltage. For all analytes a good linear regression relationship (r > 0.999) was obtained between peak area and concentration over a relatively wide range. The method was validated for repeatability, precision, and accuracy. The validated method was successfully applied to the simultaneous determination of the four analytes in S. superba.     

Quantitative screening for steroids in animal feeding water using reversed phase LC with gradient elution

Several isocratic separations for the determination of 20 steroids (STER) in animal feeding water samples (AFWS) from drinking-trough by LC using a mobile phase ACN/H(2)O (35:65 v/v) and different RP columns (Hypersil C18, Gemini C18 (GM), Purospher Star C18, Synergi Max C12, and Synergi Fusion) and UV detection were obtained. The elution order was the same: a first group of corticoids (CC) was early eluted, a second group of CC and anabolics (AAS) exhibited intermediate retention, and a third group constituted by AAS was strongly retained. To improve the separation performances of the isocratic separations an ACN gradient elution optimization was carried out for each column. The most satisfactory results were obtained using a Purospher column which allowed the separation of 19 STER in an analysis time close to 26 min. After sample preparation using SPE, method validation was performed in an AFWS spiked with STER according to the EC decision criteria established for quantitative screening methods. For this purpose calibration graphs, extraction efficiencies, decision limits, detection capabilities, precision (repeatability and within-laboratory reproducibility), accuracy, selectivity, and robustness were evaluated. The proposed method was applied to other AFWS with satisfactory results.     

Separation of alpha-, beta-, gamma-, delta-tocopherols and alpha-tocopherol acetate on a pentaerythritol diacrylate monostearate-ethylene dimethacrylate monolith by capillary electrochromatography

This work reports the first use of a monolith with method development for the separation of tocopherol (TOH) compounds by CEC with UV detection. A pentaerythritol diacrylate monostearate-ethylene dimethacrylate (PEDAS-EDMA) monolithic column has been investigated for an optimised condition to separate alpha-, beta-, gamma- and delta-TOHs, and alpha-tocopherol acetate (TAc). The PEDAS-EDMA monolith showed a remarkably good selectivity for separation of the TOH isomers including the beta- and gamma-isomers which are not easily separated by standard C8 or C18 particle-packed columns. Retention studies indicated that an RP mechanism was involved in the separation on the PEDAS-EDMA column, but polar interactions with the underlying ester and hydroxyl groups enhanced the separation of the problematic beta- and gamma-isomers. Separation of all the compounds was achieved within 25 min using 3:10:87 v/v/v 100 mM Tris buffer (pH 9.3)/methanol/ACN as the mobile phase. The method was successfully applied to a pharmaceutical sample with recoveries from 93 to 99%. Intraday and interday precisions (%RSD) for peak area and retention time were less than 2.3. LODs for all four TOHs and TAc were below 1 ppm.     

Characterization and differentiation of diverse transgenic and nontransgenic soybean varieties from CE protein profiles

Nowadays, soybeans are commercialized in a wide variety of colors and tones. Moreover, some pigmented seeds are being commercialized as soybeans while, on other occasions, these seeds are labeled as mung beans, azuki beans or soybean frijoles generating confusion on their identity. In this work, CE has been applied for the first time for the characterization and differentiation of different pigmented beans commercialized as soybeans. Other seeds commercialized as azuki, mung green soybeans or soybean frijoles were also analyzed. Borate buffer (at pH 8.5) containing 20% v/v ACN was used as the separation media and solution containing ACN/water (75:25 v/v) with 0.3% v/v acetic acid was used to solubilize the proteins from the samples. A 50 cm bare fused-silica capillary was employed for obtaining adequate separations in about 12 min. The CE protein pattern observed for yellow soybeans was different from that corresponding to green and red soybeans. The seeds commercialized as black soybean presented electropherograms identical or similar to those yielded by the yellow seeds with the exception of the sample labeled as black soybeans frijoles that presented a totally different pattern. In addition, CE protein profiles obtained for azuki and mung green soybeans were very similar to those corresponding to red soybeans and green soybeans, respectively. Finally, the CE method was also applied to differentiate transgenic and nontransgenic soybean varieties. Discriminant analysis, using several protein peak areas as variable, was used to successfully classify these samples.     

Simultaneous determination of silicon and phosphorus in soil and plants by reversed-phase ion-pair chromatography

A reversed-phase ion-pair high-performance liquid chromatographic method, using tetrabutylammonium bromide (TBABr) as ion-pair reagent, has been developed for the simultaneous analysis of silicon (Si) and phosphorus (P) as heteropoly acids in soil and plant samples. The effect of the concentrations of ion-pair reagent, acetate buffer and organic modifier as well as the pH of buffer on separation was made clear. The reaction conditions and stability of heteropoly acids were investigated. Furthermore, the phenomenon occurred in the optimized process was also further researched. The separation was performed on a reversed-phase C(18) column within 11 min with 40:60 (v/v) 0.1M acetate buffer (pH 3.9)-acetonitrile (ACN) containing 0.8 mM TBABr as a mobile phase. The linear ranges of the peak area calibration curves for Si and P were 0.08-50 mg/L and 0.40-50 mg/L, respectively. The detection limits calculated at S/N=3 were 0.0057 mg/L and 0.0280 mg/L for Si and P, respectively. The method was successfully applied to the analysis of soluble and total contents of Si and P in soil and plant samples.     

Determination of pKa values of nonsteroidal antiinflammatory drug‐oxicams by RP–HPLC and their analysis in pharmaceutical dosage forms

In this study, pK_a values were determined by using the dependence of the capacity factor on the pH of the mobile phase for four ionizable substances, namely, tenoxicam, piroxicam, meloxicam, and naproxen (I.S.). The effect of the mobile phase composition on the ionization constant was studied by measuring the pK_a at different ACN concentrations, ranging from 30 to 40%. The adequate condition for the chromatographic determination of these compounds in pharmaceutical dosage forms was established based on the different retention behaviors of the species. An octadecylsilica Nucleosil C18 column (150×4.6 mm, 5 μm) was used for all the determinations. The chromatographic separation of oxicams was carried out using acetonitrile (ACN)/water at 35% v/v, containing 65 mM phosphoric acid and UV detection at a wavelength of 355 nm. The method developed was successfully applied to the simultaneous determination of these drug compounds in laboratory‐prepared mixtures and their commercial pharmaceutical dosage forms. Each analysis requires no longer than 12 min.     

Simultaneous determination of seven flavonoids in Epimedium using pressurized liquid extraction and capillary electrochromatography

Herba Epimedii (known as Yinyanghuo in China) is one of the commonly used Chinese medicines. Flavonoids are considered as its active components. In this study, a CEC method was developed for the simultaneous determination of seven flavonoids, including hexandraside E, kaempferol-3-O-rhamnoside, hexandraside F, icariin, epimedin A, B, and C, in Epimedium using baicalein as internal standard (IS). The influence of relevant parameters such as buffer concentration, pH, and proportion of ACN was investigated and optimized. Baseline separation was obtained using a Hypersil C18 capillary (3 microm, 100 microm/25 cm) with a mixture of 20 mM phosphate buffer (pH 4.0)/ACN (70:30 v/v) as mobile phase running at 30 kV and 25 degrees C in 20 min. All calibration curves showed good linearity (r2 >0.9992) within test ranges. The LOD and LOQ were lower than 8.6 and 42.8 microg/mL, respectively. The RSDs of intra- and interday for relative peak areas of seven analytes were less than 3.1 and 4.4%, and the recoveries were 95.2-103.3%. Samples of different Epimedium species were analyzed using the validated method, which is useful for quality control of Epimedium and its medical preparations.     

Development and validation of a stability-indicating LC method for the assay of lodenafil carbonate in tablets

A stability-indicating liquid chromatographic method has been developed for the quantitative determination of lodenafil carbonate in tablets. The method employs a Synergi Fusion C18 column (250 × 4.6 mm, i.d., 4 μm particle size), with mobile phase consisting of a mixture of methanol-acetic acid 0.1% pH 4.0 (65:35, v/v) and UV detection at 290 nm, using a photodiode array detector. A linear response (r = 0.9999) was observed in the range of 10-80 μg/mL. The method showed good recoveries (average 100.3%) and also intra and inter-day precision (RSD < 2.0%). Validation parameters as specificity and robustness were also determined. Specificity analysis showed that no impurities or degradation products were co-eluting with the lodenafil carbonate peak. The method was found to be stability-indicating and due to its simplicity and accuracy can be applied for routine quality control analysis of lodenafil carbonate in tablets.     

Stability-indicating method for determination of oxyphenonium bromide and its degradation product by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed for determination of oxyphenonium bromide (OX) and its degradation product. The method was based on the HPLC separation of OX from its degradation product, using a cyanopropyl column at ambient temperature with mobile phase of acetonitrile-25 mM potassium dihydrogen phosphate, pH 3.4 (50 + 50, v/v). UV detection at 222 nm was used for quantitation based on peak area. The method was applied to the determination of OX and its degradation product in tablets. The proposed method was also used to investigate the kinetics of the acidic and alkaline degradation of OX at different temperatures, and the apparent pseudo first-order rate constant, half-life, and activation energy were calculated. The pH-rate profile of the degradation of OX in Britton-Robinson buffer solutions within the pH range 2-12 was studied.