Characterisation and quantification of medium chain and long chain triglycerides and their in vitro digestion products, by HPTLC coupled with in situ densitometric analysis

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Abstract

The development of new and simple high performance thin layer chromatography (HPTLC) assays for the quantification of medium chain triglycerides (MCT, tricaprylin) and long chain triglycerides (LCT, triolein) and their lipolytic products, bile salts (BS) and phospholipids (PL) are described. Different classes of lipids (PL, BS, fatty acids, monoglycerides, diglycerides, and triglycerides) were separated on a single silica gel 60 HPTLC plate by Automated Multiple Development (AMD) methods using a Camag AMD 2. Post-chromatographic staining of long chain lipids (triolein, diolein, monoolein, and oleic acid), PL and BS with a solution of copper sulphate-phosphoric acid and medium chain lipids (tricaprylin, dicaprylin, monocaprylin, and caprylic acid) with a solution of ammonium molybdate-perchloric acid allowed visualisation of the lipids. Lipids were quantified by in situ spectrodensitometric measurements using a Camag TLC scanner 3. The intra- and inter-assay accuracy was between 83 and 115% and the assay was precise to within a CV of less than 20% over a range of 0.1–1 and 5–50 μg for long chain lipids and medium chain lipids, respectively. The methods have been employed to study the kinetics of triolein and tricaprylin lipolysis in an in vitro lipid digestion model commonly used to assess the digestibility of novel oral lipid-based formulations.

Introduction

Whilst there has been a great deal of interest in the use of lipid-based oral delivery systems as a strategy for bioavailability enhancement for poorly water soluble drugs [1], [2], [3], [4], [5], [6], [7], [8], [9], there are relatively few examples of commercial lipid-based formulations. Constraints associated with the development of successful lipid-based formulations include a lack of understanding of the mechanisms by which lipids improve absorption, and the lack of an in vitro model that is predictive of bioavailability and therefore able to be used for rapid and systematic assessment of potential lipid-based formulations.

Triglycerides (TG) consisting of medium chain length fatty acids (MCT; C8–C14) and triglycerides of long chain fatty acids (LCT; C16–C22) are often considered for use in lipidic formulations. MCT and LCT may enhance the absorption of poorly water soluble drugs by their actions on gastric transit, intestinal permeability or drug metabolism, but are perhaps most widely used to enhance drug solubilisation and dissolution in the gastrointestinal tract (GIT) [10], [11]. TG lipids and their digestion products may enhance drug solubilisation and dissolution by stimulating bile salt (BS) and phospholipid (PL) secretion into the gastrointestinal (GI) lumen and by enhancing the solubilisation capacity of endogenous BS/PL mixed micelles in the intestine by intercalation into the micelle structures.

After oral administration, hydrolysis of TG by lingual and gastric lipase and most importantly by the pancreatic lipase/colipase complex, proceeds as a two-step reaction. Firstly, hydrolysis of TG yields a single fatty acid (FA) and diglyceride (DG). Secondly, the DG is hydrolysed to produce a second FA and the corresponding 2-monoglyceride (2-MG). Although 2-monoglyceride may undergo isomerisation to 1-monoglyceride (1-MG), which may then be hydrolysed to yield a third FA and glycerol, this process is generally believed to be limited in vivo [12].

Numerous studies of the physicochemical characteristics of lipid digestion products have been conducted in humans [13], [14], [15], [16], [17], [18], [19], [20]. However, from a formulation standpoint, the fate of a co-administered drug as digestion of an oily vehicle progresses remains a key issue. The interaction between the co-administered lipid and drug as the formulation is exposed to, and digested and dispersed by the GI environment, will depend on the type of lipid involved and the physicochemical properties of the particular drug. Elucidation of the digestion characteristics of different lipids and lipid vehicles may, therefore, provide a framework for the development of an in vitro model in which the factors that dictate drug distribution into the various phases of the GI contents, and potentially, therefore, dictate the extent of absorption, may be probed. A prerequisite for the establishment of such a model is a reliable, accurate and sensitive analytical method for the quantification of MCT and LCT (and their digestion products), PL and BS in the lipid digest.

The progress of triglyceride lipolysis is generally monitored in vitro using an automated pH-stat titration apparatus [21], [22]. A limitation of this methodology is that it quantifies the rate and extent of digestion indirectly via titration of the FA produced. Detection and quantification of the various classes of lipolytic products (i.e. parent TG, DG, MG and FA), however, is complicated by their lack of chromophoric groups. Quantification usually proceeds via extraction from the reaction mixture, separation of lipid classes by thin layer chromatography (TLC), scraping of the adsorbed lipid from the TLC plates, transesterification with methanol, and quantification of FA methyl esters by gas capillary (GC) chromatography [23], [24]. Other techniques utilised for the quantification of lipid digestion products include the use of radiolabelled lipids (although lipid extraction from the silica gel is still required for counting by liquid scintillation [25], [26], [27], [28]), TLC coupled with flame ionisation detection (FID) and most recently high performance liquid chromatography with mass spectrometry detection (HPLC-MS) [29], [30], [31]. HPLC-MS has some advantages, particularly in terms of selectivity, but has not found extensive application in lipid analysis, in part as a result of high cost and limited availability and also as a result of variability in molecular ion fragmentation patterns [31]. As a result, in situ densitometry TLC has increasingly been employed for the quantitation of LCT and its digestion products [16], [17], [32], however to this point, application to the assay of medium chain lipids has been limited.

The present study describes for the first time, high performance thin layer chromatography (HPTLC) techniques for quantifying both MCT and LCT (and their digestion products), together with PL and BS. In these studies, triolein, tricaprylin, egg phosphatidylcholine (PC) and Na taurodeoxycholate (NaTDC) were selected as model TG, PL and BS, respectively. The methodology employs a simple acidification and dilution procedure without an extraction step for sample preparation. The method has been employed to study the kinetics of in vitro lipolysis of tricaprylin and triolein by pancreatic lipase.

Section snippets

Materials

Oleic acid, 2-monoolein, 1,2-diolein, triolein, and Na taurodeoxycholate (NaTDC) were products from Sigma Chemical Co. (MO, USA). Tributyrin, tricaprylin, 1,3-dicaprylin, 1-monocaprylin and Trizma® maleate were also purchased from Sigma. Two grades of l-α-lecithin (l-α-phosphatidylcholine, PC) were purchased from Sigma. Type X-E (approximately 60% pure PC, from dried egg yolk) was used to prepare mixed micellar PC/NaTDC solutions for digestion experiments and type XI-E (approximately 99% pure

HPTLC assay characteristics

A six-step development program with differing solvent compositions was required to separate the long chain lipids (triolein, diolein, monoolein and oleic acid), PC and NaTDC in a single sample. The separation of medium chain lipids (tricaprylin, dicaprylin, monocaprylin and caprylic acid) was optimised using a 4-step development method. Fig. 1 presents scanned sections of HPTLC plates illustrating the separation of different long chain lipids, PC and NaTDC (panel A) and medium chain lipids

Conclusions

HPTLC analytical methods for the quantification of MCT and LCT (and their digestion products), PC and NaTDC and application of these methods to in vitro lipid digestion studies have been described. The HPTLC assays are simple, rapid and represent an alternative to previous TLC-gas chromatographic methods [23], [24], radiolabelled assay techniques [27] and HPLC-MS [31]. Validation experiments have shown the assays to be accurate and precise. Importantly, since an extraction step is not employed,

Acknowledgements

This work was funded in part by GlaxoWellcome, UK. L. Sek gratefully acknowledges scholarship support provided by an Australian Postgraduate Award.

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