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

A qualitative and quantitative analysis of the conjugated 1β- and 6α-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 3α-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.     

Studies on the metabolites of phylloquinone (vitamin K1) in the urine of man

The nature of the metabolites excreted in the urine was investigated up to 48 h after oral and intravenous administration of 0.3 to 1.3 mg [1′,2′-³H₂]phylloquinone. The metabolites were water-soluble of which the major fraction consisted of glucuronide conjugates. A chromatographic comparison of the aglycone fragments released by β-glucuronidase and by dilute HCl revealed the presence of at least three labelled aglycones. The major aglycones obtained by enzyme hydrolysis consisted of at least two closely related organic acids which were not separated by adsorption thin-layer chromatography but one of which on treatment with dilute acid yielded a neutral metabolite with the chromatographic properties of phylloquinone γ-lactone. The results suggest that phylloquinone γ-lactone, the only previously isolated urinary metabolite of phylloquinone, is an artifact produced by the conditions of acid hydrolysis. Although the acid labile aglycone was the minor component of the two acid metabolites, its proportion in urine extracts as measured by conversion to the lactone, increased with the time after administration of labelled phylloquinone.     

Method for the biological monitoring of hexahydrophthalic anhydride by the determination of hexahydrophthalic acid in urine using gas chromatography and selected-ion monitoring

A method for the determination of hexahydrophthalic acid, a metabolite of hexahydrophthalic anhydride, in human urine has been developed. The urine was worked-up by liquid—solid extraction, esterified with boron trifluoride—methanol, and analysed by capillary gas chromatography and selected-ion monitoring. Hexadeuterium-labelled hexahydrophthalic acid was used as the internal standard. The precision was 4% at 0.7 μg/ml and 5% at 0.07 μg/ml. The recovery of the acid for the overall method was 101% at 0.07 μg/ml of urine (with a coefficient of variation of 4%) and 95% at 0.7 μg/ml (coefficient of variation 2%). The limit of detection was 20 ng/ml urine.     

The identification of novel steroid N-acetylglucosaminides in the urine of pregnant women

A series of pregnanediols and pregnanetriols doubly conjugated with N-acetylglucosamine and glucuronic or sulfuric acid has been identified in urine from pregnant women. Steroid conjugates were separated by ion-exchange chromatography and the glucuronide and monosulfate fractions were analysed by fast atom bombardment mass spectrometry. After removal of the acid moiety, the neutral steroids were isolated, derivatized, and analysed by gas chromatography-mass spectrometry (GC-MS). The analyses revealed the presence of steroids conjugated with N-acetylhexosamine both in the glucuronide and the monosulfate fractions. Following enzyme hydrolysis, the sugar was identified by GC-MS as N-acetylglucosamine (GlcNAc). The major steroid conjugated with GlcNAc both in the glucuronide and monosulfate fractions was identified as 5α-pregnane-3α,20α-diol. 5β-Pregnane-3α,20α-diol was also present as a GlcNAc conjugate in both fractions whereas a GlcNAc conjugate of 5α-pregnane-3β,20α-diol was only found in the sulfate fraction. 5α-Pregnane-3α,20α,21-triol was a double conjugate with GlcNAc in the sulfate fraction whereas a pregnane-2,3,20-triol was a double conjugate in the glucuronide fraction. The positions of conjugation were determined by collision-induced dissociation of the pseudomolecular anions produced by fast atom bombardment ionization. The sulfate and glucuronic acid moieties were located at C-3 and N-acetylglucosamine at C-20. An alternative localization of GlcNAc at C-21 of 5α-pregnane-3α,20α,21-triol cannot be excluded. Judging from the enzymatic hydrolysis of the conjugates, the sugar was attached in β-glycosidic linkage. The mean excretion of N-acetylglucosaminides of the pregnanediols and pregnanetriols was 32.2 μmol/g creatinine (range 17.9–49.1 μmol) in five healthy women in the 38th–39th week of pregnancy. The mean excretion of 5β-pregnane-3α,20α-diol glucuronide in the same women was 71 μmol/g creatinine, (range 27–127 μmol). This indicates that conjugation with N-acetylglucosamine constitutes a quantitatively important pathway of progesterone metabolism in human pregnancy.     

Quantification of carnitine and specific acylcarnitines by high-performance liquid chromatography: application to normal human urine and urine from patients with methylmalonic aciduria, isovaleric acidemia or medium-chain acyl-CoA dehydrogenase deficiency

This paper describes the development of a high-performance liquid chromatographic method for the quantitation of free carnitine, total carnitine, acetylcarnitine, propionylcarnitine, isovalerylcarnitine, hexanoylcarnitine and octanoylcarnitine in human urine. Carnitine and acylcarnitines were isolated from 10 or 25 μl of urine using 0.5-ml columns of silica gel, derivatized with 4'-bromophenacyl trifluoromethanesulfonate and separated by high-performance liquid chromatography. Using 4-(N,N-dimethyl-N-ethylammonio)-3-hydroxybutanoate (“e-carnitine”) as the internal standard, standard curves (10–300 nmol/ml) were generated. Carnitine and acylcarnitines were quantified (when they were present) in normal human urine and the urine of patients diagnosed with one of three different disorders of organic acid metabolism: methylmalonic aciduria, isovaleric acidemia, and medium-chain acyl-CoA dehydrogenase deficiency.     

A Magnetic Bead-Based Method for Concentrating DNA from Human Urine for Downstream Detection

Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the biomarkers from PCR inhibitors. In this report, a 1 mL urine sample was mixed within the bulb of a transfer pipette containing lyophilized nucleic acid-silica adsorption buffer and silica-coated magnetic beads. After mixing, the sample was transferred from the pipette bulb to a small diameter tube, and captured biomarkers were concentrated using magnetic entrainment of beads through pre-arrayed wash solutions separated by small air gaps. Feasibility was tested using synthetic segments of the 140 bp tuberculosis IS6110 DNA sequence spiked into pooled human urine samples. DNA recovery was evaluated by qPCR. Despite the presence of spiked DNA, no DNA was detectable in unextracted urine samples, presumably due to the presence of PCR inhibitors. However, following extraction with the magnetic bead-based method, we found that ∼50% of spiked TB DNA was recovered from human urine containing roughly 5×10³ to 5×10⁸ copies of IS6110 DNA. In addition, the DNA was concentrated approximately ten-fold into water. The final concentration of DNA in the eluate was 5×10⁶, 14×10⁶, and 8×10⁶ copies/µL for 1, 3, and 5 mL urine samples, respectively. Lyophilized and freshly prepared reagents within the transfer pipette produced similar results, suggesting that long-term storage without refrigeration is possible. DNA recovery increased with the length of the spiked DNA segments from 10±0.9% for a 75 bp DNA sequence to 42±4% for a 100 bp segment and 58±9% for a 140 bp segment. The estimated LOD was 77 copies of DNA/µL of urine. The strategy presented here provides a simple means to achieve high nucleic acid recovery from easily obtained urine samples, which does not contain inhibitors of PCR.     

Drug testing data from the 2007 Pan American Games: δ C values of urinary androsterone, etiocholanolone and androstanediols determined by GC/C/IRMS

The main purpose of this article is to show the application of the CG/C/IRMS in real time during competition in the steroid confirmation analysis. For this reason, this paper summarizes the results obtained from the doping control analysis during the period of the 2007 Pan American Games held in Rio de Janeiro, Brazil. Approximately 5600 athletes from 42 different countries competed in the games. Testing was performed in accordance to World Anti-Doping Agency (WADA) technical note for prohibited substances. This paper reports data where abnormal urinary steroid profiles, have been found with the screening procedures. One 8 mL urine sample was used for the analysis of five steroid metabolites with two separate analyses by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Urine samples were submitted to GC/C/IRMS for confirmation analysis to determine the ¹³C/¹²C ratio of selected steroids. Fifty-seven urine samples were analyzed by GC/C/IRMS and the δ ¹³C values (‰) of androsterone, etiocholanolone, 5β-androstane-3α, 17β-diol (5β-diol), 5α-androstane-3α, 17β-diol (5α-diol) and 5β-pregnane-3α, 20α-diol (5β-pdiol), the endogenous reference compound are presented. One urine sample with a testosterone/epitestosterone (T/E) ratio of 4.7 was confirmed to be positive of doping by GC/C/IRMS analysis. The δ values of 5β-diol and 5α-diol were 3.8 and 10.8, respectively, compared to the endogenous reference compound 5β-pdiol, which exceeded the WADA limit of 3‰. The results obtained by CG/C/IRMS confirmation analyses, in suspicious samples, were conclusive in deciding whether or not a doping steroid violation had occurred.     

Rapid assay for γ-carboxyglutamic acid in urine and bone by precolumn derivatization and reversed-phase liquid chromatography

A reversed-phase high-performance liquid chromatographic assay for the analysis of γ-carboxyglutamic acid (Gla) in urine and bone protein hydrolyzates is described. The method employs precolumn derivatization with o-phthalaldehyde and mercaptoethanol. Gla was quantified by reference to an internal standard (β-carboxyaspartic acid). The “within-run” coefficient of variation of the assay for Gla in urine was between 2.1 and 3.4%, and that for bone protein hydrolyzates was 3.2%. The “between-run” coefficient of variation ranged from 4.1 to 5.5%. There was good agreement between the measurement of urinary Gla by high-performance liquid chromatography and amino acid analyzer. Free Gla could not be detected in serum.     

Study on measurement of δ-aminolevulinic acid in plasma by high-performance liquid chromatography

A method for the determination of δ-aminolevulinic acid in plasma of lead-exposed workers by high-performance liquid chromatography with fluorescence detection of a fluorescent δ-aminolevulinic acid derivative (2-methylidineamino-3,5-diacetyl-4,6-dimethylpropionic acid) was established. The detection limit of δ-aminolevulinic acid in plasma was 0.01 μg/ml at a signal-to-noise ratio of 5:1. A linear correlation was obtained between the amounts of δ-aminolevulinic acid injected from 0.01 to 0.5 μg/ml (r = 0.999). The recovery of 0.05 and 0.1 μg/ml of δ-aminolevulinic acid added to plasma with various concentrations of δ-aminolevulinic acid in plasma ranged from 80.0 to 100.8%. This method, combined with the use of an automatic sampler, should facilitate the routine measurement of δ-aminolevulinic acid in plasma.     

Multiresidue analysis of β2-agonists in human and calf urine using multimodal solid-phase extraction and high-performance liquid chromatography with electrochemical detection

Various β₂-agonists are used as illegal growth promoters in man and in animals. We developed a multiresidue procedure for the analysis of four β-agonists in human and calf urine. The sample was pre-extracted with an Extrelut column at alkaline pH. The β-agonists were eluted with a mixture of tert.-butylmethyl ether and hexane. Then the extract was further cleaned with a mixed mode SPE column, or with a combination of immunoaffinity chromatography (IAC) and the mixed mode SPE column. The IAC column contained antibodies against salbutamol, which were suitable for multiresidue extractions. The extract was then brought onto a mixed mode SPE column at an acidic pH. The column was washed with 70% methanol in water. Thereafter, the β-agonists were eluted with ammoniated ethanol–hexane. The extract was analysed with an HPLC method with electrochemical detection. The β-agonists were separated on a reversed-phase column using a mobile phase buffered at pH 5.5 and containing an ion-pair reagent. Recoveries were higher when the IAC procedure was not performed (90–105% vs. 65–75%), but the extracts were cleaner when the latter step was included. Detection limits in human and calf urine were in the low ng/ml range. The study indicated that β₂-agonists can be analysed in human and calf urine without the selectivity of a mass spectrometer, but that comprehensive clean-up is required to avoid the interference of urine matrix components.     

Oestrogen conjugates of human late-pregnancy urine

1. The separation of the oestrogen conjugates in late-pregnancy urine into two groups, peaks I and II, by gel filtration on Sephadex G-25 (Beling, 1963) has been shown to be affected by the presence of urate, which delays the elution of peak II conjugates. 2. By reapplication to a Sephadex column, peak I conjugates have been further separated into two groups (peaks IA and IB) and the metabolites in urine effecting this separation have been studied. 3. Further analysis of the mixed conjugates in the main groups IA, IB and II by ion-exchange and partition chromatography has led to the identification of some of the conjugates present. 4. Oestriol 3-sulphate 16α-glucuronide and 16α-hydroxyoestrone 3-sulphate 16α-glucuronide have been identified in peak IA. 5. The main components of peak IB have been identified as oestrone 3-glucuronide and oestriol 3-glucuronide. 6. The major conjugate in peak II was oestriol 16α-glucuronide and no oestriol 17β-glucuronide was found; small amounts of the ring-d monoglucuronides oestradiol 17β-glucuronide, 16-epioestriol 16β-glucuronide and 16α-hydroxyoestrone 16α-glucuronide were found in this fraction. 7. The behaviour of synthetic oestrogen monoglucuronides has been used as a guide in separation.     

Urinary metabolites of cannabidiol in dog, rat and man and their identification by gas chromatography—mass spectrometry

Urinary metabolites of cannabidiol (CBD), a non-psychoactive cannabinoid of potential therapeutic interest, were extracted from dog, rat and human urine, concentrated by chromatography on Sephadex LH-20 and examined by gas chromatography—mass spectrometry as trimethylsilyl (TMS), [²H₉]TMS, methyl ester—TMS and methyloxime—TMS derivatives. Fragmentation of the metabolites under electron-impact gave structurally informative fragment ions; computer-generated single-ion plots of these diagnostic ions were used extensively to aid metabolite identification. Over fifty metabolites were identified with considerable species variation. CBD was excreted in substantial concentration in human urine, both in the free state and as its glucuronide. In dog, unusual glucoside conjugates of three metabolites (4″- and 5″-hydroxy- and 6-oxo-CBD), not excreted in the unconjugated state, were found as the major metabolites at early times after drug administration. Other metabolites in all three species were mainly acids. Side-chain hydroxylated derivatives of CBD-7-oic acid were particularly abundant in human urine but much less so in dog. In the latter species the major oxidized metabolites were the products of β-oxidation with further hydroxylation at C-6. A related, but undefined pathway resulted in loss of three carbon atoms from the side-chain of CBD in man with production of 2″-hydroxy-tris,nor-CBD-7-oic acid. Metabolism by the epoxide-diol pathway, resulting in dihydro-diol formation from the Δ-8 double bond, gave metabolites in both dog and human urine. It was concluded that CBD could be used as a probe of the mechanism of several types of biotransformation; particularly those related to carboxylic acid metabolism as intermediates of the type not usually seen with endogenous compounds were excreted in substantial concentration.     

Analysis of the thromboxane/prostacyclin balance in human urine by gas chromatography/selected ion monitoring: abnormalities in diabetics

We microanalyzed 2,3-dinor-6-keto-prostaglandin F_1α (2,3-dinor-6-keto-PGF_1α 1) and 11-dehydrothromboxane B₂ (11-dehydro-TXB₂, 2) in human urine. Samples containing a [²H₄]-analogue as an internal standard were extracted by chromatography using Sep Pak tC18 and silica gel. The compounds were then analysed by means of the lactone ring opening reaction and dimethylisopropylsilylation. The conversion of 1 to 1-methyl ester (ME)-propylamide (PA)-9,12,15-dimethylisopropylsilyl (DMIPS) ether derivative and of 2 to 1-ME-6-methoxime (MO)-9,12,15-tris-DMIPS ether derivative was followed by gas chromatography/selected ion monitoring (GC/SIM). Interfering substances from the urine matrix were eliminated during GC/SIM analysis using a DB-5 column. We were able to detect 1 (222–1031 pg/mg creatinine) and 2 (18–155 pg/mg creatinine) in human urine. Furthermore, the thromboxane/prostacyclin (IX/PGI) ratio in the urine of diabetics was higher than that of healthy volunteers. This method can be used to determine the TX/PGI balance in human urine.     

Analysis of metabolic profiles of prostaglandins in urine using a lipophilic anion exchanger

A method is described for the fractionation of prostaglandins and their metabolites in urine. Following acidification and extraction on Amberlite XAD-2, samples were separated by chromatography on the lipophilic anion exchanger diethyl-aminohydroxypropyl Sephadex LH-20 into fractions containing neutral compounds, monocarboxylic, dicarboxylic and polycarboxylic acids. The compounds in resulting fractions were further separated by reversed phase partition chromatography. As an application, the metabolic profiles in urine of [9β-^3H]-labeled prostaglandin F_2α¹ and prostaglandin analogs 15-methyl-PGF_2α and 16,16-dimethyl-PGF_2α were investigated in the cynomolgus monkey. It was demonstrated that the resolution of individual prostaglandin metabolites by reversed phase partition chromatography was considerably simplified by initial group separation on the anion exchanger, and several metabolites were much purified. A glucuronic acid conjugate of the main metabolite of 15-methyl-PGF_2α (dinor-15-methyl-PGF_2α) was tentatively identified using computerized gas chromatography - mass spectrometry.     

Urinary excretion of C-20-reduction products of corticosterone

1. A 200 mg. portion of corticosterone was ingested by a healthy man and the urine collected. Part of the urine was treated with the gastric juice of Helix pomatia and extracted with ethyl acetate, and the extract fractionated with Girard T. Paper-chromatographic separation of the non-ketonic fraction in the Bush (1952) system B₅ revealed the presence of two unknown polar components. 2. The unknown compounds did not possess a reducing (blue tetrazolium) or a reducible (potassium borohydride) grouping. Both contained a terminal α-glycollic fragment as shown by the formation of formaldehyde, and of a non-volatile aldehyde on oxidation with sodium bismuthate. 3. Unknown compound (I) had paper-chromatographic mobilities identical with those of 5β-pregnane-3α,11β,20β,21-tetraol. The oxidation product of compound (I) had a retention time (gas–liquid chromatography) on an SE30 column identical with that of 3α,11β-dihydroxy-21-nor-5β-pregnan-20-al. The retention times of various derivatives agreed with those produced in an identical manner on the standard, and accordingly compound (I) is formulated as 5β-pregnane-3α,11β,20ξ,21-tetraol. 4. Unknown compound (II) had a higher R_F than compound (I), and its oxidation product had a longer retention time than that of compound (I). From the group effects observed in paper and gas–liquid chromatography, compound (II) is tentatively formulated as 5α-pregnane-3α,11β,20ξ,21-tetraol. The 5α/5β ratio found was about 2·0.     

An approach to the systematic analysis of urinary steroids

1. Human urine, its extracts, extracts of urine pretreated with enzyme preparations containing β-glucuronidase and steroid sulphatase or β-glucuronidase alone, and products derived from the specific solvolysis of urinary steroid sulphates, were submitted to the following sequence of operations: reduction with borohydride; oxidation with a glycol-cleaving agent (bismuthate or periodate); separation of the products into ketones and others; oxidation of each fraction with tert.-butyl chromate, resolution of the end products by means of paper chromatography or gas–liquid chromatography or both. 2. Qualitative experiments indicated the kind of information the method and some of its modifications can provide. Quantitative experiments were restricted to the direct treatment of urine by the basic procedure outlined. It was partly shown and partly argued that the quantitative results were probably as informative about the composition of the major neutral urinary steroids (and certainly about their presumptive secretory precursors) as those obtained by a number of established analytical procedures. 3. A possible extension of the scope of the reported method was indicated. 4. A simple technique was introduced for the quantitative deposition of a solid sample on to a gas–liquid-chromatographic column.     

Interactions between glycosaminoglycans and proteins, with particular reference to a new technique for isolating serum glycoproteins

1. Chondroitin sulphate–serum protein complexes (A, B and C), successively precipitated by adding chondroitin sulphate to serum at three arbitrary descending pH values (5·2, 4·3 and 3·1), were dissociated at pH 6·7 and chromatographed on DEAE-Sephadex, when the liberated serum proteins were simultaneously freed of chondroitin sulphate and separated into five fractions. Evidence that serum proteins were precipitated as a result of electrostatic interactions with dissociated carboxylate groups on the glycosaminoglycan is presented. 2. Serum proteins (fraction G), unable to form complexes with chondroitin sulphate, contained 4·4% of sialic acid and accounted for 4 and 26% of the total protein and protein-bound sialic acid in serum respectively. This fraction interacted electrostatically with chondroitin sulphate only when rendered more basic by removal of sialic acid residues with neuraminidase. The heat stability, solubility properties and high carbohydrate content of fraction G classified it as a seromucoid fraction. 3. Fraction G contained several glycoprotein and hexuronic acid-containing fractions, including a hitherto undetected brown-pigmented high-molecular-weight serum component, which migrated in starch gel between the origin and Sα₂-globulin and contained 3·1 and 4·1% of sialic acid and hexuronic acid respectively. 4. Glycosaminoglycan–protein interactions are discussed in relation to protein fractionation. By prior removal of less acidic proteins by these interactions, a new technique is available for isolating serum seromucoids in higher yields and under milder conditions than existing methods.     

Presence of immunoreactive salusin-β in human plasma and urine

Salusin-α and salusin-β are multifunctional bioactive peptides originally identified using bioinformatics analyses. Salusin-β has been shown to exert potent hypotensive, bradycardic, and pro-atherosclerotic effects. The form in which it exists in biological fluids remains undetermined due to technical difficulties originating from its unexpected physicochemical properties. Here we show that salusin-β peptide adheres to polypropylene and polystyrene, so that the aliquoted peptide dissolved in distilled water may rapidly disappear from the solution. By circumventing these features and using an antibody against C-terminal portion of salusin-β, we have successfully established a specific radioimmunoassay suitable for detection of immunoreactive human salusin-β. We have characterized the molecular form of salusin-β in human plasma and urine. The assay detected immunoreactive salusin-β concentrations as low as 5 fmol/tube and the concentration required for 50% inhibition of binding was 122 fmol/tube. Cross-reactivities with salusin-α and other bioactive peptides were negligible. Reverse-phase high performance liquid chromatography coupled with the radioimmunoassay detection after extraction from plasma and urine and using an octyl-silica column, revealed a major immunoreactive component that co-eluted with authentic salusin-β. Salusin-β-like immunoreactivity in normal human urine ranged from 0.23 to 2.22 nmol/l (mean ± SD, 1.16 ± 0.84 nmol/l, n = 10). These data present the first evidence that salusin-β circulates and is excreted in its authentic form, thereby verifying the initially predicted processing sites for salusin-β in humans.     

Determination of urinary 18β-glycyrrhetinic acid by gas chromatography and its clinical application in man

A sensitive and quantitative gas chromatographic assay for the determination of 18β-glycyrrhetinic acid (18β-GA), the main metabolite of glycyrrhizin after oral licorice consumption in human urine, has been developed and validated. For the extraction of 18β-GA from urine two Sep-Pak C₁₈ extractions, hydrolysis with Helix pomatia and three liquid–liquid extractions were performed, using 18α-glycyrrhetinic acid (18α-GA) as internal standard. Both 18β-GA and internal standard were converted into their pentafluorobenzyl-ester/trimethylsilyl-ether derivatives and detected by flame ionization detection using a WCOT-fused-silica capillary column. Good quality control data were obtained in precision and accuracy tests. The detection limit of the gas chromatographic method was 10 μg/l with a urine volume of 10 ml. A detection limit of 3 μg/l was obtained by performing GC–MS. The GC method was used to monitor the urinary excretion of 18β-GA after licorice consumption by two healthy volunteers and a patient suspected of licorice abuse. Furthermore, it was shown that this GC assay enables to detect other metabolites related to licorice consumption.     

Determination of 6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α, thromboxane B2, 2,3-dinor-thromboxane B2, PGE2, PGD2 and PGF2α in human urine by gas chromatography—negative ion chemical ionization mass spectrometry

A method for quantification of 6-keto-PGF_1α, 2,3-dinor-6-keto-PGF_1α, TXB₂, 2,3-dinor TXB₂, PGE₂, PGD₂ and PGF_2α in human urine samples, using gas chromatography—negative ion chemical ionization mass spectrometry, is described. Deuterated analogues were used as internal standards. Methoximation was carried out in urine samples which were subsequently applied to phenylboronic acid cartridges, reversed-phase cartridges and thin-layer chromatography. The eluents were further derivatized to pentafluorobenzyl ester trimethylsilyl ethers for final quantification by gas chromatography—mass spectrometry. The overall recovery was 77% for tritiated 6-keto-PGF_1α and 55% for tritiated TXB₂. Urinary levels of prostanoids were determined in a group of six volunteers before and after intake of the thromboxane synthase inhibitor Ridogrel, and related to creatinine clearance.