Differentiated quantification of human bile acids in serum by high-performance liquid chromatography–tandem mass spectrometry

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

“…For bile acids, LOQ ranged from 2nmol/l to 50nmol/l. After correction for sample volume, this was comparable or slightly better than what has been reported in recent publications providing LOQ values [16,[18][19][20][23][24] (Table 4). For C4, LOQ was 5nmol/l, which is similar to what was reported by DeBarber et al [25].…”

“…Table 4 summarizes and compares extraction recoveries of the here and previously reported methods for the measurement of bile acids and C4. 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].…”

“…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].…”

“…Bond Elut C18 cartridges from Varian, Inc. (Palo Alto, CA, USA) using a procedure previously described by our group [24], after minor adaptations (mainly reduction of sample volume). Pre-conditioning of the cartridges was achieved by activation with 2ml methanol followed by 2ml purified water and finally 2ml ammonium carbonate buffer 100mM, pH 9.3, upon which the sample was loaded onto the cartridge and allowed to pass through it by gravity.…”

“…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.…”