An improved micro-scale liquid-chromatographic assay for piperacillin in plasma and urine

An accurate, sensitive, and specific liquid-chromatographic method is described for measuring piperacillin in plasma and urine. Plasma samples deproteinized with two volumes of acetonitrile containing 1.2 mg of the internal standard, p-nitrobenzene sulfonamide, per liter are centrifuged. The clear supernate is evaporated under nitrogen, and the residue is reconstituted in 50 microL of the mobile phase (32/68 by vol acetonitrile/water, adjusted to pH 2.5 with 85% phosphoric acid), of which 10 microL is injected onto a reversed-phase (C-18) column. Urine samples are diluted 10-fold with distilled water, an equal volume of acetonitrile containing 3 mg of the internal standard per liter is added, and 20 microL is chromatographed. Stability studies indicate that storage conditions are critical for both plasma and urine. Piperacillin in plasma is stable at -70 degrees C for at least six weeks, but 100% of it is degraded during the same time at -20 degrees C. Piperacillin in urine is also stable at -70 degrees C for six weeks, but 20% is degraded during six weeks at -20 degrees C.