Determination of Rhodamine 123 in cell lysate by HPLC with visible wavelength detection.
Rhodamine 123 (R123) is widely used to quantify P-glycoprotein (P-GP) functional efflux activity in vitro. We developed a rapid and specific high-performance liquid chromatography (HPLC) method to quantify Rhodamine 123 for use in experimental cell culture studies. The R123 standards (2.5-250 ng/mL) and quality controls (QCs) (5, 75, 200 ng/mL) were prepared in cell lysis buffer consisting of 0.75% Triton 100X and 0.2% sodium chloride. The mobile phase consisted of acetonitrile, 1.5 mM tetrabutyl ammonium bromide in 20mM sodium acetate buffer (pH 4.0) (50:20:30) delivered at a rate of 1.0 mL/min. Samples (50 microl) were injected onto a C(18) reversed-phase HPLC column with detection at 500 nm. Analyte retention times were 1.4 and 4.3 min for R123 and internal standard (R6G), respectively. Intra- and inter-day coefficients of variation were < or = 4.2%. Samples were stable for at least three freeze-thaw cycles at room temperature for 24 and 48 h. This method was used to evaluate the functional activity of P-glycoprotein in renal tubule cell models including human kidney (HK-2), Madin-Darby canine kidney (MDCK) and multi-drug resistance gene-transfected MDCK cells (MDR1-MDCK).
Iqbal
Tahira
T
Renal Clinical Pharmacology Laboratory, Department of Pharmacy Practice and Science, University of Maryland, School of Pharmacy, 100 N. Penn St., AHB Rm. 540-D, Baltimore, MD 21201, USA.
Kinjo
Minori
M
Dowling
Thomas C
TC
D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004285:Dogs; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000166:cytology; D015203:Reproducibility of Results; D020112:Rhodamine 123 / Q000032:analysis*; D013053:Spectrophotometry / Q000379:methods*
D020112:Rhodamine 123