title: Does the commonly used pH-stat method with back titration really quantify the enzymatic digestibility of lipid drug delivery systems? A case study on solid lipid nanoparticles (SLN).
abstract: Enzymatic digestion of lipid drug carriers is very important. Commonly, pancreatin induced formation of fatty acids is monitored by the pH-stat method, which provides a fast, but unspecific readout. However, according to the literature, the pKa values of long chain fatty acids are strongly dependent on the local environment and might vary between 4.2 and 10.15. The high pKa values would lead to an incomplete detection of the lipid digestion and false results. In order to investigate these issues in more detail, we produced cetyl palmitate solid lipid nanoparticles (CP-SLN) stabilized with poloxamer 188 or polysorbate 80. The digestion of CP-SLN was investigated by two different and independent readouts. A HPTLC assay was used in addition to the pH-stat method (with or without back titration). An incomplete digestion of CP-SLN was observed with all methods. Partial digestion of polysorbate 80 contributed to the formation of fatty acids. Depending on the investigated system and the experimental conditions (FaSSIF or FeSSIF) the results of both readout methods were comparable or not. For example, in FeSSIF conditions, the values detected by HPTLC were roughly twice as high as the pH-stat results. Our findings on solid lipids agree with data from Helbig et al. on lipid emulsions, where a gas chromatography method detected much higher values than the pH-stat assay (Food Hydrocoll. 28 (2012) 10-19). The results of our pH-stat experiments with back titration at different pH values showed increased values for fatty acids from pH 7.5 to pH 10. The values obtained by back titration at high pH values (pH 9 or higher) did exceed the digestion values measured by HPTLC. Therefore, we conclude that the pH-stat method might give the same results as more specific reference methods, but it might also both under- (without back titration) or overestimate (with back titration) the enzymatic digestion of lipid drug delivery systems. A further outcome of our study was the proof that lipase is the main enzyme involved in the digestion of the solid wax cetyl palmitate, because CP-SLN loaded with the inhibitor orlistat were not digestible and gave similar values as the corresponding control samples. In summary, our experimental results confirm the theoretical considerations (based on published pKa values) that the pH-stat method will not detect all fatty acids quantitatively. The very strong impact of the local environment on the pKa value of long chain fatty acids is a serious limitation and might lead to misleading interpretations.
authors: Institute of Pharmacy, Faculty of Biosciences, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, D-06120 Halle (Saale), Germany. Electronic address: martha.heider@pharmazie.uni-halle.de.
authors: Martha
authors: M
authors: Heider
authors: Biocenter MLU Halle-Wittenberg, Weinbergweg 22, 06120 Halle (Saale), Germany. Electronic address: gerd.hause@biozentrum.uni-halle.de.
authors: Gerd
authors: G
authors: Hause
authors: Institute of Pharmacy, Faculty of Biosciences, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, D-06120 Halle (Saale), Germany. Electronic address: http://pharmtech.pharmazie.uni-halle.de.
authors: Karsten
authors: K
authors: Mäder
mesh_terms: D001647:Bile Acids and Salts / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D004337:Drug Carriers / Q000737:chemistry; D016503:Drug Delivery Systems; D005227:Fatty Acids / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007783:Lactones / Q000737:chemistry; D008049:Lipase / Q000737:chemistry; D008055:Lipids / Q000737:chemistry*; D008066:Lipolysis; D008823:Micelles; D008854:Microscopy, Electron; D046529:Microscopy, Electron, Transmission; D050997:Nanomedicine / Q000379:methods*; D053758:Nanoparticles / Q000737:chemistry*; D000077403:Orlistat; D010168:Palmitates / Q000737:chemistry; D010179:Pancreas / Q000201:enzymology; D010194:Pancreatin / Q000737:chemistry; D010316:Particle Size; D020442:Poloxamer / Q000737:chemistry; D011136:Polysorbates / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry; D013696:Temperature
chemical_list: D001647:Bile Acids and Salts; D004337:Drug Carriers; D005227:Fatty Acids; D007783:Lactones; D008055:Lipids; D008823:Micelles; D010168:Palmitates; D011136:Polysorbates; D013501:Surface-Active Agents; D020442:Poloxamer; C006957:cetyl palmitate; D010194:Pancreatin; D000077403:Orlistat; D008049:Lipase
keywords: Back titration; Biorelevant dissolution media; Cetyl palmitate; Digestion; HPTLC; Orlistat; Pancreatic lipase; Pancreatin; SLN; Solid lipid nanoparticles; pH-stat