Examples of Current Assay Methods

There are many possible permutations for coupling one of the chromatographic or immunoaffinity separations with one or another of the spectrometric detection technologies. HPLC with UV or fluorescence spectrometry/ and HPLC with MS/ are among the most widely used quantitative analytical methods in the pharmaceutical development of new chemical entities because of their general applicability and 

HPLC/UV and HPLC/MS Assay of New Chemical Entities — Nucleoside Drugs 

Contemporary quantitative assays require the analyst to select appropriate compounds to serve as internal standards. A fixed quantity of an internal standard is added to each sample so that the intensity of the signals from the analyte from each sample can be normalized to those from the internal standard and compared to samples analyzed during the same run, or from another analytical set on another date. Internal standards must have chemical properties similar to 

The HPLC/UV chromatograms of pre-dose and F-ddA patient plasma with added 5 -Cl-deoxyadenosine 

FIGURE 12.9 HPLC/UV analysis of plasma from a patient before (A) and after (B) receiving an F-ddA dose. 5 -Cl-dA was added to the plasma as the internal standard. The plasma analyzed in (B) was obtained 85 minutes after beginning a 100-minute intravenous infusion. Arrows indicate the time of elution for each component. The dotted line indicates the methanol concentration gradient. Data courtesy of Dr. J. Kelly, NCI, NIH. 

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