Standard additions, spectrophotometric assays

One cannot fail to note the vast expansion of the collection in the last few decades. Surely this was not fueled by additional biologic assays. Underlying the initial growth phase was the widespread utilization of spectrophotometry for identification and assay. Separation science was the second phase in pharmaceutical industry control laboratories. As a corollary, USP and NF method selection moved in the same direction. Spectrophotometric identity tests and assays are more reliable, especially for compliance testing, when performed in the relative mode, which uses a reference standard, rather than the absolute mode, which is the norm in titrimetry. There is some residual difference of opinion in other countries on this point, but that is rendered moot by the widespread adoption of separation science by the pharmaceutical industry and, thus, by the compendia. It is a characteristic of chromatographic methods that a reference standard be required, sometimes more than one for a procedure. The accumulation of modern tests and assays results in 5 to 10 uses for many reference standards. 

CPA is available commercially in pure form. Benzyloxy-carbonylglycylphenylalanine (Z-Gly-Phe) is one of the commonly used substrates.The progress of reaction is conveniently tnonitored spectrophotometrically at 220-225 nm. In addition to peptides, esters such as 0-(N-benzoylglycyI)-L-p-phenyllactate (Bz-Gly-(O)Phe) and -L-mandelate are hydrolyzed easily by CPA. Typical Km values for these artificial substrates are 0.5-5 mM with a turnover number of 20 s for amides and 100 s for esters under standard assay conditions. ... 

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