Studies on Steroids CCXXXXIV. Separation and Characterization of C-25 Epimers of Unconjugated and Conjugated Trihydroxycholestanoic Acids in Urine from a Patient with Zell Weger Syndrome by High-Performance Liquid Chromatography

Abstract

The separation and characterization of C-25 epimers of unconjugated and glycine- and taurine-conjugated 3α, 7α, 12α - trihydroxy-5β-cholestanoic acid (THCA) in biological fluids by high-performance liquid chromatography (HPLC) are described. The 5β-cholestanoic acid fraction was obtained from a urine specimen from a patient with Zellweger syndrome by passing it through a Sep-pak C₁₈ cartridge. Bile acids were derivatized quantitatively into the fluorescent compounds through the hydroxyl group at C-3 by treatment with 1-anthroyl nitrile. The derivatives were separated into the unconjugated, glycine- and taurine-conjugated fractions by ion-exchange chromatography on alipophilicgel, piperidinohydroxypropyl Sephadex LH-20. Sub-sequent resolution of each fractionin to (25s)- and (25R)-THCA was attained by HPLC on a Cosmosil 5C column. The C-25 epimers of unconjugated and conjugated Tk%A were unequivocally identified on the b asis of the irbehaviors in HPLC using mobile phases of different pHs. The ratios of the unconjugated, glycine- and taurine-conjugated (25RbTHCA to the corresponding (25S)-epimers were 16:1, 5:4 and 3:2, respectively .     

Studies on steroids. CCLII. Separation and characterization of 3-oxobile acids in serum by high-performance liquid chromatography with fluorescence detection

The separation of unconjugated and glycine- and taurine-conjugated bile acids with a C-3 oxo group has been carried out by high-performance liquid chromatography on a reversed-phase column. The chromatographic behaviour of these 3-oxobile acids was dependent on the number and positions of hydroxyl groups and the structure of the side-chain. The newly developed method has been applied to the characterization of 3-oxobile acids in biological fluids. The bile acid fraction was obtained from a serum specimen by passing it through a Sep-Pak C18 cartridge. 3-Oxobile acids were derivatized quantitatively to fluorescent oximes through the oxo group by treatment with O-(2-anthrylmethyl)hydroxylamine. The derivatives were separated into the unconjugated and glycine- and taurine-conjugated fractions by ion-exchange chromatography on a lipophilic gel, piperidinohydroxypropyl Sephadex LH-20. Subsequent resolution of each fraction into individual 3-oxobile acids was achieved by chromatography on a Nova-Pak Phenyl column using 3% methanol in 0.3% potassium phosphate buffer (pH 7.0)-acetonitrile (8:5, v/v) as the mobile phase. The derivatized 3-oxobile acids were monitored by fluorescence detection (excitation wavelength 260 nm and emission wavelength 405 nm), the limit of detection being 20 fmol. Glycine- and taurine-conjugated 7 alpha,12 alpha-dihydroxy- and 7 alpha-hydroxy-3-oxo-5 beta-cholanoic acids in human serum were unambiguously idenitified on the basis of their chromatographic behaviour using mobile phases of different pH values.     

Separation and Determination of Bile Acids in Human Bile by High-Performance Liquid Chromatography

Abstract

A method for simultaneous determination of major bile acids in human bile is described. The unconjugated, glycine- and taurine-conjugated bile acids are extracted with Sep-pak C¹⁸ and separated into groups by ion-exchange chromatography on a lipophilic gel. Subsequently, resolution of each group into ursodeoxycholate, cholate, chenodeoxycholate, deoxycholate and lithocholate is attained into two stages by high-performance liquid chromatography on a Radial-PAK A column. First, 0.3% ammonium phosphate (pH 7.7)/acetonitrile (19:8, v/v) is used for separation of the latter three as a mobile phase. Ursodeoxycholate and cholate are efficiently separated in 0.3% ammonium phosphate (pH 7.7)/acetonitrile (23:8, v/v). The present method is applicable to quantitation of bile acids in human bile with satisfactory accuracy and precision.     

Class separation of bile lipids by thin-layer chromatography

A thin-layer chromatography technique is described that permits separation of each class of bile lipid, such as cholesterol, free (unconjugated) bile acids, glycine- and taurine-conjugated bile acids and phospholipids, in a single run. The use of silica gel G-aluminium pre-coated sheets facilitates further processing, such as the extraction in situ of each class of separated bile lipids for determination by conventional methods.     

Studies on steroids. CCXXVIII Trace analysis of bile acids by gas chromatography-mass spectrometry with negative ion chemical ionization detection

A suitable derivatization method for the trace analysis of bile acids by gas chromatography (GC) in combination with negative ion chemical ionization (NICI) mass spectrometry is described. Of various derivatives for the carboxyl group, the pentafluorobenzyl (PFB) ester provided the highest value of the ratio of the negative to the positive ion current. A characteristic carboxylate anion [M - 181]- was produced as the most abundant ion by the loss of the PFB group in NICI. The PFB esters were further derivatized to the dimethylethylsilyl (DMES) ethers, whereby lithocholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid and cholic acid were distinctly separated by GC on a cross-linked methyl silicone fused-silica capillary column. The detection limit for the PFB-DMES derivatives of dihydroxylated bile acids was 2 fg when the fragment ion was monitored at m/z 563 in the NICI mode using isobutane as a reagent gas.     

Measurement of bile acid CoA esters by high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS)

The novel and rapid assay presented here combines high-performance liquid chromatography and electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS) to directly measure and quantify the CoA esters of 3alpha,7alpha,12alpha-trihydroxy- and 3alpha,7alpha-dihydroxy-5beta-cholestan-26-oic acid (THCA and DHCA). The latter are converted inside peroxisomes to the primary bile acids, cholic and chenodeoxycholic acids, respectively. Prior to MS/MS, esters were separated by reversed-phase HPLC on a C(18) column using an isocratic mobile phase (acetonitrile/water/2-propanol) and subsequently detected by multiple reaction monitoring. For quantification, the CoA ester of deuterium-labelled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid (d(4)-CA) was used as internal standard. To complete an assay took less than 8 min.To verify the validity of the assay, the effect of peroxisomal proteins on the efficacy of extraction of the CoA esters was tested. To this end, variable amounts of the CoA esters were spiked with a fixed amount of either intact peroxisomes or peroxisomal matrix proteins and then extracted using a solid-phase extraction system. The CoA esters could be reproducibly recovered in the range of 0.1-4 micromol l(-1) (linear correlation coefficient R(2) > 0.99), with a detection limit of 0.1 micromol l(-1).In summary, electrospray ionization tandem mass spectrometry combined with HPLC as described here proved to be a rapid and versatile technique for the determination of bile acid CoA esters in a mixture with peroxisomal proteins. This suggests this technique to become a valuable tool in studies dealing with the multi-step biosynthesis of bile acids and its disturbances in disorders like the Zellweger syndrome.     

Study on stereospecificity of enzyme reaction related to peroxisomal bile acid synthesis in rat liver.

We examined stereoselectivities of enzymes related to bile acid formation in hepatic peroxisomes using two stereoisomers of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) and its coenzyme A (CoA) derivatives. The activity of acyl-CoA synthetase for 25 S-THCA was 1.4-times higher than that for 25 R-THCA. The difference was also observed after clofibrate-treatment. This activity was located in microsomes, differing from palmitoyl-CoA synthetase located in mitochondria, peroxisomes and microsomes. There was no stereoselectivity in the reaction of peroxisomal fatty acyl-CoA oxidase for THCA isomers, and the activity was one tenth of that for acyl-CoA synthetase. Considering the overall reaction of peroxisomal bile acid formation, the stereoselective difference observed in THCA-CoA synthesis should be denied. Thus, the previous finding that the overall formation of bile acid from THCA was not stereoselective was further confirmed. Furthermore, the activity for THCA oxidation was not induced by clofibric acid, suggesting that there would be different isozymes of peroxisomal acyl-CoA oxidase against THCA-CoA and palmitoyl-CoA.     

Studies on the metabolism of the Δ9‐tetrahydrocannabinol precursor Δ9‐tetrahydrocannabinolic acid A (Δ9‐THCA‐A) in rat using LC‐MS/MS,LC‐QTOF MS and GC‐MS techniques

In Cannabis sativa, Δ9‐Tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A) is the non‐psychoactive precursor of Δ9‐tetrahydrocannabinol (Δ9‐THC). In fresh plant material, about 90% of the total Δ9‐THC is available as Δ9‐THCA‐A. When heated (smoked or baked), Δ9‐THCA‐A is only partially converted to Δ9‐THC and therefore, Δ9‐THCA‐A can be detected in serum and urine of cannabis consumers. The aim of the presented study was to identify the metabolites of Δ9‐THCA‐A and to examine particularly whether oral intake of Δ9‐THCA‐A leads to in vivo formation of Δ9‐THC in a rat model. After oral application of pure Δ9‐THCA‐A to rats (15 mg/kg body mass), urine samples were collected and metabolites were isolated and identified by liquid chromatography‐mass spectrometry (LC‐MS), liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and high resolution LC‐MS using time of flight‐mass spectrometry (TOF‐MS) for accurate mass measurement. For detection of Δ9‐THC and its metabolites, urine extracts were analyzed by gas chromatography‐mass spectrometry (GC‐MS). The identified metabolites show that Δ9‐THCA‐A undergoes a hydroxylation in position 11 to 11‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A (11‐OH‐Δ9‐THCA‐A), which is further oxidized via the intermediate aldehyde 11‐oxo‐Δ9‐THCA‐A to 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A‐COOH). Glucuronides of the parent compound and both main metabolites were identified in the rat urine as well. Furthermore, Δ9‐THCA‐A undergoes hydroxylation in position 8 to 8‐alpha‐ and 8‐beta‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A, respectively, (8α‐Hydroxy‐Δ9‐THCA‐A and 8β‐Hydroxy‐Δ9‐THCA‐A, respectively) followed by dehydration. Both monohydroxylated metabolites were further oxidized to their bishydroxylated forms. Several glucuronidation conjugates of these metabolites were identified. In vivo conversion of Δ9‐THCA‐A to Δ9‐THC was not observed. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of conjugated bile acids by packed-column supercritical fluid chromatography.

A rapid method has been developed for the simultaneous separation of the polar glycine- and taurine-conjugated bile acids by packed-column supercritical fluid chromatography. Samples were analysed on a cyanopropyl-bonded silica column with ultraviolet detection at 210 nm and carbon dioxide modified with methanol as the mobile phase. The influence of the stationary phase, modifier concentration, temperature, column pressure and modifier identity on retention was also studied. This new chromatographic method is applicable to the assay of conjugated bile acids in duodenal bile samples from patients with hepatobiliary diseases.     

Determination of urinary beta-phenylethylamine as its N-benzenesulphonamide derivative by gas chromatography with flame photometric detection.

A selective and sensitive method for the determination of urinary beta-phenylethylamine (PEA) by gas chromatography (GC) has been developed. After extraction of the urine sample with n-pentane, PEA was converted into its N-benzenesulphonamide derivative and then determined by GC with flame photometric detection using a DB-1301 capillary column. By using this method, nanogram amounts of PEA in urine could be accurately and precisely determined without any influence from coexisting substances. Analytical results for the determination of PEA in urine samples from normal subjects are presented.     

Extraction and fractionation of bile acids and their conjugates using pre-packed microparticulate silica cartridges (Sep-Pak Sil and Bond-Elut C18)

A new method for the extraction of bile acids from human plasma using acetonitrile precipitation of plasma protein and subsequent use of Bond-Elut C18 cartridges is described. After extraction the bile acids can be separated into three fractions: unconjugated, glycine-, and taurine-conjugated, using Sep-Pak SIL cartridges at 4 degrees C, eluting with ethanol--chloroform--water--glacial acetic acid mixtures. These extraction and fractionation procedures were evaluated in terms of recovery, reproducibility and resolution between the fractions. All these parameters were found to be satisfactory. Although the reproducibility of fractionation on Sep-Pak SIL cartridges was found to vary between batches, this did not give rise to significant difficulties. Plasmas from normals and patients with hepatobiliary disease were analysed by capillary gas-liquid chromatography after extraction and fractionation using the procedure described.     

Direct quantification of 11‐nor‐Δ9‐tetrahydrocannabinol‐9‐carboxylic acid in urine by liquid chromatography/tandem mass spectrometry in relation to doping control analysis

An accurate and precise method for the quantification of 11‐nor‐Δ⁹‐tetrahydrocannabinol‐9‐carboxylic acid (THCA) in urine by liquid chromatography/tandem mass spectrometry (LC/MS/MS) for doping analysis purposes has been developed. The method involves the use of only 200 µL of urine and the use of D₉‐THCA as internal standard. No extraction procedure is used. The urine samples are hydrolysed using sodium hydroxide and diluted with a mixture of methanol/glacial acetic acid (1:1). Chromatographic separation is achieved using a C8 column with gradient elution. All MS and MS/MS parameters were optimised in both positive and negative electrospray ionisation modes. For the identification and the quantification of THCA three product ions are monitored in both ionisation modes. The method is linear over the studied range (5–40 ng/mL), with satisfactory intra‐and inter‐assay precision, and the relative standard deviations (RSDs) are lower than 15%. Good accuracy is achieved with bias less than 10% at all levels tested. No significant matrix effects are observed. The selectivity and specificity are satisfactory, and no interferences are detected. The LC/MS/MS method was applied for the analysis of 48 real urine samples previously analysed with a routine gas chromatography/mass spectrometry (GC/MS) method. A good correlation between the two methods was obtained (r² > 0.98) with a slope close to 1. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparison of gas chromatographic and gas chromatographic/mass spectrometric techniques for the analysis of TNT and related nitroaromatic compounds

The use of capillary column gas chromatography and gas chromatography/mass spectrometry for the analysis of a series of standard solutions (0.1 to 10 μg/ml) of 2,4,6-trinitrotoluene (TNT) and eight other nitroaromatic components was evaluated. The techniques included gas chromatography with electron capture detection (GC/ECD), full scan and selected ion monitoring gas chromatography/mass spectrometry with electron impact ionization (EI/FS and EI/SIM), full scan and selected ion monitoring gas chromatography/mass spectrometry with positive ion chemical ionization using methane reagent gas (PICI/FS and PICI/SIM), and full scan and selected ion monitoring gas chromatography/mass spectrometry with negative ion chemical ionization using methane reagent gas (NICI/FS and NICI/SIM). The performance of the techniques was comapared by determining the linear response range, precision, and detection limits of the analyses.     

A rapid HPLC method for the determination of carboxylic acids in human urine using a monolithic column

A rapid HPLC method for the determination of carboxylic acids in urine samples using a Chromolith Performance RP/18e 100/4.6 with Chromolith Guard Cartridge RP/18e 10/4.6 (Merck KgaA, Darmstadt, Germany) was developed. The method facilitates the simultaneous determination of aromatic hydrocarbon metabolites mandelic acid (MA) and phenylglyoxylic acid (PGA) from styrene and ethylbenzene, hippuric acid (HA) from toluene and 2-, 3-, 4-methylhippuric acids (MHA) from xylene. 3-Hydroxybenzoic acid (3-HBA) was used as internal standard. A chromatographic run is completed within less than 5 min for styrene, ethylbenzene and toluene metabolites, and within 10 min for xylene metabolites. The detection limits are 9 mg L^–1 urine for MA, 1.25 mg L^–1 urine for PGA, 4.9 mg L^–1 urine for HA, 22 mg L^–1 urine for 2-MHA, and 18.5 mg L^–1 urine for 3-MHA.No significant differences of the MA, PGA and HA concentrations in human urine samples obtained by HPLC chromatography on LiChrosorb RP 18 and on Chromolith RP/18e columns were found. The results were evaluated by using ANOVA.Abbreviations MA mandelic acid - PGA phenylglyoxylic acid - HA hippuric acid - MHA methylhippuric acid - 3-HBA 3-hydroxybenzoic acid - ANOVA analyses of variance - GC gas chromatography - HPLC high-performance liquid chromatography     

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.     

Chromatography–mass spectrometry determination of alkyl methylphosphonic acids in urine

The potentials of different chromatography–mass spectrometry methods for the determination of alkyl methylphosphonic acids (AMPAs)—the chemical markers of nerve agents in urine—are compared. The gas chromatography–mass spectrometry (GC–MS) characteristics of various volatile AMPA derivatives are studied. The preference of perfluorobenzyl derivatives over methyl, trimethylsilyl and tert-butyldimethylsilyl esters for the GC–MS determination of AMPAs in urine is demonstrated. An optimal technique for the determination of AMPAs in urine is HPLC combined with high-resolution MS² mass spectrometry with the isotope–labeled forms of target compounds as internal standards. The detection limits of AMPAs in the proposed analytical procedures vary from 0.1 to 1.0 ng/mL.     

Determination of sucrose esters of fatty acids in food additive premixes by gas chromatography and confirmation of identity by gas chromatography/mass spectrometry

A gas chromatographic (GC) method was developed for the determination of sucrose monoesters of fatty acids (mono-SuE) and sucrose acetate isobutyrate (SAIB) in food additive premixes. Mono-SuE and SAIB fractions were prepared by column chromatography with either a C8 or a silica gel solid-phase extraction column. The mono-SuE fraction was acetylated and applied to a wide-bore GC column (0.53 mm x 15 m) by splitless injection for determination. The SAIB fraction was applied to the GC column without derivatization. Gas chromatography/mass spectrometry was used to confirm the identity of GC peaks. The detection limits for mono-SuE and SAIB were 0.005 and 0.01%, respectively. Mono-SuE (C12, C14, C16, C18, and C18:1) and SAIB were found in commercial food additive premixes and some foods.     

Determination of 13C/12C ratios of endogenous urinary steroids: method validation, reference population and application to doping control purposes

The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS)-based method for stable carbon isotopes of endogenous urinary steroids is presented. The key element in sample preparation is the consecutive cleanup with high-performance liquid chromatography (HPLC) of underivatized and acetylated steroids, which allows the isolation of ten analytes (11beta-hydroxyandrosterone, 5alpha-androst-16-en-3beta-ol, pregnanediol, androsterone, etiocholanolone, testosterone, epitestosterone, 5alpha-androstane-3alpha,17beta-diol, 5beta-androstane-3alpha,17beta-diol and dehydroepiandrosterone) from a single urine specimen. These steroids are of particular importance to doping controls as they enable the sensitive and retrospective detection of steroid abuse by athletes.Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers repeatability and reproducibility. Specificity was further demonstrated by gas chromatography/mass spectrometry (GC/MS) for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring (13)C/(12)C ratios of all implemented steroids, a reference population of n = 61 subjects was measured to enable the calculation of reference limits for all relevant steroidal Delta values.     

Artifacts in the determination of intravenous anesthetics by gas chromatography-mass spectrometry: Tramadol, the correlation between the structures of metabolites and impurity substances

The simultaneous determination of preparations used for multicomponent intravenous anesthesia (Promedol, tramadol, ketamine, diazepam, Thiopental, and phentanyl) and of their metabolites in blood and urine of surgical patients by chromatography-mass spectrometry was considered. Artifacts due to the Chromatographie interference of various preparations and their metabolites were revealed. The lability of the anesthetics and their metabolites in the course of sample preparation and analysis by gas chromatography (GC) was examined. The degradation products of the test preparations responsible for the generation of false positive results were found. Phentanyl, Promedol, ketamine, tramadol, Thiopental, diazepam, and their metabolites excreted with urine in the free forms were determined in the whole blood and urine of surgical patients. Bound forms of metabolites and the initial medicinal preparations excreted as complexes with glucuronic acid and other acids were also determined in urine. Metabolites and impurity substances in the intravenous anesthetics with similar mass spectra and retention times were distinguished. Methodological recommendations concerning the analysis of difficult-to-separate (by capillary gas chromatography) pairs of substances used for intravenous anesthesia and their metabolites are given. The following pairs of components are difficult to separate: Norpromedol-2-methylamino-5-chlorobenzophenon (a product of diazepam hydrolysis), norketamine-Promedol, and anhydrotramadol (a GC artifact)-ketamine. The cumulation of an impurity substance from the tramadol preparation, identified by us as epoxytramadol, in the body was examined.     

Sensitive determination of styrene and related compounds in human body fluids by headspace capillary gas chromatography with cryogenic oven trapping

Summary A simple and sensitive method is presented for determination of styrene, toluene, ethylbenzene, isopropylbenzene andn-propylbenzene in human body fluids by capillary gas chromatography (GC) with cryogenic oven trapping. After heating a blood or urine sample containing each compound andp-diethylbenzene (internal standard, IS) in a 7.0-mL vial at 60°C for 20 min, 5 mL of headspace vapor was drawn into a glass syringe and injected into a GC. All vapor was introduced into an Rtx-Volatile middle bore capillary column in splitless mode at oven temperature of 20°C to trap entire analytes, and the oven temperature then programmed to 280°C for GC measurements by flame ionization detection. The present conditions gave sharp peaks of each compound and IS, and low background noises for whole blood or urine samples.