Quantification of 5-azacytidine in plasma by electrospray tandem mass spectrometry coupled with high-performance liquid chromatography.
5-Azacytidine (5AC), a nucleoside analogue and hypomethylating agent, has anticancer properties and has been utilized in the treatment of various malignancies. 5AC is unstable and rapidly hydrolyzed to several by-products, including 5-azacytosine and 5-azauracil. A sensitive, reliable method was developed to quantitate 5AC using LC/MS/MS to perform pharmacokinetic and pharmacodynamic studies on 5AC combination therapy trials. Blood samples were collected in a heparinized tube and immediately processed for storage. To increase the stability of 5AC in plasma, 25 ng/mL tetrahydrouridine was added to the plasma and snap frozen. Plasma samples were extracted using acetonitrile then cleaned up by Oasis MCX ion exchange solid-phase extraction cartridges. 5AC was separated on an YMC Jsphr M80 C(18) column with gradient elution of ammonium acetate (2 mM) with 0.1% formic acid and methanol mobile phase. 5AC elutes at 5.0 +/- 0.2 min with a total run time of 30 min. Identification was through positive-ion mode and multiple reaction monitoring mode at m/z+ 244.9-->113.0 for 5AC and m/z+ 242.0-->126.0 for 5-methyl-2'-deoxycytidine, the internal standard. The lower limit of quantitation of 5AC was 5 ng/mL in human plasma, and linearity was observed from 5 to 500 ng/mL fitted by linear regression with 1/x weight. This method is 50 times more sensitive than previously published assays and successfully allows studies to characterize the pharmacokinetics and pharmacodynamics of 5AC.
Zhao
Ming
M
Division of Experimental Therapeutics, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Bunting-Blaustein Cancer Research Building, 1650 Orleans Street, Room 1M87, Baltimore, MD 21231, USA.
Rudek
Michelle A
MA
He
Ping
P
Hartke
Carol
C
Gore
Steve
S
Carducci
Michael A
MA
Baker
Sharyn D
SD
D000970:Antineoplastic Agents / Q000097:blood* / Q000493:pharmacokinetics; D001374:Azacitidine / Q000097:blood* / Q000493:pharmacokinetics; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D012015:Reference Standards; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D000970:Antineoplastic Agents; D001374:Azacitidine