Novel LC–MS/MS method for assay of 7α-hydroxy-4-cholesten-3-one in human plasma

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).…”

“…Several of them (GDCA, TDCA and TLCA) were totally absent from the circulation as a consequence of breakdown of enterohepatic circulation. The concentration obtained for C4 was 1.2nmol/l, (<LOQ) and indicates poor liver function due to cirrhosis, as C4 concentrations in healthy subjects have been reported to be between 12nmol/l and 120nmol/l (5-48ng/ml) [14].…”

“…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.…”