The fluid chromatograph mass spectrometer contained a 14 AM degasser, Shimadzu 10ADvp Pump, a higher pressure equipment, a CTO 10Avp column oven and a Shimadzu 10ATvp autoinjector built with an electrospray ionization probe. Extraction of midazolam and 1 hydroxymidazolam was done with 0. 2 ml plasma, diluted TGF-beta with 30 l of 1 M NaOH solution and 10 l of diazepam solution, to which 1 ml of ethyl acetate was added. The samples were centrifuged, evaporated and reconstituted in the mobile phase. The gradient elution, using two mobile phases: 0. 01% of methanol and ammonium acetate, was as follows: 70A : 30B to 5A : 95B in 0. 5 min, then 5A : 95B for 1 min, next 5A : 95B to 70A : 30B and for 6 min. The ow rate was 0. 2 ml min1. Separation by HPLC on a column was followed by mass spectrometric detection. This analysis had a diminished chemical library price limit of quantitation of just one. 0 ng ml1, with a calibration curve range between 1. 0 to 500. 0 ng ml1. Intra and interday CV of midazolam and 1 hydroxymidazolam were below 15%. The liquid chromatograph?mass spectrometer contained an system and a TSQ Quantum Discovery maximum system equipped with an ESI probe. Lipophilic analytes were extracted from 0. 5 ml plasma, diluted with 10 l of diazepam answer, with 4 ml ethyl acetate. The samples were evaporated, centrifuged and reconstituted in the mobile phase. Separation by HPLC on a column was adopted by tandem mass spectrometric detection. The mass spectrometer was operated in constructive ion mode and quantication was ergo performed using selected reaction tabs on the changes of m/z 295277 for tanshinone IIA, m/z 297251 for cryptotanshinone, m/z 277249 for tanshinone, and m/z 285193 for the diazepam, respectively. This analysis had a LLOQ of 0. Lymph node 1 ng ml1, with intra and interday CV of tanshinone I, tanshinone IIA and cryptotanshinone being below 15%. Hydrophilic analytes were extracted from 0. 5 ml plasma, diluted with 10 l of Hedgehog agonist protocatechuic acid solution, with 1 mol l1 HCl 30 l and then 4 ml ethyl acetate. The samples were evaporated, centrifuged and reconstituted in the mobile phase. Separation by HPLC on C18 column was followed by electrospray ionization tandom mass spectrometric detection. The mass spectrometer was operated in adverse ion style and quantication was hence performed using selected reaction track of the transitions of m/z 135. 0 for danshensu, 108. 0 for protocatechuic aldehyde and 108. 0 for IS, respectively. This analysis had a LLOQ of 0. 1 ng ml1, and intra and interday CV of danshensu and protocatechuic aldehyde were below 15%. The plasma concentration?time data of analytes obtained on 16 and days 1 were reviewed by design independent strategies.