A reversed-phase high performance liquid chromatography (RP-HPLC) method with ultraviolet detection was developed and validated for the simultaneous quantification of antiretroviral drugs lamivudine (3TC), stavudine (d4T), and zidovudine (AZT) in perfusate samples obtained from the Single-Pass Intestinal Perfusion studies. The chromatographic analysis was performed using a Gemini C18 column and didanosine as internal standard (IS). The following parameters were considered for the validation procedure: system suitability, accuracy, precision, linearity and selectivity. The limits of detection were 0.32 µg/mL for 3TC, 0.11 µg/mL for d4T and 0.45 µg/mL for AZT and the limits of quantification were 1.06 µg/mL for 3TC, 0.38 µg/mL for d4T and 1.51 µg/mL for AZT. Repeatability and intermediate precision ranged from 1.05 to 1.31 and 1.50 to 1.87, respectively, and are expressed as percent of relative standard deviation (RSD). Based on these results, the developed and validated RP-HPLC method can be used for simultaneous determination of 3TC, d4T, and AZT in perfusate samples. Furthermore, this method is simple and adequate for measurements of the antiretroviral drugs in the same sample, since those compounds are mostly co-administered. Besides, this work can be used as an initial base for the development of similar methods in the same conditions presented in our study.

ABSTRACT BCS (Biopharmaceutics Classification System) and BDDCS (Biopharmaceutics Drug Disposition Classification System) were proposed as tools for classifying drugs into four categories. Both systems consider the solubility as an important characteristic for the classification of compounds in drug development and in vivo disposition prediction. Although some results of drug solubility can be found in the literature, the aforementioned characteristic is not entirely clear when considering didanosine (ddI). Based on that, the solubility of ddI was evaluated using equilibrium and intrinsic dissolution methods. For the equilibrium method, excess amount of ddI was added to each media until obtaining a supersaturated solution and the mixture was submitted to agitation at 37 °C. For the intrinsic dissolution method, the drug was compressed into the Wood's apparatus matrix and subjected to dissolution in each media with agitation at 37 °C. The results obtained from the equilibrium method indicated that it was necessary 139.37 mL of pH 1.2 media, 87.72 mL of pH 4.5 media, 12.54 mL of pH 6.8 media, 5.03 mL of pH 7.5 media and 7.65 mL of purified water for drug solubilization. Furthermore, a very fast intrinsic dissolution rate (IDR) was obtained for each media: 0.1 mg/min/cm² (pH 1.2), 0.2 mg/min/cm² (pH 4.5), 0.2 mg/min/cm² (pH 6.8), 0.1 mg/min/cm² (pH 7.5) and 0.1 mg/min/cm² (purified water). Based on these results, ddI can be considered as a highly soluble drug for both equilibrium and intrinsic dissolution methods.

A solubilidade e a taxa de dissolução de fármacos são de grande importância em estudos de pré-formulação de formas farmacêuticas. A melhora na solubilidade permite que os fármacos sejam candidatos potenciais à bioisenção, podendo ser uma boa maneira para desenvolver formulações dose-eficientes. O comportamento de solubilidade da lamivudina, estavudina e zidovudina em solventes individuais (sob faixa de pH 1,2 a 7,5) foi estudado pelos métodos de solubilidade em equilíbrio e dissolução intrínseca. No estudo de solubilidade pelo método do equilíbrio (método de agitação de frascos), conhecidas quantidades do fármaco foram adicionadas em cada meio até atingirem a saturação e a mistura foi submetida à agitação de 150 rpm por 72 horas a 37 ºC. No ensaio de dissolução intrínseca, conhecida quantidade de cada fármaco foi comprimida na matriz do aparato de Wood e submetida à dissolução em cada meio com agitação de 50 rpm a 37 ºC. No método de solubilidade em equilíbrio, a lamivudina e a zidovudina podem ser consideradas como fármacos altamente solúveis. Embora a estavudina apresente alta solubilidade nos meios pH 4,5, 6,8, 7,5 e água, a determinação da solubilidade em pH 1,2 não foi possível devido a problemas de estabilidade. Com relação à dissolução intrínseca, a lamivudina e a estavudina apresentaram alta velocidade de dissolução. Considerando o valor limite apresentado por Yu e colaboradores (2004), todos os fármacos apresentaram características de alta solubilidade no método de dissolução intrínseca. Com base nos resultados obtidos, a dissolução intrínseca parece ser superior para os estudos de solubilidade como um método alternativo para o propósito de classificação biofarmacêutica.

Solubility and dissolution rate of drugs are of major importance in pre-formulation studies of pharmaceutical dosage forms. The solubility improvement allows the drugs to be potential biowaiver candidates and may be a good way to develop more dose-efficient formulations. Solubility behaviour of lamivudine, stavudine and zidovudine in individual solvents (under pH range of 1.2 to 7.5) was studied by equilibrium solubility and intrinsic dissolution methods. In solubility study by equilibrium method (shake-flask technique), known amounts of drug were added in each media until to reach saturation and the mixture was subjected to agitation of 150 rpm for 72 hours at 37 ºC. In intrinsic dissolution test, known amount of each drug was compressed in the matrix of Wood's apparatus and subjected to dissolution in each media with agitation of 50 rpm at 37 ºC. In solubility by equilibrium method, lamivudine and zidovudine can be considered as highly soluble drugs. Although stavudine present high solubility in pH 4.5, 6.8, 7.5 and water, the solubility determination in pH 1.2 was not possible due stability problems. Regarding to intrinsic dissolution, lamivudine and stavudine present high speed of dissolution. Considering a boundary value presented by Yu and colleagues (2004), all drugs studied present high solubility characteristics in intrinsic dissolution method. Based on the obtained results, intrinsic dissolution seems to be superior for solubility studies as an alternative method for biopharmaceutical classification purposes.