Biological fluid screening and confirmation of bile acids by use of an integrated flow-injection-LC-evaporative light-scattering system

Summary An integrated flow-injection-liquid chromatographic system is presented for the screening of biological fluids for bile acids, and for subsequent confirmation. It is based on the use of a flow-injection configuration with an evaporative light-scattering detector (ELSD), for routine screening for the total concentration of the analytes, followed by the individual determination, by liquid chromatography, of only those samples for which the total concentration is close to or above the normal level in healthy individuals. Bile acids are extracted from human serum and urine withn-hexane under acid conditions. After phase separation the organic layer is evaporated to dryness under a stream of nitrogen and redissolved in weakly alkaline solution (pH 8). The aqueous phase is then passed through a miniaturized Amberlite XAD-7 column for preconcentration and clean up, and eluted with acetonitrile-methanol, 65∶35 (v/v). The effluent is introduced directly into the ELSD for determination of the total bile-acid content of the sample. For confirmatory analysis another aliquot of the sample is processed in the screening module and the effluent from the adsorbent column is then directed to the chromatographic column to obtain the bile acid profile of the sample. Good agreement was obtained in the analysis of urine and serum samples by both procedures.     

Characterization of bile acids and fatty acids from ox bile in oil paintings by gas chromatography-mass spectrometry

Characterization of ox bile, traditionally used in painting, is of interest in the fields of archaeometry and conservation and restoration of works of art. Bile acids, fatty acids (F), and cholesterol found in ox bile have been identified using a derivatization method that combines the formation of ethyl esters from the carboxylic groups and the trimethylsilyl ethers from hydroxyl groups. This method of analysis is consistent with these others proposed by the authors to analyze drying oils, proteins, and diterpenic resins usually used as binders and varnishes by the painters. Bile acids from binary samples such as animal glue/ox bile, casein/ox bile and Arabic gum/ox bile have been successfully analyzed using the proposed method. Finally, a method of analysis of mixtures of drying oil and ox bile has been also proposed attempting to quantitatively characterize samples in which ox bile was added to the drying oil for increasing the surfactant properties.     

Capillary gas chromatography/negative ion chemical ionization mass spectrometry for the quantification of bile acids including 1 beta-hydroxylated and unsaturated bile acids in serum and urine

12 bile acids, including 1 beta-hydroxylated and unsaturated bile acids, have been quantified by capillary gas chromatography/negative ion chemical ionization mass spectrometry, using the trimethylsilyl(TMS) ether derivatives of bile acid pentafluorobenzyl(PFB) esters. The analysis time is 12 min and the minimum measurable amount is 100 fg for each bile acid. Bile acids in 200 microL of serum and 50 microL of urine from healthy human adults were measured. These small sample sizes enhance the practicality of using this method as a screening test for bile acids in the serum and urine of human infants, where small sample size is a major problem.     

A Fully Automatic Method for Analysis of Individual Bile Acids in the Serum Using High-Performance Liquid Chromatography for Clinical Use

Abstract

We have reported highly sensitive methods for the analysis of individual bile acids in the serum using high-performance liquid chromatography coupled with an enzymatic fluorometric system. In this report, a new system equipped with a sample treatment mechanism for chromatographic analysis of serum bile acids is detailed. Most of the protein and other hydrophilic components of the injected serum sample are removed in the pretreatment system, so that only the remaining bile acids are introduced into the chromatographic system and eluted with irrigants containing a coenzyme (NAD) for fluorometric detection. With this method, we are able to simultaneously determine 15 different serum bile acids in an hour without the tedious manual sample processing steps. This system opens up an approach to fully automated analysis of bile acids in the blood.     

Development and validation of a method for measuring the glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography

We developed and validated a simple method for measuring the individual glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography with a C18 reversed-phase column using an isocratic solvent system of acidified methanol--potassium phosphate. Without preliminary derivatization or purification, complete separation of the ten major conjugated bile acids present in bile could be achieved in 65 min. Total bile acid concentrations were identical when measured enzymatically and by summing the individual bile acids determined by high-performance liquid chromatography. Bile acid composition determined by gas-liquid chromatography correlated with results by high-performance liquid chromatography. Finally, measurements of individual glycine and taurine conjugates in human bile and in mixtures of bile acid standards by high-performance liquid chromatography and thin-layer chromatography gave similar results. This high-performance liquid chromatographic system permits simultaneous quantification of total and individual bile acids and their glycine and taurine conjugates in bile.     

Determination of conjugated bile acids in human urine by high-performance liquid chromatography with chemiluminescence detection.

A qualitative and quantitative analysis of the conjugated 1 beta- and 6 alpha-hydroxy bile acids, including common bile acids, in human urine using high-performance liquid chromatography with chemiluminescence detection is described. After extraction of urine with C18 silica cartridges, the bile acids were separated into non-conjugated, glycine, taurine and sulphate fractions by ion-exchange chromatography on a lipophilic gel. Solvolysis of the sulphate was carried out by treatment with trifluoroethanol in acetone containing hydrochloric acid, and the liberated amino acid conjugates were fractionated again. The individual bile acids were separated on a reversed-phase C18 column (Bile Pak II), with detection by an immobilized 3 alpha-hydroxysteroid dehydrogenase enzyme reactor and chemiluminescence reaction of the generated NADH using 1-methoxy-5-methylphenazinium methylsulphate-isoluminol-microperoxidase system. The assay method showed the detection limits ranging from 8 to 250 pmol for the bile acids tested. Analysis of urine samples obtained from newborns, non-pregnant women and women in late pregnancy showed a large difference in bile acid composition and conjugation mode, suggesting that bile acid metabolism is different during fetal and neonatal periods.     

Bile acids as stationary phase in liquid chromatography

Summary Bile acids chemically bonded onto aminopropylsilica have been employed as stationary phases in liquid chromatography. Bile acid aggregates were dynamically formed around molecules chemically anchored on the supports when the eluent contained bile salts. The bile salt aggregates achieved the separation of 1,1′-binaphthyl-2,2′-diyl-hydrogenphosphate enantiomers and dansyl amino acids.     

The use of SEP-PAKTM C18 Cartridges in the Preparation of Bile Acid Methyl Ester Acetates

Abstract

A method is described for the rapid and Quantitative extraction of bile acid derivates by Sep-Pak^TM C₁₈ cartridge. The method is used for the preparation of bile acid methyl ester acetates. The method was validated by determining the efficiency and the recovery of radiolabelled taurine-conjugated and free bile acids and of bile acid containing biological samples, by thin-layer chromatography with zonal scanning after each step and by internal standardization withing the gas-chromatographic analysis. The recovery of bile acids after hydrolysis amounted to 93.7% ± 2.7%, 97.7% ± 3.6% and 100% ± 2.3% for gallbladder bile, serum bile and mixtures of pure bile acids resp. The recovery of cholic acid methyl ester acetate and chenodeoxycholic acid methyl ester acetate after the entire procedure, including hydrolysis Sep-Pak^TM -extraction, methylation, acetylation and again Sep-Pak^TM -extraction, amounted to 85.6% ± 4.6%, 88.4% ± 5.3% and 89.3% ± 3.5% for gallbladder bile acids, serum bile acids and bile acid mixtures resp. It is concluded that Sep-Pak^TM can efficiently be used in the preparation of bile acid methyl ester acetates, thereby avoiding time-consuming and inconsistent extractions.     

Quantification of neurotransmitter amino acids by capillary electrophoresis laser-induced fluorescence detection in biological fluids

The role of neurotransmitter amino acids (NAAs) in the functioning of the nervous system has been the focus of increasingly intense research over the past several years. Among the various amino acids that have important roles as neurotransmitters, there are alanine (Ala), glutamic acid (Glu), aspartic acid (Asp), serine (Ser), taurine (Tau) and glycine (Gly). NAAs are present in plasma, cells and—at trace levels—in all biological fluids, but complex components in biological matrices make it difficult to determine them in biological samples. We describe a new capillary electrophoresis (CE) method with laser-induced fluorescence detection by which analytes are resolved in less than 12 minutes in a 18 mmol/L phosphate run buffer at pH 11.6. The use of elevated temperatures during sample derivatization leads to a drastic reduction in the reaction time, down to 20 min, compared to the 6–14 h usually described for reactions between FITC and amino acids at room temperature. In order to demonstrate its wide range of applications, the method was applied to the analysis of NAA in human plasma and in other sample types, such as red blood cells, urine, cultured cells, cerebrospinal fluid, saliva and vitreous humor, thus avoiding the typical limitations of other methods, which are normally suitable for use with only one or two matrix types.     

Determination of bile acids in pig liver, pig kidney and bovine liver by gas chromatography-chemical ionization tandem mass spectrometry with total ion chromatograms and extraction ion chromatograms

An effective method has been developed for quantitative determination of six bile acids including lithocholic acid (LCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), hydodeoxycholic acid (HDCA), cholic acid (CA) and ursodeoxycholic acid (UDCA) in biological tissues including pig liver, pig kidney and bovine liver by gas chromatography-chemical ionization/tandem mass spectrometry (GC-CI/MS/MS). Camphor-10-sulphonic acid (CSA) was proposed as effective catalyst for bile acid derivatization. Reactions were accelerated ultrasonically. The effects of different catalysts and reaction times on derivatization efficiency were evaluated and optimized. Bile acids were determined as methyl ester-trimethylsilyl ether and methyl ester-acetate derivatives. The efficiency of trimethylsilylation and acetylation was evaluated. Trimethylsilylation was done with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) as the trimethylsilyl donating reagent in a ultrasonic bath for 20 min. Acetylation was done in pyridine with acetic anhydride at 40-45°C for 4 h. The former reaction was faster than the latter. Thus, trimethylsilylation was employed for the quantitative analysis. Negligible interferences from sterols in biological matrices were observed when the biological samples were treated with solid phase extraction before GC-CI/MS/MS. The linearity, reproducibility, detection limit and recovery were evaluated under the optimized conditions. Satisfactory results were obtained when bile acid derivatives of LCA, CDCA, HDCA, and UDCA were determined with total ion chromatograms (TIC) while DCA and CA were determined with extracted ion chromatograms (EIC), respectively. The detection limits (S/N=3) for six bile acids in biological tissues were ranging from 0.40 to 1.6 ng/mL and the recoveries indicated that the proposed method was feasible for the determination of trace bile acids in the biological samples studied. The experimental results for the animal tissues purchased from five different markets were compared. Interestingly, all of the six bile acids were present in pig liver while only the dihydroxy bile acids, DCA, CDCA and HDCA were found in pig kidney. In addition to DCA and CDCA, trihydroxy bile acid, CA, are the major bile acids in bovine liver.     

Determination of individual bile acids in serum by high performance liquid chromatography

A high performance liquid chromatographic (HPLC) method for analysis of 4 free and 8 conjugated bile acids in submicromolar quantities in serum is described using precolumn derivatization with 4-bromomethyl-7-methoxycoumarin (BMC) and fluorescence detection. Bile acids were extracted from serum with 0.4 M sodium bicarbonate, adsorbed onto a Sep-Pak C18 cartridge and eluted with methanol. The extract was derivatized with BMC in acetonitrile using 18-crown-6 crown ether as catalyst and the BMC labelled glycine conjugates and free bile acids were analysed using acetonitrile + methanol + water gradient elution and detection at 320/385 nm. Using a novel and simple approach, taurine conjugates were isolated by extracting the dried, derivatized material with water, in contrast to previous methods which required column chromatography cleanup to isolate the taurine conjugates prior to derivatization. The isolated taurine conjugates were then hydrolysed enzymatically, extracted, derivatized and analysed as free-bile acids. Recoveries of individual bile acids varied from 83-96% for free and glycine conjugates and 72-83% for taurine conjugates. Coefficients of variation were in the range of 5.1-12.5%. In addition to the simpler and shorter procedure for taurine conjugates, this method has increased sensitivity over most other procedures and improved HPLC separation for the various bile acids and conjugates with equivalent recovery and reproducibility compared with other published methods.     

Determination of serum metabolic profiles of bile acids by microcolumn liquid chromatography/laser-induced fluorescence

A method for the determination of individual free and conjugated bile acids in serum using microcolumn liquid chromatography coupled with a laser-induced fluorescence detector is described. Bile acids are separated into free/glycine-conjugate and taurine-conjugate fractions using a Sep-Pak SIL cartridge. The taurine-conjugated bile acid fraction is subjected to enzymatic hydrolysis. Subsequently, free and conjugated bile acids are labeled using 4-(bromomethyl)-7-methoxycoumarin as a fluorogenic reagent, producing stable derivatives that can be excited by the 325 nm line of a He/Cd laser. Prior to their fluorimetric detection, the individual components of a bile acid serum profile are separated by reversed-phase microcolumn liquid chromatography.     

An LC-ESI-MS method for the quantitative analysis of bile acids composition in fecal materials

In this work, a liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS) method was developed and validated for quantification of bile acids in fecal materials. Co-eluting matrix impurities in fecal materials have been shown to greatly suppress the ionization of analytes in mass spectrometry, which is known as the matrix effect. To correct large quantitative errors caused by the matrix effect, we developed a scheme that combined the standard addition method with internal standard (SA-IS). The fecal sample pretreatment involved a single step of extraction with ethanol. Bile acids were separated using a Luna C(18) column (150 mm, 2 mm i.d., 5 μm) with gradient elution. The deprotonated analytes were detected in selective ion monitoring mode. Our results showed that, by using this method, the accuracy of quantification was significantly improved in comparison to the conventional internal standard method. The linearity, sensitivity, accuracy and precision of the method were within the range of 0.05-5 μmol/L. This SA-IS method was successfully applied to the analysis of bile acids in the samples collected from patients diagnosed with inflammatory bowel disease.     

Sample preparation for organic acids in biological fluids

A review of sample preparation methods for organic acids in biological fluids, in particular serum and urine, is presented. It covers techniques on organic acid determination without sample preparation, release of organic acids from binding locations, removal of proteins by protein precipitation and ultrafiltration, isolation of the organic acids by liquid-liquid and liquid-solid extraction, purification of the extract, derivatization and pre-fractionation. The various alternative sample preparation steps are compared and critically discussed. Examples of applications including profile analysis of organic acids by gas chromatography (GC), determination of particular organic acids by GC or liquid chromatography and determination of fatty acids as a distinct chemical class of acids demonstrate that the kind of sample preparation chosen depends strongly on the analytical aims.     

Bile Salts in Chiral Micellar Electrokinetic Chromatography: 2000–2020

Bile salts are naturally occurring chiral surfactants that are able to solubilize hydrophobic compounds. Because of this ability, bile salts were exploited as chiral selectors added to the background solution (BGS) in the chiral micellar electrokinetic chromatography (MEKC) of various small molecules. In this review, we aimed to examine the developments in research on chiral MEKC using bile salts as chiral selectors over the past 20 years. The review begins with a discussion of the aggregation of bile salts in chiral recognition and separation, followed by the use of single bile salts and bile salts with other chiral selectors (i.e., cyclodextrins, proteins and single-stranded DNA aptamers). Advanced techniques such as partial-filling MEKC, stacking and single-drop microextraction were considered. Potential applications to real samples, including enantiomeric impurity analysis, were also discussed.     

Improved gas chromatography methods for micro-volume analysis of haloacetic acids in water and biological matrices

A fast headspace solid-phase microextraction gas chromatography method for micro-volume (0.1 mL) samples was optimized for the analysis of haloacetic acids (HAAs) in aqueous and biological samples. It includes liquid-liquid microextraction (LLME), derivatization of the acids to their methyl esters using sulfuric acid and methanol after evaporation, followed by headspace solid-phase microextraction with gas chromatography and electron capture detection (SPME-GC-ECD). The derivatization procedure was optimized to achieve maximum sensitivity using the following conditions: esterification for 20 min at 80 degrees C in 10 microL methanol, 10 microL sulfuric acid and 0.1 g anhydrous sodium sulfate. Multi-point standard addition method was used to determine the effect of the sample matrix by comparing with internal standard method. It was shown that the effect of the matrix for urine and blood samples in this method is insignificant. The method detection limits are in the range of 1 microg L(-1) for most of the HAAs, except for monobromoacetic acid (MBAA) (3 microg L(-1)) and for monochloroacetic acid (MCAA) (16 microg L(-1)). The optimized procedure was applied to the analysis of HAAs in water, urine and blood samples. All nine HAAs can be separated in < 13 min for biological samples and < 7 min for drinking water samples, with total sample preparation and analysis time < 50 min. Analytical uncertainty can increase dramatically as the sample volume decreases; however, similar precision was observed with our method using 0.1 mL samples as with a standard method using 40 mL samples.     

Simultaneous determination of polysaccharides and 21 nucleosides and amino acids in different tissues of Salvia miltiorrhiza from different areas by UV–visible spectrophotometry and UHPLC with triple quadrupole MS/MS

Salvia miltiorrhiza, a traditional Chinese medicine, is a widely used herbal medicine to treat cardiovascular and cerebrovascular diseases. In this study, ultraviolet (UV)–visible spectrophotometry and ultra‐high performance liquid chromatography with triple quadrupole tandem mass spectrometry analytical methods were used for rapid quantification of polysaccharides and 21 nucleosides and amino acids in S. miltiorrhiza to determine 17 samples of different tissues from different areas. Based on the total contents, hierarchical clustering analysis and principal components analysis were performed to classify these samples. The established methods were validated with good linearity, precision, repeatability, stability, and recovery. Chemical analysis revealed a higher content of total analytes in the sample of inflorescence from Nanjing (34.17 mg/g), sample of root and rhizome from Shaanxi (34.13 mg/g) and sample of stem and leaf from Nanjing (31.14 mg/g), respectively, indicating that root and rhizome from Shaanxi and the aerial parts from Nanjing exhibited the highest quality due to their highest content. In addition, contents of nucleosides and amino acids in the aerial parts (14.67 mg/g) were much higher than that in roots and rhizomes (9.17 mg/g). This study suggested that UV–visible spectrophotometry and ultra‐high performance liquid chromatography with triple quadrupole tandem mass spectrometry are effective techniques to analyze polysaccharides, nucleosides, and amino acids in plants, and they provided valuable information for the development and utilization value of the aerial parts of S. miltiorrhiza. This analysis would also provide useful information for the quality control of S. miltiorrhiza.     

The Role of Bile Acids in the Human Body and in the Development of Diseases

Bile acids are specific and quantitatively important organic components of bile, which are synthesized by hepatocytes from cholesterol and are involved in the osmotic process that ensures the outflow of bile. Bile acids include many varieties of amphipathic acid steroids. These are molecules that play a major role in the digestion of fats and the intestinal absorption of hydrophobic compounds and are also involved in the regulation of many functions of the liver, cholangiocytes, and extrahepatic tissues, acting essentially as hormones. The biological effects are realized through variable membrane or nuclear receptors. Hepatic synthesis, intestinal modifications, intestinal peristalsis and permeability, and receptor activity can affect the quantitative and qualitative bile acids composition significantly leading to extrahepatic pathologies. The complexity of bile acids receptors and the effects of cross-activations makes interpretation of the results of the studies rather difficult. In spite, this is a very perspective direction for pharmacology.     

Two-dimensional electrophoresis on a microfluidic chip for quantitative amino acid analysis

Analysis of complex biological samples requires the use of high-throughput analytical tools. In this work, a microfluidic two-dimensional electrophoresis system was developed with mercury-lamp-induced fluorescence detection. Mixtures of 20 standard amino acids were used to evaluate the separation performance of the system. After fluorescent labeling with fluorescein isothiocyanate, mixtures of amino acids were separated by micellar electrokinetic chromatography in the first dimension and by capillary zone electrophoresis in the second. A double electrokinetic valve system was employed for the sample injection and the switching between separation channels. Under the optimized conditions, 20 standard amino acids were effectively separated within 20 min with high resolution and repeatability. Quantitative analysis revealed linear dynamic ranges of over three orders of magnitudes with detection limits at micromolar range. To further evaluate the reliability of the system, quantitative analysis of a commercial nutrition supplement liquid was successfully demonstrated. Figure