RESUMO
The aim of this study was to optimise a method for gentamicin determination in an agar matrix and to investigate if and how agar composition can affect the gentamicin diffusion kinetics during the agar diffusion tests for antibiotics sensitivity. Gentamicin was separated by RP-HPLC and detected at 365 nm after pre-column derivatization with 1-fluoro-2,4-dinitrobenzene. Recovery (> or = 79%), linearity (r2 > or = 0.997) and sensitivity (1 microg/ml) were assessed using four different agar matrices. The kinetics of gentamicin diffusion tested on BioMerieux and DID manufacturers' products showed in uninoculated agar plates significant differences that were even more pronounced in the presence of Pseudomonas aeruginosa metabolism.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Gentamicinas/análise , Ágar , Difusão , Gentamicinas/metabolismo , Cinética , Pseudomonas aeruginosa/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeAssuntos
Ágar/metabolismo , Ágar/normas , Gentamicinas/metabolismo , Ágar/química , Cromatografia Líquida de Alta Pressão , Meios de Cultura/química , Meios de Cultura/metabolismo , Meios de Cultura/normas , Difusão , Gentamicinas/química , Cinética , Testes de Sensibilidade Microbiana , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
BACKGROUND: The diagnosis of "factitious hypoglycemia" is essentially based on the disclosure of hypoglycemic agents in blood or urine. The aim of this study was to evaluate the performance of capillary electrophoresis (CE) as a quantitative method for determination of chlorpropamide, tolbutamide, glipizide, gliclazide, and glibenclamide in serum. METHODS: Serum samples (1 mL), with internal standard added, were purified by solid-phase extraction on OASIS(TM) HLB cartridges (Waters), dried under reduced pressure, and reconstituted with 30-60 microL of acetonitrile:H(2)O. Analysis was carried out by micellar electrokinetic capillary chromatography in 5 mmol/L borate, 5 mmol/L phosphate, 75 mmol/L sodium cholate, pH 8.5, containing 25 mL/L methanol. Separation was accomplished in a 20 cm x 50 microm (i.d.) silica capillary at 25 degrees C and a constant voltage of +10 kV. Pharmacokinetics of gliclazide (80-mg tablet) in a diabetic patient were assayed by both HPLC and CE. Two hypoglycemic patients positive by HPLC analysis for unreported gliclazide and tolbutamide overdose were also screened by CE. RESULTS: Separation of six drugs (including the internal standard) was accomplished in 5 min plus 5 min rinsing. The between-day CV of the ratio of the areas of the sulfonylurea drugs to internal standard was <1% (n = 10). Linearity (r(2) > or =0.998) and recovery (> or =80%) were good for all sulfonylurea drugs tested. Pharmacokinetic curves for gliclazide by CE and HPLC were superimposable. CE analysis confirmed the HPLC diagnosis of surreptitious abuse of gliclazide and tolbutamide. CONCLUSION: CE is a useful tool in the clinical chemistry and toxicology laboratory for drug monitoring and pharmacokinetic investigations.
Assuntos
Hipoglicemia/diagnóstico , Hipoglicemiantes/sangue , Compostos de Sulfonilureia/sangue , Adulto , Cromatografia Líquida de Alta Pressão , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Overdose de Drogas , Eletroforese Capilar , Feminino , Gliclazida/efeitos adversos , Gliclazida/sangue , Gliclazida/farmacocinética , Humanos , Hipoglicemia/sangue , Hipoglicemia/induzido quimicamente , Hipoglicemiantes/efeitos adversos , Hipoglicemiantes/farmacocinética , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Compostos de Sulfonilureia/efeitos adversos , Compostos de Sulfonilureia/farmacocinética , Tolbutamida/efeitos adversos , Tolbutamida/sangueRESUMO
The aim of this study was to evaluate, using high-performance liquid chromatography, the concentration of ceftazidime in agar released from an E test strip, sampling at the edge of the strip at different points (1, 2, 4, 8, 16, 32, 64, and 128 microg/ml) at 6, 15, and 24 h after its deposition on uninoculated plates. From 6 to 24 h, the ceftazidime concentration in agar increased at the graduations 1, 2, and 4 microg/ml (+140, +82, and +58%, respectively), remained fairly constant at 8 microg/ml (-1.9%), and decreased at 16, 32, 64, and 128 microg/ml (-25, -44, -36, and -58%, respectively). In the 6-24 h range, the ceftazidime concentrations between 16 and 1 microg/ml were +/-1 serial dilution of the values reported on the strip, confirming the accuracy of the E test in agar.
Assuntos
Ceftazidima/análise , Cefalosporinas/análise , Testes de Sensibilidade Microbiana/métodos , Fitas Reagentes , Ágar , Cromatografia Líquida de Alta PressãoRESUMO
RANTES (regulated on activation, normal T cell expressed and secreted), a C-C chemokine, is one of the major HIV-suppressive factors produced by CD8+ T cells. Wild-type RANTES and genetically modified analogues were expressed in a baculovirus system and purified from cell culture supernatants employing a multi-step strategy based on affinity and RP-HPLC. Quantification and purity control of the final proteins were carried out by capillary electrophoresis using the synthetic or the recombinant wild-type RANTES as a reference. The procedure here reported requires only three days to obtain 0.016-0.270 mg of the pure and characterised proteins, starting from 370-900 ml of culture media, and is suitable for the analysis of a large number of RANTES analogues.