RESUMO
A capillary zone electrophoretic (CZE) method has been developed for the determination of impurities (phosphyte and phosphate) in technical-grade ibandronate, which is a potent nitrogen-containing bisphosphonate. Successful separation of the drug from the impurities was achieved using 1mM tetradecyl-trimethyl-ammonium bromide (TTAB) and 5mM potassium chromate (pH 10.0) as background electrolyte with an indirect detection at 254 nm. The optimised method was validated for specificity, precision, linearity and accuracy. The limit of detection (LOD) was 2 microg/mL and the limit of quantification (LOQ) was 7 microg/mL for both phosphyte and phosphate. The developed CZE method used to determine phosphyte and phosphate as bisphosphonates impurities can be used to evaluate the quality of regular production samples of ibandronate.
Assuntos
Conservadores da Densidade Óssea/análise , Difosfonatos/análise , Contaminação de Medicamentos , Eletroforese Capilar/métodos , Cromatos/química , Eletroforese Capilar/instrumentação , Concentração de Íons de Hidrogênio , Ácido Ibandrônico , Fosfatos/análise , Fosfitos/análise , Compostos de Potássio/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrofotometria Ultravioleta , Compostos de Trimetil Amônio/químicaRESUMO
Purification of gliadin subclasses has been difficult since they share many biochemical and physicochemical properties. In this report, the optimization of a preparative electrophoretic method to fractionate gliadins is described. Separation was performed in preparative 7% polyacrylamide gels at pH 3.1. The separation performance was tested using analytical electrophoresis at pH 3.1 and capillary electrophoresis. Preparative gels of different lengths were employed. Using 5-cm preparative gels, several fractions of alpha-, beta-, and gamma-gliadins and fast-mobility and slow-mobility omega-gliadins were collected in 40 h of separation. Resolution was maintained at a protein load of up to 30 mg in each run. The highest efficiency of recovery was achieved using aluminum lactate as the collecting buffer. Fractionation with 10 cm in length gels improved resolution but increased operation times. Gels of 2 cm in length did not separate alpha/beta- and gamma-gliadins efficiently but were useful to separate the two main fractions of omega-gliadins in shorter times. In conclusion, preparative electrophoresis at low pH allowed the separation of alpha-, beta-, gamma-, and omega-gliadin fractions from crude material under nondenaturing conditions.
Assuntos
Gliadina/isolamento & purificação , Eletroforese em Gel de Poliacrilamida/métodos , Farinha/análise , Concentração de Íons de Hidrogênio , Triticum/químicaRESUMO
Gliadins are one of the major wheat grain storage proteins. They are a group of more than 50 proteins that share structural homology and similar physicochemical properties. Electrophoresis at pH 3.1 using acidic-polyacrylamide gel electrophoresis (A-PAGE) has traditionally been a way to characterize them. Gliadins were classified in alpha-, beta-, gamma-, and omega-gliadins, according to their decreasing mobility in the A-PAGE system. CE has been successfully applied to the analysis of gliadins demonstrating high efficiency and resolution for wheat cultivar identification. We compared purified gliadin fractions using CE and A-PAGE through their relative mobility. The conditions selected for the fastest CE separation employed a 25-micron-i.d. fused-silica capillary column using 0.1 M phosphate, pH 2.5; 20% acetonitrile; and 0.03% hydroxypropylmethylcellulose (HPMC) as the running buffer. The analysis of the purified gliadin fractions showed the same relative mobility pattern in both systems, allowing the characterization of cultivars commonly grown in Argentina. Because it provides efficiency, reproducibility, and short analysis times, CE is a more suitable technique for routine cultivar differentiation. In addition, the CE separation in which all gliadin fractions, even the slow-moving ones, are presented, will be useful for future applications such as control of gliadin fraction isolation or gliadin quantification.
Assuntos
Eletroforese Capilar/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Gliadina/análise , Concentração de Íons de Hidrogênio , Triticum/químicaRESUMO
In the present work, we used capillary zone electrophoresis (CZE) for the analysis of Vitamin C and inorganic cations (potassium, sodium, calcium, and magnesium) in lettuce (Lactuca sativa) during postharvest storage. The leaves of the creasphead lettuce samples were treated with 18 M omega water for cation analysis and, alternatively, with 3% wt/vol oxalic acid solution for ascorbic acid extraction. They were then homogenized and centrifuged, and the supernatants were introduced into the capillary for CE analysis. The cation analysis was performed on an uncoated fused-silica capillary column of 75 microns i.d. and 50 cm total length, using 15 mM creatinine, 15 mM alpha-hydroxyisobutyric acid, acetic acid, pH 4.00, as running buffer at an applied voltage of 20 kV. Indirect on-line UV detection was carried out at 214 nm. The content of Vitamin C was measured using an uncoated fused-silica capillary column of 50 microns i.d. and 45 cm total length; 10 mM sodium tetraborate, 10 mM sodium biphosphate, pH 9.00, as background electrolyte at an applied voltage of 20 kV. On-line UV detection was achieved at 254 nm.
Assuntos
Ácido Ascórbico/análise , Eletroforese Capilar/métodos , Lactuca/química , Metais/análise , Cálcio/análise , Cátions/análise , Magnésio/análise , Potássio/análise , Sódio/análiseRESUMO
L. tenuis and L. corniculatus seeds are morphologically very similar but their purchase prices are quite different. Chromosome number counting is the only test used thus far in laboratories for the identification of these seeds. Recently, the flavonol's pattern has been used as a criterion for differentiation. In the present work, we studied the storage protein patterns of different Lotus seed samples by capillary gel electrophoresis (CGE), as an alternative method, comparing it with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The seeds were treated according to International Seed Testing Association (ISTA) recommendations. CGE separations were performed using an uncoated capillary of 18 cm effective length and 50 microns i.d. and the Bio-Rad Protein Kit (Hercules, CA, U.S.A.). On-line detection was carried out at 220 nm.
Assuntos
Fabaceae/química , Proteínas de Plantas/análise , Plantas Medicinais , Sementes/química , Eletroforese Capilar/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Dodecilsulfato de SódioRESUMO
A reproducible, rapid procedure for the simultaneous quantitative separation of S-adenosylmethionine and S-adenosylhomocysteine by capillary zone electrophoresis has been developed. Separations were performed by using an uncoated capillary of 60 cm effective length and 50 microm ID, 40 mM sodium phosphate buffer, pH 2.50, as background electrolyte solution, and 30 kV. On-line detection was carried out at 254 nm. Under the conditions selected we resolved a standard solution containing S-adenosylmethionine and S-adenosylhomocysteine in a run time shorter than 8 min. A mass detection limit in the range of 10 fmol was achieved. Adenosyl-L-methionine, S[methyl-3H] has also been assayed under the same experimental conditions. Other related compounds did not show interference, including those derived from the hydrolysis of S-adenosylmethionine. The present method allows simultaneous determination of these compounds, which play an important role in many microbiological and enzymatic research studies.
Assuntos
Eletroforese Capilar/métodos , S-Adenosil-Homocisteína/análise , S-Adenosilmetionina/análise , Soluções Tampão , Fosfatos , Controle de Qualidade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A reproducible, rapid procedure for the determination of homocysteine, cysteine, and glutathione in human blood by micellar electrokinetic chromatography (MEKC) has been developed. Whole blood samples were deproteinized and centrifuged, and the supernatant was directly introduced into the capillary for analysis without further derivatization. Separations were performed using an uncoated capillary of 30 cm effective length and 50 microns i.d.; 20 mM phosphate, 20 mM borate, and 50 mM SDS, pH 9.00, as running buffer; and an applied voltage of 18 kV. On-line detection was carried out at 214 nm.
Assuntos
Cromatografia Capilar Eletrocinética Micelar , Homocisteína/sangue , Cisteína/sangue , Glutationa/sangue , HumanosRESUMO
A reproducible, rapid procedure for the determination of glutathione in human blood by micellar electrokinetic chromatography has been developed. Whole blood samples were deproteinized with 5% w/v sulfosalicylic acid (final concentration). After centrifugation, the supernatant was directly injected for analysis, without further derivatization. Separations were performed by using an uncoated capillary of 30 cm effective length and 50 microns internal diameter (ID), 50 mM Tris-HCl, 30 mM sodium dodecyl sulfate (SDS), pH 7.00, as running buffer, and 10-20 kV. On-line detection was carried out at 214 nm and a detection limit in the range of femtomoles was achieved. Under the same experimental conditions, we resolved a mixed standard solution containing glutathione in its oxidize and reduced forms, lipoamide and alpha-lipoic acid. The corresponding migration times were reproducible. The present method allows rapid determination of these compounds, which play a critical role in oxidative stress, in cellular defense against injurious agents and whose levels are related to the toxicology and metabolism of several toxins and drugs, such as antineoplastic agents.
Assuntos
Cromatografia/métodos , Glutationa/sangue , Micelas , Ácido Tióctico/análogos & derivados , Ácido Tióctico/sangue , Benzenossulfonatos , Eletroquímica , Humanos , Cinética , Oxirredução , SalicilatosRESUMO
2-(Aminomethyl)-4-aminobutyric acid (isoornithine), 3-methylisoornithine, and 2,3-dimethylisoornithine were not decarboxylated by liver ornithine decarboxylase (ODC, EC 4.1.1.17) of thioacetamide-treated rats but were good competitive inhibitors of the enzyme (Ki ranged from 0.72 to 1.79 mM). When assayed in vivo in the treated rats, the above mentioned isoornithines were also found to inhibit liver ODC when administered 1 h before sacrifice. When the methylputrescines formally derived from the decarboxylation of several isoornithines were assayed on rat liver ODC, it was found that only 2,3-dimethylputrescine decreased the enzymatic activity. When assayed in vivo, it was found to decrease ODC activity by 60%, when the latter was measured 1 h after administration. The effect was reverted 4 h after administration of the drug. Isoornithines were not taken up by H-35 hepatoma cells; hence they did not affect their ODC activity. 2,3-Dimethylputrescine however, was transported into the cells and significantly decreased its ODC activity.