A sensitive and specific procedure for quantitation of ADR-529 in biological fluids by high-performance liquid chromatography (HPLC) with column switching and amperometric detection.
An HPLC method using electrochemical detection (ED) has been validated for the determination of ADR-529 in plasma and urine using ICRF-192 as an internal standard (IS). Prior to storage and quantitation, both plasma and urine samples require acid stabilization. Acidified plasma samples were prepared for HPLC using a two column solid-phase extraction (SPE). An aliquot of buffered plasma (i.e., pH 6-7) was first deproteinated and desalted on a C-18 SPE column. The analytes were then eluted onto a C-8 SPE column where retention and selective cleanup were achieved in the cation-exchange mode via silanol interactions. Acidified urine samples were diluted in acetonitrile prior to injection. The HPLC system for plasma and urine samples employed two narrow-bore silica columns used in the weak cation-exchange mode and separated by a switching valve. To prohibit late-eluting peaks from passivating the glassy carbon working electrode, a heart-cut containing ADR-529 and the IS was vented from the first silica column to the second using an automated switching valve. Amperometric detection at an oxidation potential of +1050 mV vs a Ag/AgNO3 reference electrode was used. Linearity was validated between 5 and 500 ng/ml in plasma and between 2 and 100 micrograms/ml in urine. Imprecision and percentage bias were typically less than 10% for both plasma and urine controls throughout their respective dynamic ranges. The absolute recoveries for ADR-529 and the IS from plasma were greater than 95%. This method is being successfully applied to the pharmacokinetic/dynamic evaluation of ADR-529 in animals and humans.
Lewis
R C
RC
Department of Pharmacokinetics/Dynamics, Adria Laboratories, Inc., Columbus, Ohio 43216.
Phillips
B A
BA
Baldwin
J R
JR
Rossi
D T
DT
Narang
P K
PK
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004355:Drug Stability; D004563:Electrochemistry; D011786:Quality Control; D011929:Razoxane / Q000032:analysis* / Q000097:blood / Q000652:urine; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D011929:Razoxane