Trifluoroacetic acid tfa for hplc

Ammonium formate (reagent grade; 97%), ammonium bicarbonate (reagent grade), and bovine serum albumin (BSA; ≥98%) were purchased from Fluka Biochemica (Buchs, Switzerland). Trifluoroacetic acid (TFA) for HPLC (>99.9%), acetic anhydride (≥97%), ammonia solution (25%) for LC-MS, iodoacetamide (IAA; reagent grade), dithiothreitol (DTT; reagent grade), and triisopropylsilane (TIS; 98%) were obtained from Sigma Aldrich (St. Louise, WA, USA). Formic acid (FA; 98%) and acetic acid (99%) were purchased from Lach-Ner (Neratovice, Czech Republic). Methanol and acetonitrile (both OPTIMA LC-MS) were obtained from Fisher Chemical (Waltham, MA, USA). Sequencing grade chymotrypsin and Glu-C were purchased from Roche Diagnostics (Mannheim, Germany). Fmoc Arg(Pbf) WANG resin, Fmoc Lys(Boc) WANG resin, Fmoc Phe WANG, and Fmoc Glu(OtBu) WANG for peptide standards synthesis were purchased from Iris Biotech (Marktredwitz, Germany).

A preparative 2D-mini gel (7 cm IPG strip, GE Healthcare, Chicago, IL, USA) stained Coomassie (Brilliant Blue G, Sigma Aldrich, Darmstadt, Germany), containing an equal amount of each non-labeled individual sample mixed with 10 µg of IS labeled sample, in a total of 75 µg of protein, was performed for further spot cut-off for mass spectrometry (MS) protein identification. The introduction of some labeled sample into preparative gel facilitates gel match with analytical gels for spot location and picking for MS analysis. The protein spot of interest was cut off from the gel and trypsin digested, as we previously described [16 (link),17 (link)]. Tryptic peptides, suspended in 50% (v/v) ACN and 0.1% (v/v) trifluoroacetic acid (TFA, for HPLC, ≥99.0%, Sigma Aldrich, Darmstadt, Germany) and submitted to sonication in an ultrasonic bath during 15 min, were directly applied on a 100-well matrix-assisted laser desorption/ionization (MALDI) plate with 5 mg/mL α-cyano-4-nydroxycinnamic acid (α-CHCA, 1:1) prepared in 0.1% TFA/60% ACN (Merck, Darmstadt, Germany) (v/v) and allowed to co-crystallize at RT. Peptides were analysed on an Applied Biosystems SCIEX 4800 Plus MALDI Proteomics Analyser with time-of-flight/time-of-flight (TOF/TOF) ion optics exactly as described [18 (link)]. The identified proteins are shown in Table S2 (Supplementary Materials).

Nifedipine was purchased from Alfa Aesar (Lancashire, UK). Lactose monohydrate and magnesium stearate were obtained from Sigma-Aldrich (Dorset, UK), whilst microcrystalline cellulose (MCC) as Pharmacel 102 was obtained from DFE Pharma (Goch, Germany). Colloidal silica dioxide (Aerosil 200) was obtained from Evonik Industries (Essan, Germany) and grades of hydroxypropyl methylcellulose (HPMC) as METHOCEL™ were obtained from Colorcon (Dartford Kent, UK). Market extended release Nifedipine preparations including Nifedipress MR 10 and Adalat LA 30 were obtained from Dexcel Pharma (Daventry, UK) and Bayer (Reading, UK).
Dissolution media comprised of a phosphate/citrate buffer with Sodium lauryl sulphate (SLS). Dibasic sodium phosphate and citric acid were purchased from Acros Organics (Morris Plains, NJ, USA), whereas phosphoric acid and SLS were obtained from Sigma-Aldrich (Dorset, UK).
For sample analysis using HPLC: Trifluoroacetic acid (TFA) for HPLC (≥99.0%), was obtained from Sigma-Aldrich (Dorset, UK), whilst acetonitrile and methanol were purchased from Fisher Scientific (London, UK).