Quantitative determination of a chemically modified hammerhead ribozyme in blood plasma using 96-well solid-phase extraction coupled with high-performance liquid chromatography or capillary gel electrophoresis.
Versatile bioanalytical assays to detect chemically stabilized hammerhead ribozyme and putative ribozyme metabolites from plasma are described. The extraction protocols presented are based on serial solid-phase extractions performed on a 96-well plate format and are compatible with either IEX-HPLC or CGE back-end analysis. A validation of both assays confirmed that both the HPLC and the CGE methods possess the required linearity, accuracy, and precision to accurately measure concentrations of hammerhead ribozyme extracted from plasma. These methods should be of general use to detect and quantitate ribozymes from other biological fluids such as serum and urine.
Bellon
L
L
Department of Oligonucleotide Chemistry, Department of Biochemistry, Department of Pharmacology, Ribozyme Pharmaceuticals, Inc., 2950 Wilderness Place, Boulder, Colorado 80301, USA. lbellon@lifetech.com
Maloney
L
L
Zinnen
S P
SP
Sandberg
J A
JA
Johnson
K E
KE
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004983:Ethanolamines / Q000737:chemistry; D008297:Male; D051379:Mice; D009841:Oligonucleotides / Q000737:chemistry; D011786:Quality Control; D016337:RNA, Catalytic / Q000097:blood*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D015203:Reproducibility of Results
D004983:Ethanolamines; D009841:Oligonucleotides; D016337:RNA, Catalytic; C007369:2-diethylaminoethanol