ABSTRACT
OBJECTIVE: To develop a high performance liquid chromatography method with pre-column derivatization for the determination of binded and free formaldehyde in cross linked sodium hyaluronate injection. METHODS: After the samples were hydrolyzed by heating with 1.0 mol · L-1 hydrochloric acid, binded formaldehyde was released. By contrast, the concentration of free formaldehyde was measured without the hydrolysis step. Formaldehyde was derived with 2, 4-dinitrophenylhydrazine (DNPH) and then determined by HPLC. The analysis was carried out on a C18 column(4.6 mm × 250 mm, 5 μm). The mobile phase was 65% acetonitrile, the flow rate was 1.0 mL · min-1, and the wavelength was set at 365 nm. RESULTS: This method had a linear range of 0.1-50 μg · mL-1 (y =76 023x +7 069, r=1.0000). The limit of detection(LOD) and the limit of quantification(LOQ) were 0.001 μg · mL-1 and 0.005 μg · mL-1, respectively. The average recoveries of the binded and free formaldehyde were 102.9% and 105.5%, and the relative standard deviations (RSDs, n=18) were 1.3% and 3.0%, respectively. CONCLUSION: This method has a wide linear range and good accuracy, which is suitable for the determination of both binded and free formaldehyde contents in cross linked sodium hyaluronate injection.
ABSTRACT
Objective To establish an HPLC method for determination of low-molecular-weight carbonyl compounds in the oil-containing herbs. Methods After carbonyl compounds in the samples were extracted with water, the solution reacted with 2, 4-dinitrophenylhydrazine (DNPH) in an acidic medium to form 2, 4-dinitrophenylhydrazone derivatives, which were separated on Kromasil KR100-5 C18 column (250 mm× 4.6 mm, 5 μm). The mobile phase A was water-acetonitrile-tetrahydrofuran-isopropanol (59∶30∶10∶1), mobile phase B was water-acetonitrile (35∶65), gradient elution. The flow rate was 0.8 mL/min, detection wavelength was 365 nm, and column temperature was 30 ℃. Results Good linearities were obtained in corresponding concentration ranges, with correlation coefficient over 0.999. The limits of detection of the eight DNPH derivatives were 0.002-0.008 μg/mL, and the average recoveries were 88.49%-93.65%. Conclusion The method is simple, accurate and reliable, with good reproducibility.
ABSTRACT
O malondialdeído (MDA) é um importante biomarcador utilizado na avaliação do estresse oxidativo. O objetivo deste estudo foi desenvolver uma metodologia para a quantificação plasmática de MDA, através de cromatografia líquida de alta eficiência com detecção por arranjo de diodos (CLAE-DAD), após processo de derivatização com 2,4-dinitrofenilhidrazina (DNPH), avaliando as principais variáveis pré-analíticas. A curva de calibração em plasma (0 a 40 µM) apresentou elevada linearidade (r²=0,998). Os principais parâmetros de validação foram: recuperação absoluta: 78 por cento; limite de detecção: 0,11 µM e limite de quantificação: 0,38 µM. Os valores de MDA determinados em indivíduos adultos saudáveis (n=38) foram 3,31 ± 0,38 µM (média ± DP). Estudos de estabilidade do padrão de MDA, reagente derivatizante e MDA plasmáticos, indicaram que a solução padrão pode ser armazenada a -20 e 4 ºC.
Malondialdehyde (MDA) is an important biomarker for the evaluation of oxidative stress status. The aim of this study was to develop a method for plasma MDA quantification by high performance liquid chromatography with diode-array detection (HPLC-DAD), following derivatization with 2,4-dinitrophenylhydrazine (DNPH), evaluating the main preanalytical variables. The calibration curve in plasma (0 to 40 µM) presented high linearity (r² = 0.998). Main validation parameters were: recovery: 78 percent; LOD: 0.11 µM and LOQ: 0.38 µM. The MDA values obtained in healthy volunteers (n=38) were 3.31 ± 0.38 µM (mean ± SD). Stability studies of MDA standard solution and derivatizing reagent and plasma MDA indicated that the standard solution can be stored at -20 and 4 ºC, remaining stable for at least 30 days.