Development and full validation of an UPLC-MS/MS method for the quantification of the plant-derived alkaloid indirubin in rat plasma.
An UPLC-MS/MS method for the quantification of indirubin in lithium heparinized rat plasma was developed and validated according to current international guidelines. Indirubin was extracted from rat plasma by using Waters Ostro™ pass-through sample preparation plates. The method was validated with a LLOQ of 5.00ng/mL and an ULOQ of 500ng/mL. The calibration curve was fitted by least-square quadratic regression, and a weighting factor of 1/X was applied. Recoveries of indirubin and I.S. were consistent and ≥75.5%. Stability studies demonstrated that indirubin was stable in lithium heparinized rat plasma for at least 3 freeze/thaw cycles, for 3h at RT, for 96h in the autosampler at 10°C, and for 84days when stored below -65°C. Preliminary pharmacokinetic (PK) data were obtained from Sprague Dawley rats after intravenous administration of indirubin (2mg/kg b.w.) and blood sampling up to 12h after injection. PK parameters were determined by non-compartmental analysis. Indirubin had a half-life (t1/2) of 35min, and a relatively high clearance (CL) of 2.71L/h/kg.
Institute of Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.
Evelyn A
EA
Jähne
Department of Pharmaceutics, College of Pharmacy, University of Florida, 1345 Gainesville, FL, USA.
Chethan
C
Sampath
Department of Pharmaceutics, College of Pharmacy, University of Florida, 1345 Gainesville, FL, USA; School of Life Sciences Institute for Pharma Technology, Gründenstrasse 40, CH-4132 Muttenz, Switzerland.
Veronika
V
Butterweck
Institute of Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.
Matthias
M
Hamburger
Institute of Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland. Electronic address: mouhssin.oufir@unibas.ch.
Mouhssin
M
Oufir
D061605:Administration, Intravenous; D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents / Q000032:analysis* / Q000493:pharmacokinetics; D006207:Half-Life; D007202:Indicators and Reagents; D007211:Indoles / Q000097:blood / Q000493:pharmacokinetics; D057230:Limit of Detection; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
D004396:Coloring Agents; D007202:Indicators and Reagents; D007211:Indoles; C027185:indirubin
Indirubin; Method validation; Pharmacokinetics; Phospholipid removal plate; Rat plasma; UPLC-MS/MS