Validation of a rapid and sensitive liquid chromatography-tandem mass spectrometry method for free and total mycophenolic acid.
BACKGROUND
Because mycophenolic acid (MPA) is highly protein bound and because the free fraction is the pharmacologically active portion, a rapid, reliable, and sensitive procedure is required to study the relationship between free MPA and treatment efficacy/toxicity. Liquid chromatography-tandem mass spectrometry is ideally suited for such a method.


METHODS
Free MPA was isolated from plasma by ultrafiltration. An online extraction cartridge with a column-switching technique, analytical liquid chromatography over an Aqua Perfect C(18) column, and electrospray tandem mass spectrometry was used to quantify free and total MPA. To investigate ion suppression, a continuous infusion of MPA was introduced into the effluent from the HPLC column, and different ultrafiltrates and extracted plasma samples were injected on the column.


RESULTS
A chromatographic run time of 4 min separated MPA from metabolites and internal standard, thereby avoiding interference from in-source fragmentation. Ion suppression occurred well before elution of MPA and internal standard. The lower limit of quantification for free MPA was 0.5 microg/L, and the method was linear to 1000 microg/L. Interassay imprecision (CV) was <10% for free MPA (0.5-333 microg/L). Agreement was good for free MPA (n = 52) and total MPA (n = 106) between the proposed method and a validated HPLC method with ultraviolet detection. The Passing-Bablok regression line was: y = 0.95x + 0.27 microg/L for free MPA and y = 0.98x + 0.03 mg/L for total MPA.


CONCLUSIONS
The presented method allows the accurate, precise, and rapid determination of free and total MPA in plasma over a wide analytical range covering the concentrations relevant to pharmacokinetic studies and routine monitoring of this drug.
Streit
Frank
F
Department of Clinical Chemistry, George-August University Gottingen, D-37075 Gottingen, Germany.
Shipkova
Maria
M
Armstrong
Victor William
VW
Oellerich
Michael
M
D001798:Blood Proteins / Q000378:metabolism; D002853:Chromatography, Liquid; D006801:Humans; D009173:Mycophenolic Acid / Q000097:blood* / Q000378:metabolism; D010949:Plasma; D011485:Protein Binding; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D014462:Ultrafiltration
D001798:Blood Proteins; D009173:Mycophenolic Acid