title: Development of an on-line extraction turbulent flow chromatography tandem mass spectrometry method for cassette analysis of Caco-2 cell based bi-directional assay samples.
abstract: Caco-2 cells are frequently used for screening compounds for their permeability characteristics and P-glycoprotein (P-gp) interaction potential. Bi-directional permeability studies performed on Caco-2 cells followed by analysis by HPLC-UV or LC-MS method constitutes the "method of choice" for the functional assessment of efflux characteristics of a test compound. A high throughput LC-MS/MS method has been developed using on-line extraction turbulent flow chromatography coupled to tandem mass spectrometric detection to analyze multiple compounds present in Hanks balanced salt solution in a single analytical run. All standard curves (P-gp substrates: quinidine, etoposide, rhodamine 123, dexamethasone, and verapamil and non-substrates: metoprolol, sulfasalazine, propranolol, nadolol, and furosemide) were prepared in a cassette mode (ten-in-one) while Caco-2 cell incubations were performed both in discreet mode and in cassette mode. The standard curve range for most compounds was 10-2500 nM with regression coefficients (R(2)) greater than 0.99 for all compounds. The applicability and reliability of the analysis method was evaluated by successful demonstration of efflux ratio greater than 1 for the P-gp substrates studied in the Caco-2 cell model. The use of cassette mode analysis through selected reaction monitoring mass spectrometry presents an attractive option to increase the throughput, sensitivity, selectivity, and efficiency of the model over discreet mode UV detection.
authors.affiliation: Pharmaceutical Candidate Optimization, Pharmaceutical Research Institute, Bristol-Myers Squibb, P.O. Box 5400, Princeton, NJ 08543-5400, USA. james.smalley@bms.com
authors.forename: James
authors.initials: J
authors.lastname: Smalley
authors.forename: Pathanjali
authors.initials: P
authors.lastname: Kadiyala
authors.forename: Baomin
authors.initials: B
authors.lastname: Xin
authors.forename: Praveen
authors.initials: P
authors.lastname: Balimane
authors.forename: Timothy
authors.initials: T
authors.lastname: Olah
mesh_terms: D001692:Biological Transport; D018938:Caco-2 Cells; D002463:Cell Membrane Permeability; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D003907:Dexamethasone / Q000493:pharmacokinetics; D005047:Etoposide / Q000493:pharmacokinetics; D005665:Furosemide / Q000493:pharmacokinetics; D006801:Humans; D008790:Metoprolol / Q000493:pharmacokinetics; D009248:Nadolol / Q000493:pharmacokinetics; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011433:Propranolol / Q000493:pharmacokinetics; D011802:Quinidine / Q000493:pharmacokinetics; D020112:Rhodamine 123 / Q000493:pharmacokinetics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D012460:Sulfasalazine / Q000493:pharmacokinetics; D014700:Verapamil / Q000493:pharmacokinetics
chemical_list: D004364:Pharmaceutical Preparations; D020112:Rhodamine 123; D012460:Sulfasalazine; D009248:Nadolol; D005047:Etoposide; D005665:Furosemide; D003907:Dexamethasone; D011433:Propranolol; D014700:Verapamil; D008790:Metoprolol; D011802:Quinidine