Progress in analytical methods

The classical method used for the determination of allantoin in urine was developed by Young & Conway in 1942 [211]. The method is still being widely used today since it requires only a spectrometer. The disadvantage of this method is the procedure is rather lengthy and requires critical timing of operation. Thus only a small number of samples (20-30) can be analysed in a day. Accuracy of the analytical results depends heavily on whether the analysis is carried out routinely. Pentz [212] automated the procedure by adapting it to the AutoAnalyzer. Subsequently the automatic method was adopted and improved by Lindberg & Jansson [213] in Sweden and Chen et al. [214] in the UK, and became a useful aid in their studies on urinary PD in ruminant since large numbers of samples could be processed.

There are many reports on methods for determination of allantoin by HPLC based on reversed-phase chromatography. On a C18 reversed-phase column, allantoin is not retained long enough for good separation from the polar solutes, thus the column length often needs to be extended by using two columns [215-222]. Yamamoto et al. [223] used ion-pairing agents to slow down the elution of allantoin. The advantage of the direct determination is its simplicity and the bonus of the possibility to measure uric acid, xanthine and hypoxanthine in the same run (if the concentration range for all compounds are right). The weakness is that when the concentration is low, the accuracy of allantoin measurement is affected since the allantoin peak is not well separated from the unidentified background noise when monitored at around 200 nm wavelength. Pre-column derivatization is an alternative, i.e. to change allantoin into a derivative which is detected at a unique wavelength [224-226]. In general, with the analysis of derivatized allantoin, the run time is short and analysis robust, but other purine derivatives are not included within the same run.

Methods for determination of allantoin up to 1995 were reviewed by Chen et al [227] . Since then, there has been development of newer methods. These are:

1) Methods based on GC-MS and LC-MS [228-231].
2) Methods based on capillary zone electrophoresis (CZE) [232-234]
3) Methods based on electrokinetic chromatography [235-236].
4) Method based on near infrared spectrometry [237].

The development of GC-MS and LC-MS methods will provide powerful tools for future research into the metabolism of purines and the kinetics of PD in plasma using stable isotopic tracers. On the other hand, from the other new methods and more to come in the future, the long-sought after techniques for routine PD analysis which are accurate, fast, robust and inexpensive may finally evolve.

Compared to allantoin, the measurements of uric acid, xanthine and hypoxanthine are much simpler. These three together could be determined as “total uric acid” after enzymatic conversion of xanthine and hypoxanthine to uric acid by xanthine oxidase [7,32]. A more recent account of the procedure is given in IAEA-TECDOC-945 [27].