Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS.
We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 mul of dried serum were derivatized into picolinyl esters (3beta-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously. Quantitative analyses for the picolinyl esters of 11 available sterols were performed, and detection limits were found to be less than 1 pg on-column. Reproducibilities and recoveries of 8 noncholesterol sterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.6% to 8.2% and 2.5% to 16.5%, respectively. The recovery experiments were performed using 1 mul aliquots of normal human serum spiked with 1 ng to 6 ng of sterols, and recoveries of the sterols ranged from 88.1% to 102.5% with a mean recovery of 98.1%. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.
Honda
Akira
A
Center for Collaborative Research, Tokyo Medical University, Kasumigaura Hospital, Ami, Ibaraki 300-0395, Japan.
Yamashita
Kouwa
K
Miyazaki
Hiroshi
H
Shirai
Mutsumi
M
Ikegami
Tadashi
T
Xu
Guorong
G
Numazawa
Mitsuteru
M
Hara
Takashi
T
Matsuzaki
Yasushi
Y
D018938:Caco-2 Cells; D002806:Chondrodysplasia Punctata / Q000097:blood; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D010848:Picolinic Acids / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012855:Sitosterols / Q000097:blood; D019082:Smith-Lemli-Opitz Syndrome / Q000097:blood; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013261:Sterols / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D019294:Xanthomatosis, Cerebrotendinous / Q000097:blood
D010848:Picolinic Acids; D012855:Sitosterols; D013261:Sterols; C030614:picolinic acid