Quantitation and Identification of Peaks

The radioisotope detector will give a readout of the cpm of the tRA, which was added to the initial tissue prior to extraction. By knowing how much was added, one can thus work out a recovery ratio that will allow a correction factor to be introduced when working out the endogenous levels of retinoids. Typical values are in the 60-80% range. The UV detector will give a readout of the peak areas and the amount in nanograms for each peak it has recorded. It can do this, because when setting up the machine, one establishes a calibration curve for each retinoid, which the machine uses to give a value in nanograms for each of the UV peaks.

The identification of individual peaks can be done in several ways. First, an indication is given simply by comparing the elusion time of each peak with the known elution time of each standard. This one has done when setting up the machine to establish calibration curves as described above. Thus, in Fig. 2A, the elution profile of a set of retinoid standards is shown. The elution time of peak 2 (IRA) is 19.25 min. In Fig. 2B, the retinoids extracted from a 10.5-d mouse embryo is shown, and in this chromatograph, a peak is present at exactly the same elution time (peak 2) suggesting that tRA is present in mouse embryos. By comparing the standards with the mouse embryo extract, it can be seen seen that some peaks are present (2 and 3) and the others are absent. Second, a further identification is provided by coelution of the radioactivity in the spiked sample with an endogenous UV peak (Fig. 2B). This is shown here for tRA, but can clearly be done for all the other retinoids of interest, or a cocktail of radioactive retinoids can be added to the sample. Third, the sample can be spilt into two, and a spike of one cold retinoid, e.g., tRA is added to one of the samples. They are then run on the HPLC, one after the other, and in the spiked case, only one peak should increase in height, i.e., the tRA one. Fourth, the individual peaks can be collected in a fraction collector and then peaks of interest can be rechromatographed on a different system. For example, if this was done with the retinol peak obtained from the reverse-phase chromatograph in Fig. 2B (peak 3) and the sample was then run on a normal-phase column, this would reveal whether there really were two peaks here (retinol and 9-cis), since the elution times of each retinoid changes on a different chromatography system. Fifth, the peaks can be collected and then derivatized. This involves methylating the material in the peak, e.g., the suspected tRA and then running it on a normal-phase system to confirm that it now coeluted with authentic tRA methyl ester or performing gas chromatography/mass spectorscopy to the same end (3).

Amounts as low as 1 ng of tRA can readily be detected by the method described above. However, it is to be emphasized that the figures one generates after quantitation of retinoids in tissue are only estimates. In addition to the corrections described above (recovery ratio, comparison with calibration curves), there is the further problem of UV detection. The suggested wavelength of 351 nm is an average value for several retinoids. In fact, each retinoid has a different absorption maximum, e.g., all-trans-retinol = 325 nm; all-trans-retinal = 383 nm; tRA = 350 nm; 9-cis- RA = 345 nm; ddRA = 370 nm; 4-oxo-RA = 360 nm. So were the wavelength to be set differently then a different value for the quantitation of each retinoid would be obtained. The use of a scanning wavelength detector would solve this problem, but such detectors are an order of magnitue less sensitive than fixed-wavelength detectors. Nevertheless, within these limitations, good results have been obtained, particularly where differences in retinoid content between different parts of the embryo have been identified.

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