一种新的喷他脒高效液相色谱分析法：肌酐和菊粉对离体灌注大鼠肾脏中肾小球滤过率估计及喷他脒肾排泄的比较影响

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.

作者信息

Poola Nagaraju R, Bhuiyan Dider, Ortiz Stephan, Savant Ishani A, Sidhom Madiha, Taft David R, Kirschenbaum Harold, Kalis Michelle

机构信息

Division of Pharmaceutics and Industrial Pharmacy, Long Island University, Brooklyn, New York, USA.

出版信息

J Pharm Pharm Sci. 2002 May-Aug;5(2):135-45.

PMID:12207866

Abstract

PURPOSE

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.

摘要

目的

开发并验证一种采用反相高效液相色谱法测定喷他脒（PTM）的分析方法。2. 比较肌酐和菊粉对离体灌注大鼠肾脏中PTM排泄的影响。

方法

高效液相色谱法使用一根5μm的碱钝化C18柱和一种流动相，该流动相包含乙腈（24%）和0.025M磷酸二氢盐缓冲液，pH值为3.2（76%）。流动相流速和紫外检测波长分别为1mL/min和270nm。磺胺嘧啶（SDZ）用作内标。该方法用于测量来自离体灌注大鼠肾脏（IPK）模型研究产生的灌注液和尿液样本中的喷他脒。在两种不同的肾小球滤过率标志物存在的情况下进行灌注实验：肌酐和菊粉（PTM剂量800μg）。肌酐和菊粉均采用比色法测定。

结果

高效液相色谱测定法快速、灵敏且可重复。该方法在两个标准浓度范围内得到验证：0.1至1μg/mL和1至10μg/mL。在对照（未用药）的IPK灌注中，肌酐清除率比菊粉清除率大约高15%（0.80±0.21mL/min对0.69±0.17mL/min，p>0.05）。然而，在存在PTM的情况下，肌酐清除率降至0.56±0.27（与对照相比，p<0.05）。给予PTM后菊粉清除率未改变（0.76±0.26mL/min）。在存在肌酐和菊粉的情况下，PTM的累积尿排泄率（%剂量）分别为3.0±0.47%和9.6±4.2%。肌酐使PTM清除率显著降低（0.06±0.01mL/min对0.13±0.01mL/min，p<0.05），并且使肾脏累积率显著提高（66±4.7%对37±9.7%，p<0.05）。