Espectrofotometria derivada: uma contribuição prática para o desenvolvimento de métodos / A contribution to the derivative spectrophotometry method

A espectrofotometria derivada (ED) tem sido utilizada como uma importante ferramenta no controle de qualidade de medicamentos para a determinação simultânea de fármacos em sistemas multicomponentes. Esta técnica oferece uma alternativa para melhorar a sensibilidade e a seletividade na análise de misturas e está acessível à maioria dos laboratórios. O procedimento é simples, rápido e não necessita extração prévia da amostra. Este trabalho tem como objetivo fornecer subsídios para o desenvolvimento de método por espectrofotometria derivada, utilizando a técnica do ponto de anulação, visando utilizá-la como um método alternativo no controle de qualidade de fármacos associados e, em especial, nos estudos de dissolução. Muitos subsídios foram retirados da literatura, outros de experiências vivenciadas no laboratório durante o desenvolvimento do método por ED aplicado na análise de dois inibidores da protease do vírus da imunodeficiência humana. Várias ordens de derivadas, diferentes valores de lambda delta e diferentes velocidades de varredura foram avaliados.

Derivative spectrophotometry has been successfully used as a quality control tool in pharmaceutical analysis for the simultaneous determination of drugs in multicomponent formulations. This technique, accessible to most laboratories, offers an alternative means of enhancing the sensitivity and specificity in mixture analysis. The procedure is simple, rapid and does not require any preliminary separations or treatment of the samples. The aim of this study is to provide pointers for the development of methods of analysis by derivative spectrophotometry (DS), using the zerocrossing technique, and to encourage professionals and researchers to use DS as an alternative method for quality control of drug combinations, especially in the study of dissolution. Much information, extracted from the literature, has been assembled here, together with the laboratory experience gained while developing a DS method to analyze a combination of two human immunodeficiency virus protease inhibitors. Various orders of derivatives, values of delta lambda and scan speeds were tested.

Desenvolvimento e validação de método cromatográfico para o dosamento do cloridrato de bupropiona / Development and validation of a chromatographic method for the determination of bupropion hydrochloride

O cloridrato de bupropiona é utilizado no tratamento da depressão e também indicado no tratamento da dependência à nicotina.O objetivo deste estudo foi desenvolver e validar um método por cromatografia a líquido de alta eficiência (CLAE),alternativo ao oficial preconizado pela Farmacopéia Americana para a determinação quantitativa do cloridrato de bupropiona.O método difere do oficial por apresentar uma fase móvel simples, na qual não é necessária a utilização do solvente tetraidrofurano nem da solução tampão.Outra vantagem do método proposto é a de apresentar um tempo de retenção do fármaco relativamente curto.A estabilidade do fármaco frente a determinadas condições de estresse (hidrólise, oxidação e fotólise), também foi avaliada.O método mostrou-se específico, preciso, linear, exato e robusto podendo ser utilizado como alternativa ao método oficial preconizado pela Farmacopéia Americana em ensaios de controle de qualidade.Os resultados obtidos na avaliação do fármaco quando submetido a determinadas condições de estresse sugerem que o fármaco é susceptível à fotólise

Controle de qualidade do minoxidil em laboratórios de pequeno porte / Quality control of minoxidil in compounding pharmacies

O minoxidil é um fármaco empregado no tratamento de hipertensão e da alopécia androgenética em homens e mulheres. Comercialmente, encontra-se sob a forma de comprimidos e de loção tópica. Observou-se que, para a preparação de loções com concentração superior a 2 por cento, tem sido empregado o sal do fármaco, devido à sua maior solubilidade. No entanto, esta molécula não possui patente e tampouco é citada na literatura. Objetivou-se propôr métodos alternativos para o controle de qualidade do minoxidil e confirmar a molécula descrita no laudo do fornecedor/fabricante do sulfato de minoxidil. Os testes qualitativos desenvolvidos (solubilidade, reações de cor/formação de precipitado e cromatografia em camada delgada) possibilitaram a identificação do minoxidil, bem como sua diferenciação do sulfato de minoxidil. Alterações na metodologia oficial do minoxidil permitiram a substituição da titulação potenciométrica pela detecção visual do ponto final, mantendo-se as características de exatidão e precisão. A molécula de sulfato de minoxidil foi devidamente caracterizada por testes de espectrometria de massas e espectrometria no infravermelho, sendo que a molécula informada pelo laboratório produtor foi confirmada. No entanto, os teores encontrados na análise quantitativa do fármaco não puderam ser confirmados, pela impossibilidade de comparação com métodos analíticos instrumentais, por inexistência de substância de referência.

Assay of sertraline in tablets and drug substance by liquid chromatography.

A high-performance liquid chromatography isocratic procedure was developed for the assay of sertraline in drug substance and tablets. The chromatographic system consists of a RP-8 column (125 x 4 mm, 5 microm), a mobile phase composed of acetonitrile and sodium phosphate buffer, pH 5.5 (7:3), flow rate of 1.0 ml.min(-1) and UV detection at 270 nm. The method validation yielded good results. The coefficient of variation varied between 0.19 and 1.04% and accuracy of 99.18% was found. Calibration curve was linear between 0.5 and 2.5 mg x ml(-1); its correlation coefficient was 0.9999.

Assay of fluoxetine hydrochloride by titrimetric and HPLC methods.

Two alternative methods were proposed to assay Fluoxetine Hydrochloride: a titrimetric method and another by HPLC using as mobile phase water pH 3.5: acetonitrile (65:35). These methods were applied to the determination of Fluoxetine as such or in formulations (capsules). The titrimetric method is an alternative for pharmacies and small industries. Both methods showed accuracy and precision and are an alternative to the official methods.

Affinity labeling of aryl sulfotransferase IV. Identification of a peptide sequence at the binding site for 3'-phosphoadenosine-5'-phosphosulfate.

2'-O-[(R)-Formyl(adenin-9-yl)-methyl]-(S)-glyceraldehyde 3'-triphosphate (also designated as ATP dialdehyde or ATPDA) was utilized as an affinity label for the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding site of an aryl sulfotransferase. The sulfotransferase employed in these studies was rat hepatic aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase, EC 2.8.2.9), for which a cDNA had been previously cloned and expressed in Escherichia coli and the resulting enzyme purified to homogeneity. ATPDA was a time-dependent irreversible inhibitor of the recombinant AST IV, and this inhibition was prevented by including either PAPS or adenosine 3',5'-diphosphate (PAP) in the incubation of AST IV with ATPDA. Experiments relating covalent binding of [2,8-3H]ATPDA with catalytic activity indicated that 1 nmol of the affinity label was bound per nmol of AST IV subunit. Incubation of [2,8-3H]ATPDA with the enzyme followed by reduction with sodium cyanoborohydride, proteolysis with trypsin, and separation of the resulting peptides by high pressure liquid chromatography yielded two labeled peptide fractions. Automated sequence analysis showed that both modified peptide fractions were derived from the same sequence in AST IV: 63-Leu-Glu-Lys-Cys-Gly-Arg-68. Both the sequencing results and examination of the two peptide fractions by matrix-assisted laser desorption ionization mass spectrometry indicated that the ATPDA affinity label was bound to the hexapeptide at both lysine 65 and cysteine 66. These affinity labeled amino acids are located within a region of sequence in AST IV that shows considerable homology with various sulfotransferases that possess diverse specificities for acceptor substrates, and this may provide insight into PAPS binding in other sulfotransferases.

Insulin-like growth factor binding protein (IGFBP)4 accounts for the connective tissue distribution of endothelial cell IGFBPs perfused through the isolated heart.

Insulin-like growth factor binding protein 4 (IGFBP4) was purified to homogeneity from conditioned media of bovine pulmonary artery endothelial cells and shown to have the N-terminal amino acid sequence DEAIHCPPCS, a sequence unique to IGFBP4. The IGFBP4 was separated into predominantly glycosylated and nonglycosylated fractions, with each fraction separately perfused through isolated, beating rat hearts. Both forms of IGFBP4 crossed the capillary boundary of the heart and distributed primarily in subendothelial connective tissue components with a connective tissue/cardiac muscle distribution ratio of 20:1 for the glycosylated fraction and 27:1 for the nonglycosylated fraction. Perfused IGFBP1, 2, 3, and IGF-I also crossed the capillary boundary but in contrast to IGFBP4, preferentially localized in cardiac muscle with a connective tissue/muscle ratio of approximately 1:3. We conclude that the connective tissue distribution previously reported for IGFBPs in conditioned media of pulmonary artery endothelial cells is due to IGFBP4.

Improved resolution of glycoproteins by chromatography with concanavalin A immobilized on microparticulate silica via temperature-programmed elution.

The ability of the column temperature to control elution in the affinity chromatography of glycoproteins (e.g., ovalbumin and horseradish peroxidase) on silica immobilized concanavalin A has been studied. Column temperature programs can be achieved by placing a small HPLC column within a commercial mobile phase preheater assembly. It is shown that elution of adsorbed proteins can be initiated by changing the column temperature without altering the chemical composition of the mobile phase. Further, due to the enhancement in the rate of dissociation of the sample from the ligand, the peaks are narrowed. The resolution can be controlled by changing the initial temperature, dwell time at the initial temperature, and the rate of change of the temperature program. Addition of a competitive binding agent to the mobile phase decreases the temperature needed to elute strongly retained proteins. The effect of heating the column through many thermal cycles is assessed by periodically measuring the retention of a small monosaccharide that binds to the immobilized concanavalin A. The effect of two different immobilization procedures (glutaraldehyde and carbonyldiimidazole), as well as the effect of including a monosaccharide in the mobile phase, on the stability of the column is easily monitored by thermal elution chromatography. The effect of column temperature on the above glycoproteins has been assessed through studies of enzyme activities and anion exchange and isoelectric focusing patterns before and subsequent to temperature-programmed elution affinity chromatography.