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OBJECTIVE In order to reduce animal use and alleviate animal pain in in-life procedures, the potential application for micro-sampling in pharmacokinetic (PK) studies was investigated in mice.METHODS CD-1 or BALB/c mice were orally or intravenously administered with model compounds at the dose of 1 mg·kg-1 iv or 10 mg·kg-1 po.Blood samples were collected at 0.083 (only for iv dosing), 0.25, 0.5, 1,2, 4, 7 and 24 h post-dose (n =3 per time-point) via the saphenous vein for PK evaluation.In micro-sampling method, only 30 μl/sample of blood were collected using capillary tubes (Drummond Scientific).A 150 μl/sample of blood was collected using the normal sampling method.The LC/MS/MS system consists of a Shimadzu Nexera UHPLC, RP-18 column at 25℃, and AB SCIEX TRIPLE QUAD 5500.A binary gradient program with a flow rate of 0.8 ml· min-1 was used to analyze the plasma samples.Injection volume was 1 μl.Analytes and internal standards (0.2 iμmol· L-1 tolbutamide) were detected by the MS using multiple reaction monitoring in positive electrospray ionization mode (Analyte 1.5.2).RESULTS For each of the five compounds, plasma concentration vs.time curves and PK parameters (AUC, Cmax, Tmax, clearance, T1/2, bioavailability, etc.) were compared between the micro-sampling and normal sampling methods.Plasma PK profiles of each compound generated by both methods were well matched.In addition, compared to the normal sampling methods (e.g.tail-vein bleeding, retro-orbital puncture), micro-sampling via the saphenous vein significantly alleviated pain in animals.The volume of blood withdrawn was reduced by 80%, and the number of mice required for a PK study was decreased by 65%.CONCLUSION The micro-sampling method can replace normal blood sampling methods.Using this method, the animal welfare will be significantly improved in terms of alleviating animal pain and reducing animal use.