Quantification and Validation Paper II

There are no detailed recommendations for analytical quantification procedures in the field of biotechnological production of drugs, in contrast to the recommendation made by the FDA [16] for bioanalytical methods. The aim of this paper was therefore to investigate whether the latter detailed guidelines (given by the FDA for bioanalytical methods) also could be used in the field of biotechnological synthesis. Validated methods for quantification are important also in biotechnological synthesis for the proper calculation of rate coefficients. 

As mentioned in section 2.7, the internal standard (I.S.) method is useful for quantification when the matrices are complicated and sample pre-treatment is necessary. However, it is generally difficult to choose an I.S. that meets all the necessary requirements (se above in section 2.7). Thus, 4-androsten-3,17-dione (AD) was chosen as I.S. because of its great structural similarity with both substrate and product. However, it soon appeared that 

The tolerances (C.V.-values) of the validation terms can be much narrower in this work 10% is suggested. This value is between the accepted tolerances for bioanalytical methods and those for pharmaceutical product analysis. 

The internal standard (I.S.) must be introduced in the process liquid at the start of the process together with the substrate. 

must be sufficiently different in structure from the substrate so that it will not participate in the enzymatic process. 

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