Standardization of HPLC measurements of pyridinium crosslinks

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Abstract
Measurement of the total amount of the two crosslinks, pyridinoline and deoxypyridinoline, can be made simultaneously by high pressure liquid chromatography preceded by an acidic hydrolysis of the sample and an extraction on cellulose columns. To compensate for the losses and variation in the extraction procedure and increase precision and accuracy a suitable internal standard should be used. A simple procedure to isolate a compound to be used as an internal standard is discussed. With this compound a total, long term coefficient of variation of 7% and 5% for the two crosslinks is achieved. The present inter-laboratory variation reflected by the big differences in reference intervals is probably due to the presence of several different ways to purify and quantify crosslinks to be used as calibrators. Recently the molar absorptivity for both pyridinoline and deoxypyridinoline in the ultra violet range has been defined. This may be the first step towards the creation of a primary international reference material.