Lichrospher 100 rp 18 5 μm

NRG was assayed by HPLC using a system consisting of a quaternary LC-10AT VP pump equipped with UV/VIS detector, SPD-10AVP column oven (Shimadzu), a Rheodyne injector, and chromatography data system software (CLASS-VP Ver 6.14 SP1). Chromatographic separation was carried out on column (LiChrospher®100 RP-18 (5 μm), Merck, Darmstadt, Germany) with a mobile phase consisting of a 7:3 ratio of methanol and water with 0.1 % glacial acetic acid at ambient temperature (25 ± 0.5°C) having a flow rate of 1 ml/min. Samples (20 μl) were filtered through microfilters having 0.45 μm pore size and Rheodyne injector was used for injecting the samples which were then examined at 289 nm [22 (link)].

Insulin in the SFD particles was quantified by HPLC using a Waters 2695 HPLC System equipped with a 996 photodiode array detector (Waters, Milford, MA, USA) according to the human insulin monograph of the European Pharmacopoeia 9.0. Analysis was carried out with a 250 mm RP-C18 column (LiChrospher^® 100 RP-18 5 μm, Merck, Darmstadt, Germany). The flow rate was 1 mL/min, the injection volume was 100 μL, and the UV detection wavelength was 214 nm. Each sample was analyzed in triplicate. Size exclusion chromatography was performed with a Zorbax GF-250 column (Agilent Technologies, Santa Clara, CA, USA), where the mobile phase was 0.2 M pH 7.0 phosphate buffer containing 0.005% sodium azide at a flow rate of 1.0 mL/min. The UV detection wavelength was 275 nm. Each sample was analyzed in triplicate in comparison with a chromatogram of a reference fresh insulin sample.

A 10 μL sample was directly injected in the equipment and separated using a LiChrospher^® 100 RP-18 (5 μm) (Merck) analytical column with a flow of 0.35 ml min^-1 and oven temperature of 30°C. Solvents used for separation were: solvent A: formic acid 0.1% v/v. Solvent B: methanol.