Practical Considerations in the Pharmaceutical Analysis of Methyl and Hexyl Ester Derivatives of 5-Aminolevulinic Acid

Photodynamic therapy (PDT) using topical 5-aminolevulinic acid (ALA), a water-soluble precursor of the potent endogenous photosensitiser, protoporphyrin IX (PpIX), is a treatment and diagnostic tool for premalignant and malignant skin cancers. However, to improve drug delivery to deeper skin lesions, more lipophilic ALA esters have been investigated. Owing to the necessity in drug delivery research for efficient and validated assays for ALA esters in solution, this paper aims to describe optimised protocols to quantify the methyl and hexyl esters of ALA. ALA esters were derivatised using acetyl acetone and formaldehyde reagents and analysed using reversed phase HPLC. For the first time, the significance of ALA-impurities in ester samples has been highlighted. Furthermore, it was shown that for a given concentration, peak areas obtained for ALA-esters were significantly smaller than those obtained for ALA (p < 0.05). This may be due to parent drug undergoing the derivatisation reaction more efficiently or because the ALAderivate is inherently more fluorescent than the ester derivatives. The method was optimised to give acceptable intraand inter-day variability (CV values < 5%) and the limits of detection and quantification were determined for both drugs. The validated methods were used to determine the release profiles of ALA, m-ALA and h-ALA from an o/w cream formulation. The percentage of drug loading released after five hours across a model membrane was in the order of ALA (45.2%) > m-ALA (38.3%) > h-ALA (33.9%). These findings may explain why, historically, some of the benefits seen with ALA-esters using cell culture models have not been demonstrated in vivo.