Highly sensitive quantification of 7α-hydroxy-4-cholesten-3-one in human serum by LC-ESI-MS/MS

Abstract

We describe a highly sensitive and specific method for the quantification of serum 7a-hydroxy-4-cholesten-3-one (C4), which has been used as a biomarker for bile acid biosynthesis. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). C4 was extracted from human serum (2-50 ml) by a salting-out procedure, derivatized into the picolinoyl ester (C4-7a-picolinate), and then purified using a disposable C 18 cartridge. The resulting picolinoyl ester derivative of C4 was quantified by LC-MS/MS using the electrospray ionization mode. The detection limit of the C4 picolinoyl ester was found to be 100 fg (signal-to-noise ratio 5 10), which was z1,000 times more sensitive than the detection limit of C4 with a conventional HPLC-ultraviolet method. The relative standard deviations between sample preparations and between measurements by our method were calculated to be 5.7% and 3.9%, respectively, by one-way layout analysis. The recovery experiments were performed using serum spiked with 20.0-60.0 ng/ml C4 and were validated by a polynomial equation. The results showed that the estimated concentration with 95% confidence limit was 23.1 6 2.8 ng/ml, which coincided completely with the observed X 0 6 SD 5 23.3 6 1.0 ng/ml with a mean recovery of 93.4%. This method provides highly reliable and reproducible results for the quantification of C4, especially in small volumes of blood samples.

Quantification of the 15 major human bile acids and their precursor 7α-hydroxy-4-cholesten-3-one in serum by liquid chromatography–tandem mass spectrometry

“…Extraction recoveries for bile acids were quite similar to those reported for other existing methods [16][17][18]20,[22][23][24]. The extraction recovery for C4 was found to be approximately 62%, which was markedly lower than for bile acids and for other C4 methods [12,14,[25][26]. This is presumably explained by its lipophilic properties and by the fact that the others methods used different sample preparation techniques (protein precipitation, liquid-liquid extraction followed by SPE, derivatisation and salting-out respectively).…”

“…In contrast to gas chromatography, which requires laborious sample preparation including hydrolysis and derivatisation, liquid chromatography coupled with mass spectrometry (LC-MS(/MS)) nowadays represents the most convenient alternative for this type of analysis, as demonstrated by the number of new quantification methods developed in the past years for bile acids [16][17][18][19][20][21][22][23][24] and C4 [12,14,[25][26].…”

“…Our new methods each required only 100μl of serum sample, which was less than for many of the existing methods [12,14,16,20,[22][23][24] although methods for quantification of C4 in even smaller sample volumes exist [25][26]. A low sample volume represents an important advantage if the method is to be adapted for analysis of mouse samples (muricholic acids), since mice have a very small blood volume.…”

“…A low sample volume represents an important advantage if the method is to be adapted for analysis of mouse samples (muricholic acids), since mice have a very small blood volume. In addition, our C4 method offers the advantage of requiring a rather short sample preparation in comparison to other existing methods [14,[25][26] and no derivatization [25][26]. However, in order for these methods to be improved, efforts should be made to find a combined extraction procedure for bile acids and C4, which would reduce sample volume to only 100μl instead of twice 100μl and would lead to a shorter total analysis time.…”

Proteomics Analysis of Porcine Serum Proteins by LC-MS/MS after Foot-and-Mouth Disease Virus (FMDV) Infection

“…Peptides were desalted using a peptide microtrap and eluted using a solution of 0.1% trifluoroacetic acid and 95% acetonitrile [17]. Desalted peptides were dried in a vacuum centrifuge and resuspended in 20 ml 0.1% formic acid for analysis by LC-MS/MS as described by Honda [9].…”