A suitable diluent ratio should not only keep the sample chemically and physically stable but should also allow the drug to be detected accurately and precisely by HPLC. Ideally, the drug should be able to analyse at the lowest level you want to detect (typically about 5% of the drug concentration if fully released) above the limit of detection. Typical dilution ratios range from 1:2 to 1 :10.

Prepare working standards (WS) with varying drug concentrations (highest 120% and lowest 5%) by diluting secondary standard (SS) with biorelevant medium. Examine the chromatograms and explore different dilution ratios (for example 1:2, 1:5 or 1:10).

An example of 1: 2 dilution ratio of biorelevant medium with drug is shown below

Determine the drug peak area and quality of the chromatograms and estimate the limit of Quantification and Limit of Detection at the lowest concentration (ideally 5% of drug release in biorelevant medium) of WS.

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