How do you select the concentration of standard and sample during related substances method development

 The purpose of method development is to get simple, selective, precise, accurate and robust method in place. Among many factors, the concentration of analyte plays vital role in sensitivity, precision, accuracy and linearity of method. And hence it becomes very important to define optimum concentration for standard and sample to yield optimum response!

Most of the method developers prefer to use diluted standard for evaluation of system suitability and for quantification of impurities. The system suitability is determined with the help of peak tailing, theoretical plates and or %RSD of diluted standard. The concentration of standard solution has major role in determining %RSD of the replicate standard injections. More is the concentration-less will be the %RSD and vise a versa. So, one can understand that the higher concentration of standard is good to yield lower %RSD for replicate standard injections. But the %RSD for standard is not the only factor which needs to be considered during method development and there are other factors such as accuracy, sensitivity, precision, linearity etc. which must be attended seriously. Though the increased concentration may result into improved precision but it may result into poor extraction of active and its impurities from sample matrix. And hence, the balance approach needs to be adopted while selecting concentration for standard and sample solution.

Most of the method developers prefer to select the concentration of diluted standard at the level of specification of highest unknown impurity (HUI). For example if the limit for HUI is NMT 0.2%, the concentration of standard solution will be selected as 0.2% with respect to sample concentration. To make it simpler, let us assume that the concentration of active substance in sample solution is 500 ppm, hence 0.2% of 500ppm will become 1.0ppm. Therefore diluted standard with 1.0 ppm can be used to determine system suitability and to quantify impurities.

Having said this, few method developers tends to select concentration for standard solution little higher than the HUI specification. For example in the above case, 1.0 ppm is HUI concentration. So, instead of 1.0 ppm, someone may select concentration of standard solution as 5.0 ppm. The higher concentration further provides higher precision and lower %RSD for replicate standard injections made during evaluation of system suitability. But one must be careful while selection of highest level for linearity during method validation. The levels for linearity must be selected in such way that both HUI and standard concentrations will be covered.

Let us assume, 150% is the highest level of linearity study. The concentration of HUI and standard at 150% will be 1.5 ppm and 7.5 ppm respectively. It is evident that 150% of HUI concentration does not cover the 150% of the standard concentration. But inversely, 150% of standard concentration will cover HUI concentration at 150%. And hence, in this case, linearity concentrations must be decided by considering diluted standard concentration.

LOQ is yet another important attribute of related substances method, and it is closely associated with sample concentration. Method developer must confirm whether the requirement of LOQ is met before deciding on sample concentration. The LOQ of the method shall not be less than reporting threshold given by ICH guideline Q3A(R2) (Impurity in new drug substances) or Q3B(R2) (Impurity in new drug products). For example if the reporting threshold for API is 0.05%, then the LOQ above 0.05% (such as 0.07% or 0.09%) will not be acceptable. The LOQ less than 0.05% (such as 0.03% or 0.02%) will be well acceptable.

So, these are important points method developer must consider during deciding on standard and sample concentration. There may be few more points which I might have missed here- kindly mention them in comment box.

Comment on how do you select standard and sample concentration? Do you select standard concentration at the level of specification of HUI? Or do you select standard concentration more than HUI specification? What are your top 3 takeaways from this article?

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