Impact of excipients in optimum drug extraction conditions during analysis of fed state gastric media

Purpose The objective of the study was to determine the role of excipients in the % drug extracted from milk based biorelevant media which simulate the in vivo properties of the fed state stomach; [i.e. Fed State Simulated Gastric Fluid (FeSSGF)] when protein precipitation is selected as an extraction technique. The role of selection of protein precipitation reagent for drug extraction in the presence of excipients inside commercial formulations and the effect of each excipient in the drug amount recovered were assessed. Methods Appropriate quantity of drug powder (30- 200 mg) was pulverised with related excipient using mortar and pestle (3 min) at a ratio according to the range used in the formulations commercially available (Table 1). The percentage of drug was arbitrarily set as 30% w/w of the formulation. Three samples were taken from the centre, middle and edge of the mortar, placed in volumetric flasks which were then filled with FeSSGF to concentrations equivalent to the solubility of each drug in the medium. The flasks were left to equilibrate for 90 min at 37 °C. Protein precipitation: In 1 mL of FeSSGF, each reagent (methanol, acetonitrile, or trichloroacetic acid (10% w/v)) was added at a 1:2 ratio, vortexed (30 sec), centrifuged (8000 rpm, 4 °C, 15 min), filtered (RC 0.45 μm) and injected in the HPLC. Drug recovery values were expressed as: [(amount of drug in drug-excipient FeSSGF solution)/ amount of drug in standard FeSSGF solution] × 100% based on drug’s calibration standards in FeSSGF after precipitation with each reagent. Results In all cases (three drugs and for all excipients), extraction with acetonitrile gave slightly higher recovery values than with methanol, which in turn gave higher values than the use of trichloroacetic acid with the difference being statistically significant at the 95% confidence level for the majority of excipients (p < 0.05). In the case of paracetamol, none of the excipients present prohibited the effective extraction of the drug, with the exception of HPMC, under the presence of which, recovery was lower, but still > 80%. In the extraction of propafenone HCl and furosemide, the presence of HPMC and magnesium stearate in the solution inhibited the effective extraction of drug giving recovery values < 60% in the presence of magnesium stearate and values down to 20% in the presence of HPMC. These lower recovery values can be attributed to the forces developed between the drugs and the excipients during their mixing and the different extracting ability of each reagent when the drug is dissolved in a milk based medium in the presence of other compounds interfering in its complete extraction. Conclusion The presence of certain excipients was shown to affect the percentage of drug recovered from milk based fed state gastric biorelevant media, when protein precipitation was used as extraction technique, indicating their possible interference when present in formulations. The choice of the protein precipitation reagent was proven crucial for maximising the % recovery of the drug in such media.