Simultaneous determination of 3'-azido-2',3'-dideoxyuridine and novel prodrugs in rat plasma by liquid chromatography.
3'-Azido-2',3'-dideoxyuridine (AZDU) is a nucleoside analog structurally similar to zidovudine (AZT) with proven activity against human immunodeficiency virus (HIV). The purpose of this study was to develop and validate a high-performance liquid chromatographic (HPLC) method to quantitatively determine AZDU and its novel prodrugs in rat plasma simultaneously. A reversed-phase gradient elution HPLC method was developed to quantitate AZDU and its prodrugs, N3-pivaloyloxymethyl-3'-azido-2',3'-dideoxyuridine (I), 5'-pivaloyloxymethyl-3'-azido-2',3'-dideoxyuridine (II), 5'-O-valinyl-3'-azido-2',3'-dideoxyuridine hydrochloride (III) and 5'-O-phenylalanyl-3'-azido-2',3'-dideoxyuridine hydrochloride (IV), in rat plasma. AZDU and its prodrugs were analyzed using an octadecyl silane column with a mobile phase consisting of 0.04 microM sodium acetate buffer, pH 5.0, and acetonitrile, running in a segmented gradient manner at a flow rate of 2 ml/min. Acetonitrile was increased from 10 to 50% during the first 8 min by 5% per min, followed by 10% per min until it reached 90% acetonitrile. 3'-Azido-2',3'-dideoxy-5-ethyluridine (CS-85) was used as an internal standard (25 microg/ml). Compounds were detected by UV absorption at 261 nm. Extraction recoveries for all compounds were greater than 80%. Retention times of AZDU, CS-85, prodrugs I, II, III and IV were 3.3, 5.2, 9.1, 8.8, 6.3 and 7.3 min, respectively. Calibration plots were linear over the range of 0.25-100 microg/ml for AZDU and prodrugs II, III, and IV and 0.5-100 microg/ml for prodrug I. The limit of quantitation was 0.25 microg/ml for prodrugs II, III and IV and 0.5 microg/ml for prodrug I. The intra- and inter-day variations were less than 10% and accuracies were greater than 90%. This method is rapid, sensitive and reproducible for the determination of AZDU and prodrugs in rat plasma.
Kong
Linghui
L
Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602-2352, USA.
Cooperwood
John S
JS
Oh
Chang H
CH
Chu
Chung K
CK
Boudinot
F Douglas
FD
D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D011355:Prodrugs / Q000032:analysis*; D051381:Rats; D012015:Reference Standards; D018894:Reverse Transcriptase Inhibitors / Q000097:blood* / Q000493:pharmacokinetics; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000097:blood* / Q000493:pharmacokinetics
D011355:Prodrugs; D018894:Reverse Transcriptase Inhibitors; D015215:Zidovudine; C058803:3'-azido-2',3'-dideoxyuridine