Development and Validation of Micellar Liquid Chromatographic Method for Lamivudine and Derivatives.

GUALDESI, MARÍA S.; RAVIOLO, MÓNICA A.; ESTEVE ROMERO, JOSEP S.; BRIÑÓN, MARGARITA C.

Simposio; 36th International Symposium of High-Performance Liquid Phase and Related Techniques.; 2011

The search for an effective chemotherapeutic treatment against HIV infection has led to the development of agents that target specific and critical events in the HIV replicative cycle. Lamivudine (3TC) has been shown to be somewhat less toxic than other antiviral drugs but its therapeutic value is limited by the resistance that is generated within a few weeks of treatment. To overcome this problem, we used a 5’-O-carbonate substitution strategy by linking different aliphatic alcohols on the side chains 5´-O position of 3TC. Among them, that of associate with methanol (3TC-Metha), ethanol (3TC-Etha), n-propanol (3TC-nPro), n-Butanol (3TC-Buta), n-Pentanol (3TC-Penta), n-Hexanol (3TC-Hexa) and n-Octanol (3TC-Octa) demonstrated to exhibit important biological activity against HIV and HBV.1,2 In the present study, a simple and sensitive method by micellar liquid chromatographic (MLC) was optimized and validated for the simultaneous analysis of 3TC and their derivatives. Analytical separation was performed in less than 12.5 min, with these retention times (tR, min): 3TC: 2.4; 3TC-Metha: 3.1; 3TC-Etha: 4.0; 3TC-nPro: 4.9; 3TC-Buta: 5.8; 3TC-Penta: 6.9; 3TC-Hexa: 8.1 and 3TC-Octa: 10.4. For the study we using C18 ODS column with UV detection (272 nm), at 30ºC, the flow rate was 1.0 mL min-1, and a micellar solution of 0.15 M sodium dodecyl sulphate, 4 % butanol buffered at pH 7 as mobile phase. The analytical parameters studied include linearity (r = 0.9995), intra- and inter-day precision (RSD, %: 0.02-1.48 and 0.07-1.66, respectively) and robustness. This method was sensitive with limit of detection (LOD) between 1.6 x 10-7 and 5.1 x 10-6 M and limit of quantification (LOQ) between 1.1 x 10-6 and 1.4 x 10-5 M for all compounds, with recoveries ranging from 92.92 to 118.26%. Zidovudine (AZT) was used (tR: 2.8 min) as internal standard for a major statistical analysis. Thus, the method was fully validated according to the Food and Drug Administration guideline, and is suitable, sensitive and simple for routine analysis of 3TC derivatives.