Hplc grade methanol and formic acid

Analytical grade Na₂CO₃ (≥99%), CaCl₂ (≥93%), Ca(NO₃)₂·4H₂O (≥99%), LiBr (≥99%), and LiCl (≥99%) (Sigma Aldrich, Steinheim, Germany). HPLC-grade methanol and formic acid (99%) (Merck, Modderfontein, South Africa) and trifluoroacetic acid (99%) (Sigma Aldrich, Steinheim, Germany).

Pure compounds of racemic ALO, pioglitazone (PIO), R-ALO and S-ALO (Figure 1) were received as gift sample from the Sun Pharmaceutical Industries Limited (Gurugram, Haryana, India) containing 99.82%, 99.59%, 99.39% and 99.65%, respectively as per the certificate of analysis obtained. HPLC grade methanol and formic acid was purchased from Merck (Darmstadt, Germany). ALO tablets (Perrigo Company plc, Dublin Ireland) of 25 mg strength were bought from a retail chemist shop. The C-18 solid phase extraction cartridges (150 mg, 6 mL) were purchased from Waters (India) Private Limited (DLF Tower-B, Jasola, New Delhi). Blood of 6 healthy rats from retroorbital plexus (behind the eyes) venous sinus bleed were collected and centrifuged. The pooled supernatant plasma (control) was then stored at -20°C for further use.