High-performance liquid chromatographic analysis of phosphorothioate analogues of oligodeoxynucleotides in biological fluids.
Phosphorothioate oligodeoxynucleotides (S-ODNs) have potential as anti-viral agents and are being investigated for the chemotherapy of AIDS. A high-performance liquid chromatographic method is described for the analysis, in urine and plasma, of a 28-unit deoxycytidine homopolymer (S-dC28) and a 28-unit S-ODN "antisense" to the rev gene of the human immunodeficiency virus. This method employs ion-pairing HPLC with a polymeric column. Tetrabutylammonium is used as the ion-pairing agent in a mobile phase of acetonitrile in pH 7.0 phosphate buffer. Analysis of the S-ODNs is relatively rapid (20 min) and sensitive (20 nm) and is accomplished by a gradient elution (22.5-30.0% acetonitrile) followed by ultraviolet (266 or 272 nm) absorption detection. This method is likely applicable, with appropriate modifications, to all S-ODNs of similar molecular weight regardless of sequence. The S-ODNs bind very strongly to plasma proteins but are readily prepared for analysis by a phenol extraction procedure. In a preliminary pharmacokinetic study in mice with S-dC28, very rapid elimination of the oligomer from plasma was observed (half-time, 11.6 min). Estimates for the apparent volume of distribution and total body clearance were 3 ml and 0.2 ml/min, respectively. It appears that the majority of the oligomer is eliminated by renal clearance (glomerular filtration), a property likely shared by all S-ODNs of similar molecular mass.
Bigelow
J C
JC
Department of Pharmacology, University of Vermont, Burlington 05405.
Chrin
L R
LR
Mathews
L A
LA
McCormick
J J
JJ
D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D003845:Deoxycytosine Nucleotides / Q000032:analysis; D015757:Genes, rev / Q000235:genetics; D006678:HIV / Q000235:genetics; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000032:analysis* / Q000097:blood / Q000652:urine; D009946:Organothiophosphorus Compounds / Q000032:analysis* / Q000097:blood / Q000652:urine
D004279:DNA, Viral; D003845:Deoxycytosine Nucleotides; D009841:Oligonucleotides; D009946:Organothiophosphorus Compounds