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A simple and reliable bile acid assay in human serum by LC‐MS/MS

Abstract Background Bile acids, as important signaling molecules and regulatory factors acting on glucose, lipid, and energy metabolism, are always involved in liver, biliary, and intestinal diseases. Development and validation of a simple liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for determination of bile acids is significant for the routine clinical testing. Methods Fifty microlitre of serum was mixed with 10 μl of the internal standard working solution and then 140 μl of methanol for protein precipitation. After centrifuged, the supernatant was directly used for LC‐MS/MS analysis. Results Good separation of all bile acid species was achieved. The method was validated with consistent linearity for individual bile acids, good recovery, low carryover, satisfactory sample stability, and analytical specificity against hemolysis, lipemia, and bilirubinemia. The intra‐day and the inter‐day imprecision values were in the range of 1.53%–10.63% and 3.01%–13.98%, respectively. No obvious matrix effect was observed. The reference intervals of bile acids in adults have been established for the clinical testing. Conclusions The low sample volume, simple sample preparation, good separation of all species, and satisfying validation results make this LC‐MS/MS approach suitable for usage as a high‐throughput assay in routine clinical laboratories.

bile acid synthesis is tightly controlled by feedback mechanisms in health, and bile acids are implicated in many disease states, such as cholestasis, hepatic and intestinal cancers, liver cirrhosis, and diabetes mellitus. 9 Their multiple physiological, pathological, and pharmacological functions have kept bile acids as a research focus all the time. There are many differences in bile acid composition in various fluids and tissues due to the recycling of bile acids in the body. In human blood, CA, CDCA, DCA, LCA, and UDCA, as primary and secondary bile acids, are dominated. Among these, CA, CDCA, and DCA are mainly unconjugated. 6 The conjugated ones primarily exist in the amidated (mostly glycine and taurine) forms, 10 among which GCA is well-known for diagnosis of intrahepatic cholestasis of pregnancy, 11 and TCA is correlated well with liver injury. 12 As a result, simultaneous determination of CA, CDCA, DCA, LCA, UDCA, GCA, and TCA levels in human body is significant for monitoring of many diseases.
Up to now, many analytical methods have been developed to quantify BA concentrations in different kinds of biological samples.
Enzymatic methods, due to their great simplicity and availability, have been predominant in the determination of bile acids up to the present day but may underestimate the total bile acid concentration. 13,14 Considering that the study of BA functions requires methods which cover the structurally diverse group of molecules, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has drawn great attention to quantify bile acids owing to its high sensitivity and specificity for distinguishing structural analogues. [15][16][17][18][19][20][21][22][23][24] However, most of these studies necessitated a dryness process in sample preparation by protein precipitation or solid phase extraction (SPE). This dryness process is time-consuming not favorable for rapid analysis in routine laboratories and is a source of potential errors due to the uncertainty in the dryness and re-dissolution process. As a result, sample preparation for determination of bile acids as simple as possible is significant in clinical testing for the characterization of the role that bile acids play in pathology.
The aim of this study was to validate a simple and reliable LC-MS/MS method in our laboratory for determination of serum bile acids. The primary reference intervals in a reference population of 130 volunteers have been further established.  2022/08/04) was supplied by Sysmex Co., LTD. When purity and expire date were not found on the reagent boxes or in the reagent instructions, they were not indicated here. Deionized water was freshly prepared by a water purification system (ELGA).

| LC-MS/MS conditions
The LC-MS/MS analysis was performed on a AB Sciex Exion LC coupled to a Qtrap 5500 (Applied Biosystems/Sciex). The LC system was consisted of a gradient pump, a vacuum degasser, a temperaturecontrolled autosampler, and a temperature-controlled column oven.
A Thermo Hypersil Gold C18 (2.1 × 100 mm, 1.9 μm) column was used for separation. The mobile phase consisted of solvent A (water containing 0.1% FA) and solvent B (methanol and acetonitrile at a ratio of 2:1 v/v containing 0.1% FA). Gradient elution was achieved as follows: 0-6.0 min, 50%B-72%B; 6.0-14.00 min, 72%B-80%B; 14.01-15.50 min, keeping 100%B and finally running 50%B for reequilibration of the column with 1.5 min. The flow rate was 0.3 ml/ min. The column temperature was 40°C, and the sample injection volume was 10 µl. Between injections, the autosampler syringe was washed with methanol.
The Qtrap 5500 was operated in negative mode. The optimal MS conditions were as follows: ion spray voltage of −4500 V, temperature of 450°C, curtain gas of 30.0 psi, ion source gas 1 of 40 psi, and ion source gas 2 of 50 psi. The specific selected ion monitoring (SIM) or multiple reaction monitoring (MRM) transitions for quantification under optimal conditions were monitored as shown in Table S1. 23 The MS system was controlled by a Analyst 1.6.1 software. in methanol were equally mixed to generate a IS working solution.

| Preparation of solutions, calibrators, and quality controls (QCs)
All above working solutions were stored at −40°C until analysis. It is noted that all set-ups used for standard solution preparation including glassware, pipettes, and analytical balance are calibrated annually according to the College of American Pathologists checklist for our lab.  25 The level of QCL for each analyte was below the reference interval and QCL was denoted as the normal range control, while the level of QCH was above the reference interval and QCH was denoted as the pathological range control.
In addition, a pooled serum specimen prepared by mixing real human serum samples, as a pooled quality control (PQC), was used as an additional quality control for good daily monitoring. The PQC was aliquoted and stored at −80°C until use. The target concentra-

| Sample collection and preparation
To investigate the influence of serum collection tubes on testing of bile acids, five subjects were selected for blood collection in both serum separator tubes with and without separator gel, and the concentrations of bile acids were compared. For clinical testing, blood samples were collected into yellow tubes with separator gel. After 1 h at room temperature for clotting, the samples were centrifuged at 850 × g for 5 min.
Calibrators/QCs/serum specimens (50 μl) were mixed with 10 μl of the IS working solution, and then, 140 μl of methanol was added into tubes for protein precipitation. The tubes were vortexed for 3 min and then centrifuged for 10 min at 20238 × g. The supernatants were used for direct analysis.

| Method validation
The LC/MS/MS method was validated emphasizing on linearity, the lower limit of quantification (LLOQ), the limit of detection (LOD), precision, accuracy, sample stability, carryover, and analytical specificity according to the European Medicines Agency guideline on bioanalytical method validation (European Medicines Agency 2011).
Linearity was evaluated by analysis of a 9-point calibration curve.
The calculated concentrations of the calibration standards should be within ±15% of the nominal value, except for the LLOQ for which it should be within ±20%. At least 75% of the calibration standards must fulfill this criterion. The LLOQ, defined as the lowest concentration with a signal-to-noise ratio >10, was determined by assaying a series of low-concentration calibrators in five replicates and established based on the criteria that a coefficient of variation (CV) of <20% and an accuracy within 80%-120%. The LOD represents the concentration that gives a signal-to-noise ratio >3.
Quality controls and PQC were used to evaluate imprecision.
Five replicates of each level were assayed over five days to evaluate the intra-day and inter-day precision. The CVs were within the accepted validation criteria of less than 20% at the LLOQ and less than 15% at all other tested concentrations.
Accuracy of the method was evaluated by a recovery study. In

| Serum levels of bile acids in the reference population
Serum specimens were obtained from healthy subjects (n = 130, 65 females and 65 males) with normal renal and hepatic function, normal lipid levels, and no underlying diseases. Study subjects had fasted overnight. The concentrations of bile acids for these subjects were measured using the new method on their collecting day.

| Statistics
Statistical analyses were carried out using SPSS Statistics 20.0 software package. Values below LOD were defined as LOD. 27 The test results from different types of blood collection tubes were compared by paired t test, and statistical significance was defined as p < 0.05. 28 The normality of the data was tested by a one-sample Kolmogorov-Smirnov test. The outermost 5% of observations were used to define limits for one-side reference intervals (95th for bile acids).

| LC-MS/MS conditions
In recent years, LC-MS/MS has become the most suitable method for the quantification of bile acids due to its selectivity and specific- In company of the SIM or MRM parameters displayed in Table S1, the target bile acids in the present study could be quantified reproducibly and accurately.

| Sample collection and preparation
For clinical testing of bile acids by LC-MS/MS, types of blood collection tubes were investigated. Serum separator tubes with or without separator gel had no obvious influence on concentrations of bile acids (p all above 0.05, paired t test). In addition, the percentage differences of the testing results between the two collection tubes were in the range from −12.69% to 21.25% as displayed in Table S2.
All these indicated that both types of blood collection tubes might be used in clinical tests. However, the type of blood collection tubes should be consistent in the routine clinical testing.

| Method validation
Linearity was determined by analysis of a 9-point calibration curve.
Charcoal stripped serum after removal of bile acids was used as the blank serum matrix for preparation of calibrators. The linear correlation coefficients (r 2 ) of the calibration curves for all analytes above 0.9945 exhibited excellent linearity for quantification. A high linearity response over a sufficient dynamic concentration range was observed as summarized in Table 1. The inter-assay variability of the calibration data was ≤15.88% as shown in Table S3.

Analysis of a series of low-concentration calibrators resulted in
an accuracy within 80%-120% and a CV value <20% at the concen-  Table 1.
Imprecision was calculated as the CV for QCs prepared in the blank serum and PQC prepared from a serum pool. Intra-day CVs  Table 3.  (Table 4). All these results demonstrated that there was no significant ion suppression or ion enhancement at different analyte concentrations.
In evaluation of carryover, the high concentration sample and the blank sample were alternated for six times. At the retention time of individual bile acids, the average peak area associated with the blank sample was below average peak area of the LOD as shown in Table S4, indicating that no significant carryover was found.
Stability of human serum was assessed freshly and after storage under a certain condition at different time points (Day 1, 2, 3, 7, 14, 21, and 28). As displayed in Figure 3, the percentage differences,

| Serum levels of bile acids in the reference population
Bile acids are formed in the liver from cholesterol, conjugated primarily to glycine and taurine, stored and concentrated in the gallbladder, and secreted into the intestine after the ingestion, and then Note: *100% is used as the recovery baseline.
The recovery was evaluated at four levels. At the lowest level, the recoveries of UDCA, GCA, CDCA and CA were between 11.28% and 18.32%. often been used for the extraction of bile acids from complex matrix. However, unsatisfying recovery rates and poor reproducibility values were observed in previously described SPE procedures. 20 As a result, protein precipitation (PP) method has become an alternative to SPE method. However, in most PP procedures, the supernatants should be collected for dryness and re-dissolution which is time-consuming. Herein, a simple one-step PP process based on good separation of bile acids and high sensitivity of the instrument has been developed for successful measurement of bile acids, even though the LCA has a lower physiological concentration. 25 In this pre-treatment process, dryness was not needed and the supernatant could be used for direct LC-MS/MS analysis. This simple sample pre-processing step could ensure its robust application in clinical laboratories.

TA B L E 4 Matrix effect
A potential beneficial use of our method for the investigation of bile acid levels in human serum in clinical laboratories was further ensured by full validation of this LC-MS/MS methods with consistent linearity (r 2 between 0.9945 and 0.9958), satisfactory imprecision (within 1.53%-13.98%), and accuracy (between 82% and 117%). The established reference intervals of bile acids in adults were comparable to that in the Mayo Clinic Laboratories. 25 Despite of the satisfactory analytical performance as discussed above, our study does suffer from the following limitations: firstly, the number of bile acid species is limited; secondly, no commercial quality controls were available and rigorous concordance study was not performed to verify inter-laboratory accuracy.

| CON CLUS ION
In summary, a simple and reliable LC-MS/MS method for the si-

ACK N OWLED G EM ENT
None.

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data can be available if needed.