A milk coffee sample (10 mL) was added to a centrifuge tube (50 mL), followed by ethanol (10 mL), gently but thoroughly mixed and allowed to stand (15 min) until precipitation of solids occurs. The tube was centrifuged at 2000 rpm for 3 min. The supernatant (2 mL) was placed in a 13 mm × 100 mm culture tube and the ethanol was evaporated under a stream of nitrogen at room temperature. To the resultant residue of around 1 mL was added approximately 2 mL of deionized water followed by 0.75 mL of 0.1 M H3PO4, 0.75 mL 0.05 M sodium dodecyl sulphate and 0.5 mL dehydroacetic acid as internal standard (500 ppm) and finally made up to 5.0 mL with deionized water. The solutions were mixed thoroughly and filtered through a polyvinylidenedifluoride (Millex HV) membrane (0.45 μm) before analysis.