Development and validation of an LC-MS/MS method for tyrphostin A9
|
摘要
Here we have presented a sensitive and selective LC-MS/MS method for the quantification of tyrphostin A9, which is a selective inhibitor for platelet derived growth factor receptor tyrosine kinase and has been investigated in vitro as a potent oxidative phosphorylation uncoupler. The murine analytical method was developed for three biological matrices: cell culture media, 3 T3-L1 cell lysate, and murine plasma. For each matrix the limit of detection and the limit of quantification were found to be 0.5 ng/mL and 1.0 ng/mL, respectively. The range of standard curve for each matrix was 1.0 e100 ng/mL, linearity was >0.99,and the precision and accuracy were within 20%. 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoic acid was found to be the most suitable internal standard. The validated LC-MS/MS method was used to investigate stability and in vitro pharmacokinetics of tyrphostin A9. It was found that tyrphostin A9 is susceptible to hydrolysis, and the degradation product was identified as 3,5-di-tert-butyl-4-hydroxybenzaldehyde. Tyrphostin A9 was not stable in biological matrices, and the rate of its degradation in murine plasma was faster than that in cell culture media. In vitro pharmacokinetic studies revealed that tyrphostin A9 concentrations in the cell culture media declined in a bi-exponential manner and the concentrations inside the adipocytes remained constant, suggesting tyrphostin A9 has an intracellular binding site and is retained within the cell. The LC-MS/MS method presented here paves the way for further quantitative investigations involving tyrphostin A9.
Here we have presented a sensitive and selective LC-MS/MS method for the quantification of tyrphostin A9, which is a selective inhibitor for platelet derived growth factor receptor tyrosine kinase and has been investigated in vitro as a potent oxidative phosphorylation uncoupler. The murine analytical method was developed for three biological matrices: cell culture media, 3 T3-L1 cell lysate, and murine plasma. For each matrix the limit of detection and the limit of quantification were found to be 0.5 ng/mL and 1.0 ng/mL, respectively. The range of standard curve for each matrix was 1.0 e100 ng/mL, linearity was >0.99,and the precision and accuracy were within 20%. 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoic acid was found to be the most suitable internal standard. The validated LC-MS/MS method was used to investigate stability and in vitro pharmacokinetics of tyrphostin A9. It was found that tyrphostin A9 is susceptible to hydrolysis, and the degradation product was identified as 3,5-di-tert-butyl-4-hydroxybenzaldehyde. Tyrphostin A9 was not stable in biological matrices, and the rate of its degradation in murine plasma was faster than that in cell culture media. In vitro pharmacokinetic studies revealed that tyrphostin A9 concentrations in the cell culture media declined in a bi-exponential manner and the concentrations inside the adipocytes remained constant, suggesting tyrphostin A9 has an intracellular binding site and is retained within the cell. The LC-MS/MS method presented here paves the way for further quantitative investigations involving tyrphostin A9.
Here we have presented a sensitive and selective LC-MS/MS method for the quantification of tyrphostin A9, which is a selective inhibitor for platelet derived growth factor receptor tyrosine kinase and has been investigated in vitro as a potent oxidative phosphorylation uncoupler. The murine analytical method was developed for three biological matrices: cell culture media, 3 T3-L1 cell lysate, and murine plasma. For each matrix the limit of detection and the limit of quantification were found to be 0.5 ng/mL and 1.0 ng/mL, respectively. The range of standard curve for each matrix was 1.0 e100 ng/mL, linearity was >0.99,and the precision and accuracy were within 20%. 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoic acid was found to be the most suitable internal standard. The validated LC-MS/MS method was used to investigate stability and in vitro pharmacokinetics of tyrphostin A9. It was found that tyrphostin A9 is susceptible to hydrolysis, and the degradation product was identified as 3,5-di-tert-butyl-4-hydroxybenzaldehyde. Tyrphostin A9 was not stable in biological matrices, and the rate of its degradation in murine plasma was faster than that in cell culture media. In vitro pharmacokinetic studies revealed that tyrphostin A9 concentrations in the cell culture media declined in a bi-exponential manner and the concentrations inside the adipocytes remained constant, suggesting tyrphostin A9 has an intracellular binding site and is retained within the cell. The LC-MS/MS method presented here paves the way for further quantitative investigations involving tyrphostin A9.
引文
[1]A.Gazit,P.Yaish,C.Gilon,et al.,Tyrphostins I:synthesis and biological activity of protein tyrosine kinase inhibitors,J.Med.Chem.32(1989)2344e2352.
[2]M.L.Bond,A.Azzolina,E.F.Craparo,et al.,Entrapment of an EGFR inhibitor into nanostructured lipid carriers(NLC)improves its antitumor activity against human hepatocarcinoma cells,J.Nanobiotechnol.12(2014)21.
[3]K.Iida,K.Nakayama,M.T.Rahman,et al.,EGFR gene amplification is related to adverse clinical outcomes in cervical squamous cell carcinoma,making the EGFR pathway a novel therapeutic target,Br.J.Canc.105(2011)420e427.
[4]A.Novogrodsky,M.Weisspapir,M.Patya,et al.,Tyrphostin 4-nitrobenzylidene malononitrile reduces chemotherapy toxicity without impairing efficacy,Cancer Res.58(1998)2397e2403.
[5]Q.Chen,A.A.Luo,H.Qiu,et al.,Monocyte interaction accelerates HCl-induced lung epithelial remodeling,BMC Pulm.Med.14(2014)135.
[6]N.Kumar,Y.Liang,T.G.Parslow,et al.,Receptor tyrosine kinase inhibitors block multiple steps of influenza A virus replication,J.Virol.85(2011)2818e2827.
[7]N.Kumar,N.R.Sharma,H.Ly,et al.,Receptor tyrosine kinase inhibitors that block replication of influenza A and other viruses,Antimicrob.Agents Chemother.55(2011)5553e5559.
[8]S.J.Park,Y.J.Park,J.H.Shin,et al.,A receptor tyrosine kinase inhibitor,tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation,Biochem.Biophys.Res.Commun.408(2011)465e470.
[9]Y.Sagara,K.Ishige,C.Tsai,et al.,Tyrphostins protect neuronal cells from oxidative stress,J.Biol.Chem.277(2002)36204e36215.
[10]A.R.Anand,A.Prasad,R.R.Bradley,et al.,HIV-1 gp120-induced migration of dendritic cells is regulated by a novel kinase cascade involving Pyk2,p38 MAPkinase,and LSP1,Blood 114(2009)3588e3600.
[11]C.M.Won,Kinetics and mechanism of hydrolysis of tyrphostins,Int.J.Pharm.104(1994)29e40.
[12]H.Terada,Uncouplers of oxidative phosphorylation,Environ.Health Perspect.87(1990)213e218.
[13]K.Zebisch,V.Voigt,M.Wabitsch,et al.,Protocol for effective differentiation of3T3-L1 cells to adipocytes,Anal.Biochem.425(2012)88e90.
[14]G.Tiwari,R.Tiwari,Bioanalytical method validation:an updated review,Pharm.Meth.1(2010)25.
[15]A.G.Ellis,E.C.Nice,J.Weinstock,et al.,High-performance liquid chromatographic analysis of the tyrphostin AG1478,a specific inhibitor of the epidermal growth factor receptor tyrosine kinase,in mouse plasma,J.Chromatogr.B Biomed.Sci.Appl.754(2001)193e199.
[16]C.C.Willhite,R.P.Smith,The role of cyanide liberation in the acute toxicity of aliphatic nitriles,Toxicol.Appl.Pharmacol.59(1981)589.
[17]S.J.Gee,C.E.Green,C.A.Tyson,Cyanide-induced cytotoxicity to isolated hepatocytes,Toxicol.In Vitro 4(1990)37e45.
[2]M.L.Bond,A.Azzolina,E.F.Craparo,et al.,Entrapment of an EGFR inhibitor into nanostructured lipid carriers(NLC)improves its antitumor activity against human hepatocarcinoma cells,J.Nanobiotechnol.12(2014)21.
[3]K.Iida,K.Nakayama,M.T.Rahman,et al.,EGFR gene amplification is related to adverse clinical outcomes in cervical squamous cell carcinoma,making the EGFR pathway a novel therapeutic target,Br.J.Canc.105(2011)420e427.
[4]A.Novogrodsky,M.Weisspapir,M.Patya,et al.,Tyrphostin 4-nitrobenzylidene malononitrile reduces chemotherapy toxicity without impairing efficacy,Cancer Res.58(1998)2397e2403.
[5]Q.Chen,A.A.Luo,H.Qiu,et al.,Monocyte interaction accelerates HCl-induced lung epithelial remodeling,BMC Pulm.Med.14(2014)135.
[6]N.Kumar,Y.Liang,T.G.Parslow,et al.,Receptor tyrosine kinase inhibitors block multiple steps of influenza A virus replication,J.Virol.85(2011)2818e2827.
[7]N.Kumar,N.R.Sharma,H.Ly,et al.,Receptor tyrosine kinase inhibitors that block replication of influenza A and other viruses,Antimicrob.Agents Chemother.55(2011)5553e5559.
[8]S.J.Park,Y.J.Park,J.H.Shin,et al.,A receptor tyrosine kinase inhibitor,tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation,Biochem.Biophys.Res.Commun.408(2011)465e470.
[9]Y.Sagara,K.Ishige,C.Tsai,et al.,Tyrphostins protect neuronal cells from oxidative stress,J.Biol.Chem.277(2002)36204e36215.
[10]A.R.Anand,A.Prasad,R.R.Bradley,et al.,HIV-1 gp120-induced migration of dendritic cells is regulated by a novel kinase cascade involving Pyk2,p38 MAPkinase,and LSP1,Blood 114(2009)3588e3600.
[11]C.M.Won,Kinetics and mechanism of hydrolysis of tyrphostins,Int.J.Pharm.104(1994)29e40.
[12]H.Terada,Uncouplers of oxidative phosphorylation,Environ.Health Perspect.87(1990)213e218.
[13]K.Zebisch,V.Voigt,M.Wabitsch,et al.,Protocol for effective differentiation of3T3-L1 cells to adipocytes,Anal.Biochem.425(2012)88e90.
[14]G.Tiwari,R.Tiwari,Bioanalytical method validation:an updated review,Pharm.Meth.1(2010)25.
[15]A.G.Ellis,E.C.Nice,J.Weinstock,et al.,High-performance liquid chromatographic analysis of the tyrphostin AG1478,a specific inhibitor of the epidermal growth factor receptor tyrosine kinase,in mouse plasma,J.Chromatogr.B Biomed.Sci.Appl.754(2001)193e199.
[16]C.C.Willhite,R.P.Smith,The role of cyanide liberation in the acute toxicity of aliphatic nitriles,Toxicol.Appl.Pharmacol.59(1981)589.
[17]S.J.Gee,C.E.Green,C.A.Tyson,Cyanide-induced cytotoxicity to isolated hepatocytes,Toxicol.In Vitro 4(1990)37e45.