SUN-212 Interference in Serum Androstenedione Measured by LC-MS/MS in Newborns Samples

Abstract

Introduction: Liquid chromatography followed by mass spectrometry (LC-MS/MS) is considered the gold standard method to measure steroids. Newborn screening for congenital adrenal hyperplasia (CAH) involves measurement of 17α-hydroxyprogesterone (17-OHP) in blood dried spots by immunoassay. Because this testing has high false-positive rates, serum samples to measure 17-OHP, androstenedione, 21-desoxicortisol and cortisol simultaneously by liquid chromatography-tandem mass spectrometry (LC-MS/MS) are used for confirmatory test in our laboratory. Objective: To report an interference in androstenedione levels measured by LC-MS/MS assay in serum samples from newborns. Patients and methods: The method for androstenedione measurements was based on protein precipitation followed by a semi-automated and multiplexed on-line solid phase extraction coupled reverse phase separation and detection of underivatized analyte by tandem mass spectrometry. Among 312 samples 82 presented unexpected androstenedione results considering that 17OHP levels were <5 ng/mL. These samples presented a high variability among 4 replicates (CV ranged from 20 to 133%). These samples also showed an inadequate ion ratio resulting in pseudo-elevated androstenedione, indicating a coeluition of an isobaric interferent. In routine samples from other patients this problem was not observed. Results: Since this fact suggests a possible interference in LC-MS/MS measurements and modification in chromatographic method was unable to resolve from the interference, alternative method for sample preparation was developed. Liquid-liquid extraction with diethyl ether was performed and eliminated the interference and provided substantial decrease in androstenedione values (9.3+12.94 ng/mL vs 5.2+9.59 ng/mL after extraction) with ion ration normalization and CV less 10% between replicates. The identity of this compound is still unknown. Therefore, it will be necessary additional studies to clarify this artifact. Conclusions: Although the measurement of androstenedione by reverse phase chromatography without derivatization followed by tandem mass spectrometry is the simplest and commonest approach to determine androstenedione, it is susceptible to interferences causing falsely elevated androstenedione levels in newborns.