A novel HPLC assay for pentamidine: comparative effects of creatinine and inulin on GFR estimation and pentamidine renal excretion in the isolated perfused rat kidney.

PURPOSE 1. To develop and validate an analytical method for pentamidine (PTM) by reversed-phase HPLC. 2. To compare the effects of creatinine and inulin on PTM excretion in the isolated perfused rat kidney. METHODS The HPLC method utilized a base deactivated, 5 micro, C18 column and a mobile phase containing acetonitrile (24%) and 0.025 M monobasic phosphate buffer, pH 3.2 (76%). Mobile phase flow rate and UV detection wavelength were 1 mL/min and 270 nm, respectively. Sulfadiazine (SDZ) was used as the internal standard. The method was used to measure pentamidine in perfusate and urine samples generated from studies with the isolated perfused rat kidney (IPK) model. Perfusion experiments were conducted in the presence of two different GFR markers: creatinine and inulin (PTM dose 800 micro g). Both creatinine and inulin were assayed using colorimetric methods. RESULTS The HPLC assay is rapid, sensitive and reproducible. The method was validated over two standard concentration ranges: 0.1 to 1 micro g/mL, and 1 to 10 micro g/mL. In control (drug-naïve) IPK perfusions, creatinine clearance was approximately 15% greater than inulin clearance (0.80+/- 0.21 mL/min vs. 0.69+/-0.17 mL/min, p > 0.05). In the presence of PTM, however, creatinine clearance was reduced to 0.56+/-0.27 (p < 0.05 compared to control). Inulin clearance was not altered by PTM administration (0.76+/-0.26 mL/min). Cumulative urinary excretion of PTM (% dose) was 3.0+/-0.47% and 9.6+/-4.2% in the presence of creatinine and inulin, respectively. PTM clearance was significantly reduced (0.06+/-0.01 mL/min vs. 0.13+/-0.01 mL/min, p < 0.05) and % kidney accumulation significantly enhanced (66+/-4.7% vs. 37+/-9.7%, p < 0.05) by creatinine. CONCLUSIONS Creatinine overestimated GFR in the IPK. The altered renal excretion of PTM by creatinine is consistent with inhibition of PTM tubular secretion. Because of increased kidney accumulation, detrimental effects of PTM on renal function were observed. Based on these findings, creatinine should be used cautiously as an indicator of GFR in IPK experimentation.