Metabolic Profiling of Bile Acids in Human and Mouse Blood by LC–MS/MS in Combination with Phospholipid-Depletion Solid-Phase Extraction

Abstract

To obtain a more comprehensive profile of bile acids (BAs) in blood, we developed an ultrahigh performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC-MRM-MS) method for the separation and detection of 50 known BAs. This method utilizes phospholipid-depletion solid-phase extraction as a new high-efficiency sample preparation procedure for BA assay. UPLC/scheduled MRM-MS with negative ion electrospray ionization enabled targeted quantitation of 43 and 44 BAs, respectively, in serum samples from seven individuals with and without fasting, as well as in plasma samples from six cholestatic gene knockout mice and six age- and gender-matched wild-type (FVB/NJ) animals. Many minor BAs were identified and quantitated in the blood for the first time. Method validation indicated good quantitation precision with intraday and interday relative standard deviations of ≤9.3% and ≤10.8%, respectively. Using a pooled human serum sample and a pooled mouse plasma sample as the two representative test samples, the quantitation accuracy was measured to be 80% to 120% for most of the BAs, using two standard-substance spiking approaches. To profile other potential BAs not included in the 50 known targets from the knockout versus wild-type mouse plasma, class-specific precursor/fragment ion transitions were used to perform UPLC-MRM-MS for untargeted detection of the structural isomers of glycine- and taurine-conjugated BAs and unconjugated tetra-hydroxy BAs. As a result, as many as 36 such compounds were detected. In summary, this UPLC-MRM-MS method has enabled the quantitation of the largest number of BAs in the blood thus far, and the results presented have revealed an unexpectedly complex BA profile in mouse plasma.

Quantitative analysis of endogenous compounds

“…Various approaches ( Figure 2) are followed to address the lack of blank matrices for the quantification of endogenous compounds by LC-MS/MS including, background subtraction [26-31], the standard addition method [24,25,[32][33][34][35][36][37][38][39][40][41][42][43][44], neat solutions [45][46][47][48][49][50][51][52], artificial matrices of biological fluids [23,, stripped matrices [8,10,11,[74][75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91], and surrogate analytes [66,[92][93][94][95][96][97]…”

“…In its simplest form, mobile-phase solvents (neat) [48,49,51] or pure water [45,47,50,52] can be used as a surrogate matrix. To use neat solutions as surrogate matrices, it is required to demonstrate that extraction recovery and matrix effect are comparable with the original matrix.…”

The mysterious relationship between reproduction and longevity

“…Also, as discussed above, the NHR-8 ligand involved in the DR response is not known. The secretion of most bile acids is induced upon feeding in mammals, but some are overproduced upon fasting as well 35 which makes it difficult to predict which steroid hormone might function analogously to dafachronic acid. However, it is interesting to note that FXR was recently described to affect autophagy, 36,37 as does NHR-8.…”

Sample preparation for polar metabolites in bioanalysis

“…5,8 When high selectivity towards polar metabolites or improved sample clean-up is required, LLE is performed with water immiscible organic solvents such as dichloromethane or hexane (addition of a cosolvent can be performed to slightly modify the selectivity) to remove most of the lipophilic interferents and retain the hydrophilic metabolites in the original aqueous phase. 10 SPE is also used to remove proteins and sphingolipids 11,12 and is generally followed by conventional LLE. 13 These multi-step approaches feature the advantage of compartmentalizing the sample: proteins, lipophilic metabolites, and hydrophilic metabolites are all recovered in different fractions, which can be separately analyzed and detected with appropriate analytical techniques.…”