Sample Preparation Techniques for Bile Acid Analysis

Bile acids (BAs) are amphipathic molecules composed of a hydroxylated steroid nucleus and a hydrocarbon chain containing a terminal carboxyl group. They are the end products of cholesterol metabolism and an important component of bile. Additionally, BAs serve as significant signaling molecules in the body, participating in glucose and lipid metabolism through various signaling pathways. They play a crucial role in maintaining normal physiological functions and homeostasis.

Types of Samples in Bile Acid Analysis

Serum/Plasma

Serum and plasma are minimally invasive, information-rich samples frequently used in metabolomics studies, making them a staple for bile acid analysis. Research has shown that BA concentrations do not differ significantly between serum and plasma, but plasma's anticoagulants can interfere with analysis. As a result, serum is generally preferred as a sample matrix for BA detection.

Urine

Urine is a common sample type for clinical diagnostics, containing a mix of water, inorganic salts, urea, uric acid, and other compounds. As an amphipathic compound class, BAs exist in measurable concentrations in urine, making it suitable for metabolic fingerprinting. Urine allows for analyzing both free and conjugated bile acid species, providing insight into various metabolic and pathological conditions.

Saliva

Saliva has emerged as a promising, non-invasive sample type for bile acid analysis. It offers several advantages, including ease of collection and the potential for real-time monitoring of physiological changes. BAs in saliva are derived from the circulation and can reflect the physiological state of the body, including variations in metabolic pathways. The concentration of BAs in saliva, although generally lower than in other biological fluids, can provide valuable information on the systemic status of bile acid metabolism. For analysis, saliva samples are typically processed through centrifugation to remove cellular debris, followed by protein precipitation or solid-phase extraction to isolate the BAs for quantification.

Bile

Bile is a complex, greenish-brown fluid, rich in BAs, with concentrations and profiles that closely reflect the physiological state of the organism. Primary bile acids are typically abundant, requiring dilution before UPLC-MS/MS analysis to adjust for their high concentrations. Bile sample analysis is particularly useful for evaluating physiological changes in liver and biliary health.

Liver Tissue

BAs play a significant role in liver-related conditions, such as intrahepatic cholestasis, non-alcoholic fatty liver disease, chronic hepatitis, and liver cancer. Liver samples are less accessible than other biological matrices and are thus often obtained from laboratory animals. Due to the dense matrix, sample preparation for liver tissue demands rigorous purification methods to isolate BAs accurately.

Intestinal Contents and Feces

BAs in intestinal contents and feces reflect the microbial conversion and enterohepatic recycling of these metabolites, offering a window into host-gut microbiota interactions. Fecal and intestinal samples, being compositionally complex, necessitate extensive sample treatment, including homogenization and extraction, to ensure accurate BA quantification.

Sample Preparation Techniques for Bile Acid Analysis

The accurate quantification of bile acids in biological matrices necessitates meticulous sample preparation techniques tailored to the specific characteristics of each matrix. These techniques aim to isolate BAs from complex biological fluids while minimizing interference from other matrix components. The following methodologies are commonly employed for the preparation of samples used in bile acid analysis:

Organic Solvent Extraction

Organic solvent extraction remains a foundational technique for isolating BAs from biological samples such as serum, plasma, and bile. This method typically involves the addition of an organic solvent (e.g., methanol, acetonitrile) to the sample, facilitating the precipitation of proteins and the extraction of BAs into the organic phase.

Procedure:

• Sample Preparation: An aliquot of serum or plasma is mixed with a predefined volume of organic solvent, usually in a ratio of 1:3 or 1:4 (sample to solvent).
• Vortexing: The mixture is vortexed for a set duration (e.g., 10 minutes) to ensure thorough mixing and protein precipitation.
• Centrifugation: Following vortexing, the sample is subjected to centrifugation (e.g., at 10,000 rpm for 10 minutes) to separate the precipitated proteins from the supernatant, which contains the extracted BAs.
• Evaporation: The organic phase is evaporated under a stream of nitrogen or using a vacuum concentrator, followed by reconstitution in a suitable mobile phase (e.g., 0.1% formic acid in water or acetonitrile) for analysis.

Organic solvent extraction is advantageous for its simplicity and effectiveness in removing proteins while achieving good recovery rates for BAs. However, careful selection of the solvent and optimization of the extraction conditions are critical to minimizing losses of target analytes and avoiding the co-extraction of interfering substances.

Liquid-Liquid Extraction (LLE)

Liquid-liquid extraction (LLE) is particularly effective for isolating BAs from more complex matrices, such as liver and fecal samples. This technique capitalizes on the differences in solubility of BAs in various solvents to achieve separation.

Procedure:

• Homogenization: For liver samples, tissues are homogenized in a mixture of deionized water and an organic solvent (e.g., ethyl acetate, dichloromethane).
• Phase Separation: The homogenate is subjected to centrifugation to separate the aqueous phase from the organic phase.
• Extraction: The organic phase is collected and may undergo a second extraction step with a fresh portion of organic solvent to enhance the recovery of BAs.
• Concentration: The combined organic phases are evaporated under reduced pressure or nitrogen, and the residue is reconstituted in an appropriate solvent for further analysis.

LLE allows for the effective separation of BAs from other biological components, but it may require multiple extraction steps to achieve optimal recovery. Additionally, LLE is sensitive to the choice of solvents and the ratios employed, which necessitates thorough method development for specific applications.

Solid Phase Extraction (SPE)

Solid phase extraction (SPE) is a highly versatile and widely used method for the purification and concentration of BAs from various biological samples, including urine, bile, and liver homogenates. SPE employs a solid sorbent material to selectively retain BAs while allowing other matrix components to wash through.

Procedure:

• Column Conditioning: The SPE column is preconditioned with a suitable solvent (often methanol followed by water) to ensure optimal binding of BAs.
• Sample Loading: The prepared sample is then passed through the column, allowing BAs to bind to the sorbent while unwanted compounds are washed away.
• Washing: A washing step is performed using a buffered solution or an organic solvent to further remove impurities without eluting the bound BAs.
• Elution: BAs are eluted from the column using a stronger solvent (e.g., methanol or a mixture of methanol and water), which is then collected for analysis.

SPE is favored for its ability to concentrate BAs while effectively removing interfering substances, leading to improved sensitivity in subsequent analytical methods. This technique can be adapted by selecting specific sorbent materials tailored to the properties of the target analytes, further enhancing extraction efficiency.

Solid-Phase Microextraction (SPME)

Solid-phase microextraction (SPME) is a novel technique that has gained traction for the extraction of trace levels of BAs from various biological matrices, including serum and urine. SPME involves the use of a coated fiber to adsorb analytes directly from the sample matrix.

Procedure:

• Fiber Exposure: The SPME fiber is exposed to the sample for a specified period, allowing BAs to partition onto the fiber coating.
• Desorption: After extraction, the fiber is inserted into the injection port of a gas or liquid chromatograph, where the absorbed BAs are desorbed and introduced into the analytical system.

SPME offers several advantages, including minimal sample handling, reduced solvent usage, and the ability to perform extraction under near-physiological conditions. This technique is particularly beneficial for analyzing volatile or semi-volatile compounds, but it may require careful calibration to achieve consistent recovery rates.

Sample Preparation for Serum/Plasma and Urine Samples

Protein Precipitation

Protein precipitation is a robust and widely applied method in bile acid (BA) analysis for serum, plasma, and urine samples. This technique aims to effectively separate proteins that could interfere with the analysis by binding or trapping analytes, ensuring cleaner sample profiles for downstream quantification. To precipitate proteins, a precipitating agent—commonly methanol or acetonitrile—is added to the sample in volumes approximately 3–4 times that of the biological fluid. An internal standard, often isotope-labeled to control for matrix effects, may also be incorporated to improve quantification accuracy.

Following the addition of the organic solvent, the sample is vortexed thoroughly to enhance protein denaturation and facilitate the precipitation of proteins. The mixture is then centrifuged at a low temperature (e.g., 4°C) and high speed to ensure complete separation of the precipitated proteins from the supernatant, which contains the BAs. The supernatant is collected and subsequently evaporated to dryness under nitrogen or vacuum to remove residual solvent. This dried extract is finally reconstituted in a suitable solvent, typically a mobile phase compatible with UPLC-MS/MS analysis, ensuring that BAs are in optimal condition for accurate detection and quantification.

Dilution in Urine Analysis

Due to its less complex matrix compared to serum or plasma, urine is amenable to simpler preparation steps for BA analysis. In some cases, urine samples can be analyzed after a straightforward dilution, circumventing more labor-intensive protein precipitation steps. Dilution not only simplifies sample preparation but also reduces potential matrix effects that might otherwise interfere with the ionization of BAs in mass spectrometric detection.

When investigating both free and conjugated BAs, enzyme hydrolysis is often employed as an additional step. Enzyme incubation allows for the release of conjugated BAs, providing a comprehensive profile of both free and conjugated species. This method involves the addition of specific enzymes to the urine sample, followed by a controlled incubation period. Once hydrolysis is complete, the sample can be diluted to a suitable concentration and directly injected for analysis.

Sample Preparation for Saliva Samples

Centrifugation and Filtration

Saliva samples require careful handling to ensure the accurate analysis of bile acids (BAs). Initially, saliva is collected in sterile containers, preferably after fasting to reduce contamination. Following collection, the samples should be centrifuged at low temperature (4°C) for approximately 10-15 minutes at high speed (e.g., 10,000 rpm). This step is crucial for removing cellular debris and other particulate matter, resulting in a clear supernatant for further processing.

Protein Precipitation

To eliminate potential interferences from proteins present in the saliva, a precipitating agent—commonly methanol or acetonitrile—is added to the supernatant in a volume about 3-4 times that of the saliva sample. The mixture is vortexed thoroughly to ensure complete mixing and denaturation of proteins. Following vortexing, a subsequent centrifugation step is performed to separate the precipitated proteins from the supernatant, which contains the bile acids.

Evaporation and Reconstitution

The supernatant is then evaporated to dryness under a nitrogen stream or using a vacuum concentrator to remove any residual solvent. The dried extract is reconstituted in an appropriate mobile phase (e.g., 0.1% formic acid in water or acetonitrile) to prepare it for analysis, ensuring optimal conditions for accurate detection and quantification of bile acids.

Enzyme Hydrolysis (Optional)

For comprehensive profiling of both free and conjugated bile acids, an enzyme hydrolysis step may be incorporated. Specific enzymes (e.g., glucuronidases) are added to the saliva sample prior to protein precipitation, and the mixture is incubated for a controlled period. This hydrolysis facilitates the release of conjugated bile acids, enhancing the overall profile obtained during analysis.

Sample Preparation for Bile Samples

Dilution

Given the high BA concentration in bile, dilution with purified water or organic solvents is often necessary to prevent saturation during analysis. This method also helps in reducing matrix complexity, allowing more precise quantification of BAs.

Solid Phase Extraction (SPE)

For more advanced bile sample preparation, solid phase extraction (SPE) is employed. The sample is diluted, mixed with internal standards, and passed through a C18 SPE column preconditioned with methanol. The column is sequentially washed with water and methanol to elute purified BAs, which are then collected and concentrated before UPLC-MS/MS analysis. SPE not only enhances BA purity but also improves recovery rates, with typical values ranging from 89.1% to 100.2%.

Sample Preparation for Liver Samples

Liver sample preparation for BA analysis often relies on liquid-liquid extraction (LLE) due to the matrix complexity of tissue samples. After homogenizing liver tissue in deionized water, a solvent (e.g., acetonitrile) is added to extract the BAs. The extraction is usually performed twice to ensure maximum recovery. Following centrifugation, the supernatant is evaporated to dryness, and the residue is reconstituted in a suitable solvent. This technique ensures high selectivity and sensitivity for BA quantification.

Sample Preparation for Intestinal Contents and Fecal Samples

Freeze-Drying and Single-Phase Extraction

Preparation of intestinal and fecal samples typically starts with freeze-drying to remove water content, followed by homogenization to ensure uniformity. Single-phase extraction involves diluting the sample in an organic solvent and centrifuging it at low temperatures to remove non-analyte proteins and sediment. This simple yet effective approach helps isolate BAs for further analysis.

Biphasic Extraction

Biphasic extraction, designed for more comprehensive profiling, entails treating samples with a mixture of solvents (e.g., methyl tert-butyl ether, methanol, and water) to extract polar and non-polar BA components. After ultrasonication and centrifugation, ultrapure water is added to induce phase separation, which isolates the different BA types for a broader characterization of fecal BAs.

Extraction and quantitative determination of bile acids in feces (Shafaei et al., 2021)

References

1. Dosedělová, Věra, et al. "Optimization of saliva sampling methods for analysis of bile acids by UHPLC-MS." Journal of Chromatography A 1736 (2024): 465354.
2. Shafaei, Armaghan, et al. "Extraction and quantitative determination of bile acids in feces." Analytica Chimica Acta 1150 (2021): 338224.