Quantitative Analysis of Modified Antisense Oligonucleotides in Biological Fluids Using Cationic Nanoparticles for Solid-Phase Extraction

Development and validation of a sensitive, specific, and rapid hybridization-ELISA assay for determination of concentrations of a ribozyme in biological matrices

“…As polynucleotides like ribozymes and antisense oligonucleotides are developed as therapeutics, the need arises to measure concentrations of these drugs in blood and other biological matrices. Conventional approaches to bioanalytical quantification of polynucleotides have included HPLC, electrophoresis, or capillary gel electrophoresis [1][2][3][4]6]. Overall, these approaches have limited sensitivity (50-100 ng/ml) and are time intensive because they require extraction of the polynucleotide of interest from a biological matrix.…”

“…Following hybridization of ribozyme to target, the complimentary target RNA species is cleaved, resulting in a decrease in target expression. Previous bioanalytical methodology used in the determination of ribozyme or antisense oligonucleotide concentrations required extraction of the drug from the biological matrix followed by chromatography, electrophoresis, or capillary gel electrophoresis for quantitation [1][2][3][4][5][6]. These methods are time-consuming and lack sensitivity; plasma lower-limits of quantitation typically range from 50 to 100 ng/ml.…”

A combined solid phase extraction/capillary gel electrophoresis method for the determination of phosphorothioate oligodeoxynucleotides in biological fluids, tissues and feces

“…However, one common problem in developing CGE methods for quantitating PS-ODNs is the need for rather extensive sample clean-up for the minimization of endogenous interference with ASOs during electrokinetic injection. Typical plasma and urine sample clean-up procedures for CGE analysis involve two-step SPE followed by additional desalting via dialysis, while the sample preparation from tissue and cellular fractions normally requires liquid/liquid extraction prior to analysis, and additional clean-up with solid-phase extraction for CGE analysis [6][7][8][9][10][11][12][13][14][15]23,24]. Recently, many attempts have been made to simplify and speed up these laborious sample clean-up procedures.…”

“…To date, a variety of bioanalytical methods, such as capillary gel electrophoresis (CGE) [6][7][8][9][10], CGE-MS [11,12], capillary zone electrophoresis (CZE) [13], high-performance liquid chromatograph (HPLC) [14], liquid chromatography-mass spectrometry (LC-MS) [15][16][17], liquid chromatography-tandem mass spectrometry (LC-MS/MS) [18][19][20][21] and hybridization-based enzyme-linked immunosorbent assay (ELISA) [22], have been developed and used in quantitation of PS-ODNs and metabolites. Among these analytical techniques, CGE is widely employed in preclinical and clinical studies for ASOs and allows the separation of the parent compound from chain-shortened metabolites at good resolutions and reasonable sensitivities.…”

Prooligonucleotide metabolism in a crude cell extract followed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

“…Generally oligonucleotides and metabolites are separated from the endogenous proteins after phenol/ chloroform extraction and ethanol precipitation or solidphase extraction. 8 Our studies have been performed by MALDI-MS because this technique provides an advantage over ESI-MS when direct infusion of oligos into the ESI source is used. Indeed, in MALDI-MS only singly charged ions are generated, thereby reducing the complexity of the spectra obtained for mixtures.…”