Determination of thyroid hormones by solid-phase extraction using high performance liquid chromatograph/diode array detector/electro-spray ionization mass spectrometry in urine samples

An analytical method for the determination of thyroid hormones in urine samples has been studied by using solid-phase extraction and high-performance liquid chromatography/diode array detector/electro-spray mass spectrometry. Seven thyroid hormones were successfully separated by gradient elution on the reverse phase Hypersil ODS column (4.6 mm I.D., 100 mm length, particle size $5{\mu}m$) with ammonium formate buffer and acetonitrile, and UV spectra and mass fragment could be confirmed. The extraction recoveries of thyroid hormones in the urine samples (pH 3) were in the range of 89.0-113.1% with solid-phase extraction by C18, followed by elution with 4 ml of methanol/ammonium hydroxide (9 : 1). The calibration curves showed good linearity with the correlation coefficients ($r^2$) varying from 0.992 to 0.998 and the detection limits of all analytes were obtained in the range of 2-4 ng/ml (3.8-13.0 pmol/ml).

HPLC/DAD/ESI-MS 및 고체상 추출법을 이용한 뇨시료중 갑상선 호르몬 분석

본 연구에서는 뇨시료를 고체상 추출법으로 추출한 후 HPLC/DAD/ESI-MS(high-performance liquid chromatograph/diode array detector/electro-spray ionitation mass spectrometry)를 사용하여 분석하였다. 7종의 thyroid hormones의 HPLC 분리조건은 Hypersil ODS(octadecylsilica) 컬럼(4.6mm I.D., 100 mm length, particle size $5{\mu}m$)을 사용하고 ammonium formate buffer와 acetonitrile을 이동상으로 하여 기울기 용리한 결과 완전 분리가 가능하였으며, UV spectra 및 질량스펙트럼을 확인할 수 있었다. 고체상 추출법에 의한 전처리 최적 조건을 조사한 결과 시료를 pH 3으로 한 후 C18 고체상을 사용하여 4 mL의 methanol/ammonium hydroxide(9:1) 혼합용액으로 용리할 경우 회수율이 89.0-113.1%로 나타났다. HPLC/DAD를 이용하여 10-1000 ng/mL범위에서 검량선을 작성한 결과 $r^2$값은 0.992-0.998 으로 나타났으며 검출한계는 2-4 ng/mL(3.8-13.0 pmol/mL)로 계산되었다.