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Analysis of various nucleosides in plasma using solid phase extraction and high-performance liquid chromatography
D L Walters1, D L Jacobs, J E Tomaszewski
1Battelle, Columbus, OH 43201, USA.
Abstract:
The National Cancer Institute (NCI) has screened many nucleosides for antiviral activity to the HIV-1 virus. Drugs demonstrating antiviral activity are tested in animal models to evaluate their toxicity and pharmacokinetic characteristics. These drugs are subsequently evaluated for efficacy in human clinical trials. Sensitive analytical methodology is needed to quantify nucleosides in plasma and other biological matrices in support of these studies. Battelle has modified and validated a reversed phase high-performance liquid chromatography (HPLC) method for several of these nucleosides that could be easily adapted for similar compounds. Methods have been validated for 6-chloro-2',3'-dideoxyguanosine (6ClddG), 6-chloro-2',3'-dideoxyinosine (6ClddI) and their primary metabolites 2',3'-dideoxyguanosine (ddG) and 2',3'-dideoxyinosine (ddI) in both rat and dog plasma containing EDTA. The method has also been validated for 2'-fluoro-2',3'-dideoxyara-adenosine (betaFlddA) and its primary metabolite 2'-beta-fluorodideoxyinosine (betaFddI) in rat plasma containing heparin. Calibration plasma standards were prepared over ranges of 0.1-10 microg ml(-1) for betaFlddA and betaFddI, 0.1-50 microg ml(-1) for 6ClddG and ddG, and 0.25-50 microg ml(-1) for 6ClddI and ddI in plasma containing 4 microg ml(-1) pentostatin. The addition of pentostatin to the plasma samples inhibits in-vitro deamination of the drug after collection. Quality control (QC) standards were prepared containing the appropriate anticoagulant and 4 microg ml(-1) pentostatin at concentrations within each of the bracketed calibration ranges in plasma. These methods have been successfully applied to plasma samples generated during various animal studies.
Insights
A validated reversed-phase high-performance liquid chromatography (HPLC) method quantifies HIV-1 antiviral nucleosides and their metabolites in animal plasma. This analytical method supports drug development by enabling accurate measurement in biological matrices.
Area of Science:
- Analytical Chemistry
- Pharmacology
- Biochemistry
Background:
- The National Cancer Institute (NCI) screens nucleoside analogs for human immunodeficiency virus type 1 (HIV-1) antiviral activity.
- Antiviral drug candidates undergo preclinical testing in animal models to assess toxicity and pharmacokinetics.
- Accurate quantification of nucleosides in biological samples is crucial for supporting drug development studies.
Purpose of the Study:
- To modify and validate a sensitive reversed-phase high-performance liquid chromatography (HPLC) method for quantifying specific nucleosides and their metabolites.
- To support preclinical antiviral drug development by providing reliable analytical methodology for plasma samples.
- To adapt the validated HPLC method for similar nucleoside compounds.
Main Methods:
- Validation of a reversed-phase HPLC method for quantifying 6-chloro-2',3'-dideoxyguanosine (6ClddG), 6-chloro-2',3'-dideoxyinosine (6ClddI), 2',3'-dideoxyguanosine (ddG), 2',3'-dideoxyinosine (ddI), 2'-fluoro-2',3'-dideoxyara-adenosine (betaFlddA), and 2'-beta-fluorodideoxyinosine (betaFddI).
- Analysis performed in rat and dog plasma using appropriate anticoagulants (EDTA or heparin).
- Inclusion of pentostatin (4 microg ml(-1)) in plasma samples to inhibit in-vitro deamination, with calibration standards prepared across defined concentration ranges.
Main Results:
- Validated HPLC methods were established for the target nucleosides and their metabolites in rat and dog plasma.
- Calibration standards were prepared within specific ranges (e.g., 0.1-10 microg ml(-1) for betaFlddA and betaFddI).
- The validated methods were successfully applied to analyze plasma samples from various animal studies.
Conclusions:
- The modified and validated reversed-phase HPLC method provides a sensitive and adaptable analytical tool for quantifying antiviral nucleosides and their metabolites in plasma.
- This methodology is essential for supporting pharmacokinetic and efficacy studies in preclinical drug development.
- The method's successful application in animal studies demonstrates its utility and reliability.