Elimination of liquid-liquid extraction. More reproducible recovery in a shorter time. Use of conventional apparatus (switching valve, isocratic HPLC pump). Congenital Adrenal Hyperplasia (CAH) is a genetic defect of the adrenal glands. It originates by 21-hydroxylase deficiency causing abnormal production of 17 alpha-hydroxyprogesterone (17-OHP) and androstenedione. In the neonatal period it can lead to salt wasting crisis that, without a timely diagnosis, could be dangerous up to death. The average occurrence of this kind of pathology is one case out of 15000 newborns, whereas in our medical district it is considerably higher. Its recognition can be obtained by plotting the ratio 17-OHP plus androstendione divided by cortisol Vs. 17-OHP concentration. Sample preparation involves the extraction of the analytes from dried blood spots (DBS) by methanol, containing the internal standard, and on-line SPE, which makes use of a perfusion column Applied Biosystems POROS 2.1 x 30 mm. HPLC separation is carried out by a Waters Simmetry C18 2.1 x 50 mm column, by a methanol/water gradient, with a flow rate of 300 µl/min. MS-MS MRM detection is performed on an Applied Biosystems API 4000 triple quadrupole mass spectrometer, equipped with a turbo ionspray source. Several samples were analyzed in order to setup and test the method, as far as its straightforwardness, reliability, reproducibility, and ruggedness. The influence of potential interferences, typical with this kind of matrices and analytes, has been evaluated as well, resulting not critical. Moreover, the comparison between normal and pathologic samples was carried out.

Congenital Adrenal Hyperplasia: On-line Cleanup in the Assessment of Involved Steroids out of Dried Blood Spots

SABA, ALESSANDRO;GHIRRI, PAOLO;
2007

Abstract

Elimination of liquid-liquid extraction. More reproducible recovery in a shorter time. Use of conventional apparatus (switching valve, isocratic HPLC pump). Congenital Adrenal Hyperplasia (CAH) is a genetic defect of the adrenal glands. It originates by 21-hydroxylase deficiency causing abnormal production of 17 alpha-hydroxyprogesterone (17-OHP) and androstenedione. In the neonatal period it can lead to salt wasting crisis that, without a timely diagnosis, could be dangerous up to death. The average occurrence of this kind of pathology is one case out of 15000 newborns, whereas in our medical district it is considerably higher. Its recognition can be obtained by plotting the ratio 17-OHP plus androstendione divided by cortisol Vs. 17-OHP concentration. Sample preparation involves the extraction of the analytes from dried blood spots (DBS) by methanol, containing the internal standard, and on-line SPE, which makes use of a perfusion column Applied Biosystems POROS 2.1 x 30 mm. HPLC separation is carried out by a Waters Simmetry C18 2.1 x 50 mm column, by a methanol/water gradient, with a flow rate of 300 µl/min. MS-MS MRM detection is performed on an Applied Biosystems API 4000 triple quadrupole mass spectrometer, equipped with a turbo ionspray source. Several samples were analyzed in order to setup and test the method, as far as its straightforwardness, reliability, reproducibility, and ruggedness. The influence of potential interferences, typical with this kind of matrices and analytes, has been evaluated as well, resulting not critical. Moreover, the comparison between normal and pathologic samples was carried out.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11568/117884
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