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DESENVOLVIMENTO DE MÉTODOS ANALÍTICOS EM UHPLC-MS PARA QUANTIFICAÇÃO DE IMPUREZAS EM SUBSTÂNCIAS FARMACÊUTICAS
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Na presente dissertação recorreu-se à técnica de cromatografia líquida de ultra-alta efi-
ciência acoplada à espetrometria de massa (UHPLC-MS) para desenvolvimento de métodos
analíticos e quantificação de espécies químicas, ácido etilenodiaminotetracético (EDTA), ácido
oxálico e azodicarboxilato de dibenzilo (DBAD), em produtos farmacêuticos.
A impossibilidade de utilização de detetores tradicionais, como os de absorvância (UV-
Vis) em compostos químicos que não apresentam grupos cromóforos ativos nessa gama, evi-
dencia a necessidade de recorrer a abordagens alternativas. O recurso a detetores por espetro-
metria de massa (MS) tende a melhorar a seletividade e a sensibilidade exigidas para quanti-
ficação vestigial, embora possam persistir limitações associadas à complexidade do sistema
analítico e da matriz.
A otimização dos parâmetros cromatográficos revelou-se determinante para o sucesso
do desenvolvimento metodológico e adaptação a uma ampla variedade de compostos. Entre
os fatores avaliados destacam-se a composição da fase móvel e do diluente, o volume de inje-
ção e o recurso a coluna cromatográfica ou apenas injeção direta com pré-filtro.
Os resultados demonstraram que os teores de EDTA e de ácido oxálico se encontraram
abaixo do limite de quantificação determinado. Na oxitetraciclina dihidratada, o EDTA variou
entre 0,0019 e 0,011 % (m/m) e o ácido oxálico entre 0,0024 e 0,0051 % (m/m), consoante o lote.
Já na oxitetraciclina base, o ácido oxálico foi quantificado em 0,018 % (m/m).
A quantificação do DBAD no 7-HT não foi possível, pois o método não é específico para
este composto, inviabilizando uma quantificação precisa. No cloreto de minociclina foi quan-
tificado cerca de 0,0069 % (m/m) de DBAD.
Relativamente ao desempenho do método, os limites de deteção (LoD) e quantificação
(LoQ) variaram entre 0,07 e 0,48 ppm e 0,22 e 1,46 ppm, respetivamente. As curvas de calibra-
ção apresentaram linearidade dentro das gamas de concentração testadas com coeficientes de
determinação superiores a 0,99, confirmando a fiabilidade do método desenvolvido.
In the present dissertation, ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) was employed for the development of analytical meth- ods and the quantification of chemical species, namely ethylenediaminetetraacetic acid (EDTA), oxalic acid, and dibenzyl azodicarboxylate (DBAD), in pharmaceutical products. The impossibility of using traditional detectors, such as absorbance detectors (UV-Vis), in chemical compounds that lack active chromophore groups within this range highlights the need for alternative analytical approaches. The use of mass spectrometry (MS) detectors tends to improve the selectivity and sensitivity required for trace-level quantification, although lim- itations associated with the complexity of the analytical system and the matrix may persist. The optimization of chromatographic parameters proved to be crucial for the methodo- logical development and adaptation to a wide variety of compounds. Among the evaluated factors, the composition of the mobile phase and the diluent, the injection volume, and the choice between using a chromatographic column or direct injection with a pre-filter were par- ticularly relevant. The results demonstrated that the levels of EDTA and oxalic acid were below the estab- lished quantification limit. In oxytetracycline dihydrate, EDTA ranged from 0.0019 to 0.011 % (m/m), while oxalic acid varied between 0.0024 and 0.0051 % (m/m), depending on the batch. In oxytetracycline base, oxalic acid was quantified at 0.018 % (m/m). The quantification of DBAD in 7-HT was not possible, as the method was not specific for this compound, preventing accurate determination. In minocycline chloride, approxi- mately 0.0069 % (m/m) of DBAD was quantified. Regarding method performance, the detection (LoD) and quantification (LoQ) limits ranged between 0.07 and 0.48 ppm and 0.22 and 1.46 ppm, respectively. Calibration curves exhibited linearity within the tested concentration ranges, with determination coefficients (R²) above 0.99, confirming the reliability of the developed method.
In the present dissertation, ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) was employed for the development of analytical meth- ods and the quantification of chemical species, namely ethylenediaminetetraacetic acid (EDTA), oxalic acid, and dibenzyl azodicarboxylate (DBAD), in pharmaceutical products. The impossibility of using traditional detectors, such as absorbance detectors (UV-Vis), in chemical compounds that lack active chromophore groups within this range highlights the need for alternative analytical approaches. The use of mass spectrometry (MS) detectors tends to improve the selectivity and sensitivity required for trace-level quantification, although lim- itations associated with the complexity of the analytical system and the matrix may persist. The optimization of chromatographic parameters proved to be crucial for the methodo- logical development and adaptation to a wide variety of compounds. Among the evaluated factors, the composition of the mobile phase and the diluent, the injection volume, and the choice between using a chromatographic column or direct injection with a pre-filter were par- ticularly relevant. The results demonstrated that the levels of EDTA and oxalic acid were below the estab- lished quantification limit. In oxytetracycline dihydrate, EDTA ranged from 0.0019 to 0.011 % (m/m), while oxalic acid varied between 0.0024 and 0.0051 % (m/m), depending on the batch. In oxytetracycline base, oxalic acid was quantified at 0.018 % (m/m). The quantification of DBAD in 7-HT was not possible, as the method was not specific for this compound, preventing accurate determination. In minocycline chloride, approxi- mately 0.0069 % (m/m) of DBAD was quantified. Regarding method performance, the detection (LoD) and quantification (LoQ) limits ranged between 0.07 and 0.48 ppm and 0.22 and 1.46 ppm, respectively. Calibration curves exhibited linearity within the tested concentration ranges, with determination coefficients (R²) above 0.99, confirming the reliability of the developed method.
Descrição
Palavras-chave
Espetrometria de Massa Cromatografia Líquida de Ultra-Alta Eficiência Limites de Deteção e Quantificação Curvas de Calibração Condições Cromatográficas