Simultaneous determination of plasma estrogens, androgens, and progesterone during the human menstrual cycle

A method is described for the purification and quantitation of estradiol-17β (E₂), estrone (E₁), testosterone (T), androstenedione (A) and progesterone (P) from a single plasma sample extraction. A combination of Sephadex LH-20 column chromatography and thin layer chromatography is used to separate the phenolic and neutral steroids; quantitation of the purified steroids is by radioimmunoassay (E₂, E₁) and competitive protein binding (T,A,P) techniques. The precision and accuracy of this method is comparable with that reported by others. A preliminary investigation of the temporal relationship between estrogens androgens, progesterone and serum LH during five normal and one short luteal phase menstrual cycle is presented.     

Method for quantitative analysis of 16, 16-dimethyl-prostaglandin E2 from plasma using deuterated carrier and gas chromatography-mass spectrometry.

The preparation of 3,3,4,4-²H₄16,16-dimethyl-prostaglandin E₂ (PGE₂), 16,16-dimethyl-5,6-didehydro-PGE₂, and 5,6-³H₂16,16-dimethyl-PGE₂ is described. These compounds have been used for development of a gas-liquid chromatography-mass spectrometry method for quantitative determination of corresponding nondeuterated prostaglandin. The method is based on addition of a known amount of carrier to the sample and after purification and derivation the ratio between the protium and deuterium form is measured in the mass spectrometer. With this technique 40 pg of 16,16-dimethyl-PGE₂ can be determined with a precision of ±2.6%.     

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.     

Simultaneous determination of cortisol and cortisone in urine by reversed-phase high-performance liquid chromatography clinical and doping control applications

A reversed-phase high-performance liquid chromatography (HPLC) method for the simultaneous determination of cortisol and cortisone in human urine samples using methylprednisolone as the internal standard is decribed. The method involves the systematic use of isocratic mobile phases of water and methanol, acetonitrile or tetrahydrofuran and a reversed-phase Hypersil C₁₈ column. A water-acetonitrile mixture used as the mobile phase proved to be the most adequate one for analyzing urine samples purified by solvent extraction. The proposed method is sensitive, reproducible and selective. It was applied to the determination of cortisol and cortisone in several human urine samples: healthy subjects, sportsmen before and/or after stress for doping control purposes, and patients with Cushing's syndrome.     

A comparison of sex steroid hormone excretion and metabolism by psittacine species

The metabolism, excretory rates, and excretory patterns of carbon 14 (¹⁴C) radiolabeled estradiol (E₂) and testosterone (T) were studied in female budgerigars (Melopsittacus undulatus) and orange‐winged Amazon parrots (Amazona amazonica). Radiolabeled E₂ and T were injected intramuscularly into six budgerigars and two orange‐winged Amazon parrots. Serial fecal/urine samples were collected for 168 h post‐radiolabel injection. Peak radiolabeled E₂ excretion was observed at 4 h post‐injection, and by 24 h, 93.3 ± 6.3 and 65.9% (range, 59.1–72.7%) of the injected radiolabel was recovered in the fecal/urine matter of budgerigars and orange‐winged Amazon parrots, respectively. Similarly, peak radiolabeled T excretion was observed at 4 h post‐injection with 92.7± 3.6 and 66.2% (range, 57.5–75.2%) of the injected radiolabel recovered in the fecal/urine matter by 24 h in the budgerigars and orange‐winged Amazon parrots, respectively. High‐performance liquid chromatography (HPLC) analysis of the fecal/urine material revealed that both parrot species excreted >80% of the radiolabel in the form of complex steroid conjugates. Immunoreactive E₂ and T metabolites were detected using estrone (E₁) and C‐21/C‐19 conjugate enzyme immunoassays, respectively. Hydrolysis of the E₂ metabolites and HPLC analysis of the ether extracts revealed that E₂ and E₁ were the major steroid moieties. Hydrolysis of the T metabolites and HPLC analysis of the ether extracts revealed two and three major unconjugated peaks for the budgerigars and the orange‐winged Amazon parrots, respectively. Zoo Biol 18:247–260, 1999. © 1999 Wiley‐Liss, Inc.     

Profiling oestrogens and testosterone in human urine by stable isotope dilution/benchtop gas chromatography–mass spectrometry

Oestrogens, such as oestrone (E₁), 17β-oestradiol (E₂), oestriol (E₃) and their biologically active metabolites 2-methoxyoestrone (2-MeOE₁), 2-hydroxyoestradiol (2-OHE₂) 16-ketooestradiol (16-OE₂), 16-epioestriol (16-epiE₃), as well as testosterone (T) play an important role in physiological and pathological developmental processes during human development. We therefore aimed at developing an isotope dilution/bench top gas chromatography–mass spectrometry (ID/GC–MS) method, based on benchtop GC–MS, for the simultaneous determination (‘profiling’) of the above analytes in children. The method consisted of equilibration of urine (5 ml) with a cocktail containing stable isotope-labelled analogues of the analytes as internal standards ([2,4-²H₂]E₁, [2,4,16,16-²H₄]E₂, [2,4,17-²H₃]E₃, [16,16,17-²H₃]T, [1,4,16,16-²H₄]2-MeOE₁, [1,4,16,16,17-²H₅]2-OHE₂, [2,4,15,15,17-²H₅]16-OE₂ and [2,4-²H₂]16-epiE₃). Then, solid-phase extraction (C₁₈ cartridges), enzymatic hydrolysis (sulphatase from Helix pomatia (type H-1)), re-extraction, purification by anion exchange chromatography and derivatisation to trimethylsilyl ethers followed. The samples were analysed by GC–MS (Agilent GC 6890N/5975MSD; fused silica capillary column 25 m × 0.2 mm i.d., film 0.10 μm). Calibration plots were linear and showed excellent reproducibility with coefficients of determination (r²) between 0.999 and 1.000. Intra- and inter-assay coefficients of variation (CV) were <2.21% for all quantified metabolites. Sensitivity was highest for 2-OHE₂ (0.25 pg per absolute injection: signal-to-noise ratio (S/N) = 3) and lowest for 16-epiE₃ (2 pg per absolute injection: S/N = 2.6), translating into corresponding urine sample analyte concentrations of 0.025 ng ml^?1 and 0.2 ng ml^?1, respectively. Accuracy – determined in a two-level spike experiment – showed relative errors ranging between 0.15% for 16-OE₂ and 11.63% for 2-OHE₂. Chromatography showed clear peak shapes for the components analysed. In summary, we describe a practical, sensitive and specific ID/GC–MS assay capable of profiling the above-mentioned steroids in human urine from childhood onwards.     

Structure, gas chromatographic measurement, and function of suberin synthesized by potato tuber tissue slices

The polymeric material (suberin) of the wound periderm of potato tuber slices was analyzed after depolymerization with LiAIH₄ in tetrahydrofuran or BF₃ in methanol with the use of thin layer chromatography, chemical modification, and combined gas-liquid chromatography and mass spectrometry. Fatty acids (C₁₆ to C₂₆), fatty alcohols (C₁₆ to C₂₆), octadec-9-ene-1, 18-dioic acid, and 18-hydroxy-octadec-9-enoic acid were identified to be the major components. Based on the structural information that the two bifunctional C₁₈ molecules constituted a major portion of suberin, a gas chromatographic method of measuring suberization was developed. This method consisted of hydrogenolysis of powdered tissue followed by thin layer chromatography and gas chromatographic measurement of octadecene-1, 18-diol as the trimethylsilyl ether. With this assay it was shown that the development of resistance to water loss by the tissue slices was directly proportional to the quantity of the bifunctional C₁₈ molecules, thus providing evidence that a function of suberin is prevention of water loss.     

Synthesis of prostaglandins by subpopulations of human peripheral blood monocytes

The ability of monocytes/macrophages to regulate various aspects of immunologic responses may in part depend on their release of soluble substances such as prostaglandins. Using quantitative gas-liquid chromatography/mass spectrometry, prostaglandin E₂ was found to be the major prostaglandin synthesized in culture by human peripheral blood monocytes. Subjecting these cells to discontinuous density gradient fractionation demonstrated significant differences in the synthesis of prostaglandins E₂ and E₁ among the resulting monocyte subpopulations.     

Use of lysed human platelets to study prostaglandin E2 production

The conversion of 1-¹⁴C-arachidonic acid into prostaglandin E₂ was studied in lysed human platelets. Optimum production of the labeled reaction product was obtained when reduced glutathione and hydroquinone were included in the incubations. The labeled product was characterized by silicic acid column chromatography, thin-layer chromatography, and gas-liquid chromatography and was found to behave as standard prostaglandin E₂. The results indicate that the prostaglandin synthetase in the human blood platelet is similar to prostaglandin synthetases found in other tissues.     

Enzymic preparation of dioxygen-18 labelled leukotriene E4 and its use in quantitative gas chromatography—mass spectrometry

A simple and rapid method is described for the preparation of a stable isotope oxygen-18 labelled leukotriene E₄ (LTE₄). Oxygen-18 labelling of LTE₄ methyl ester in oxygen-18 water catalysed by a pig liver esterase resulted in the incorporation of two oxygen-18 atoms in the carboxylic group of LTE₄ to the extent of 89.8% ([¹⁸O₂]LTE₄) and one oxygen-18 atom to the extent of 9.4% ([¹⁶O¹⁸O]LTE₄), with only 0.7% remaining unchanged ([¹⁶O₂]LTE₄). [¹⁸O₂]LTE₄ was found not to back-exchange following incubation in acidified urine (pH 4.0) at 4°C for up to 20 h. [¹⁸O₂]LTE₄ was demonstrated to be a useful internal standard in a method for the quantitative determination of LTE₄ in human urine involving high-performance liquid chromatography and gas chromatography with negative-ion chemical ionization tandem mass spectrometry: the concentration of LTE₄ in a 24-h urine sample of a healthy subject was determined to be 68.1 pg/ml.     

Direct estimation of urine estrone conjugate for a rapid pregnancy diagnosis in sows

A rapid and direct radioimmunoassay of urine estrone conjugate (E₁C) was developed. Urine samples were taken from Large White sows by inserting sponges in the vagina which were expelled during micturition. Analysis of samples collected daily from 13 sows between 2 and 40 days after service showed that high urine E₁C levels occurred between day 20 and 30 after insemination in pregnant sows; maximum concentrations were observed on day 25 (79.4 ± 11.7 ng/ml, $\text{X}\text{± s.e.m.}$). In this period E₁C levels never exceeded 1 ng/ml in nonpregnant sows. A single urine sample was then taken from 84 sows, 25 days after insemination, to check the accuracy of E₁C estimation as a test for early pregnancy diagnosis. Seventy-six of the examined animals were correctly diagnosed, giving an overall accuracy of 90.4%. No relationship was found between litter size and urine E₁C levels.     

Determination of structure and composition of suberin from the roots of carrot, parsnip, rutabaga, turnip, red beet, and sweet potato by combined gas-liquid chromatography and mass spectrometry

Suberin from the roots of carrots (Daucus carota), parsnip (Pastinaca sativa), rutabaga (Brassica napobrassica), turnip (Brassica rapa), red beet (Beta vulgaris), and sweet potato (Ipomoea batatas) was isolated by a combination of chemical and enzymatic techniques. Finely powdered suberin was depolymerized with 14% BF₃ in methanol, and soluble monomers (20-50% of suberin) were fractionated into phenolic (<10%) and aliphatic (13-35%) fractions. The aliphatic fractions consisted mainly of ω-hydroxyacids (29-43%), dicarboxylic acids (16-27%), fatty acids (4-18%), and fatty alcohols (3-6%). Each fraction was subjected to combined gas-liquid chromatography and mass spectrometry. Among the fatty acids very long chain acids (>C₂₀) were the dominant components in all six plants. In the alcohol fraction C₁₈, C₂₀, C₂₂, and C₂₄ saturated primary alcohols were the major components. C₁₆ and C₁₈ dicarboxylic acids were the major dicarboxylic acids of the suberin of all six plants and in all cases octadec-9-ene-1, 18-dioic acid was the major component except in rutabaga where hexadecane-1, 16-dioic acid was the major dicarboxylic acid. The composition of the ω-hydroxyacid fraction was quite similar to that of the dicarboxylic acids; 18-hydroxy-octadec-9-enoic acid was the major component in all plants except rutabaga, where equal quantities of 16-hydroxyhexadecanoic acid and 18-hydroxyoctadec-9-enoic acid (42% each) were found. Compounds which would be derived from 18-hydroxyoctadec-9-enoic acid and octadec-9-ene-1, 18-dioic acid by epoxidation, and epoxidation followed by hydration of the epoxide, were also detected in most of the suberin samples. The monomer composition of the six plants showed general similarities but quite clear taxonomic differences.     

Plasma estrogens,progesterone and chorionic gonadotropin in pregnant rhesus monkeys (Macaca mulatta) after ovariectomy

Maternal peripheral plasma concentrations of estrone (E₁), estradiol-17β (E₂), and progesterone (P) were determined in 4 rhesus monkey ovariectomized in early pregnancy (22–24 days). After ovariectomy, plasma concentrations of E₁ and E₂ were basal for 1 to 2 weeks. In contrast, slightly higher estrogen levels, which may be attributed to the ovaries, were found in intact pregnant monkeys. E₂ levels increased rapidly after this and exceeded those of E₁ until the 5th month of gestation. From that time until parturition, E₁ levels equaled or exceed those of E₂ in most instances. The pattern of P concentrations was similar to that observed in intact monkeys. Urinary chorionic gonadotropin (MCG) levels in ovariectomized monkeys were not significantly differen from those found in normal pregnancies. Thus, the patterns for circulating E_l, E₂ and P, as well as for the excretion of MCG,. after ovari-ectomy were remarkably similar to those found in intact, pregnant rhesus monkeys, indicating minimal ovarian influence.     

Analysis of nitrate,nitrite, and [15N]nitrate in biological fluids

A new automated system for the analysis of nitrate via reduction with a high-pressure cadmium column is described. Samples of urine, saliva, deproteinized plasma, gastric juice, and milk can be analyzed for nitrate, nitrite, or both with a lower limit of detection of 1.0 nmol NO₃^? or NO₂^?/ml. The system allows quantitative reduction of nitrate and automatically eliminates interference from other compounds normally present in urine and other biological fluids. Analysis rate is 30 samples per hour, with preparation for most samples limited to simple dilution with distilled water. The application of gas chromatography/mass spectrometry for the analysis of ¹⁵NO₃^? in urine after derivatization to ¹⁵NO₂-benzene is also described.     

Simultaneous quantification of 8-iso-prostaglandin-F2α and 11-dehydro thromboxane B2 in human urine by liquid chromatography-tandem mass spectrometry

Both F₂-isoprostanes (8-iso-PGF_2α), a well-known marker of oxidative stress, and thromboxanes A₂ (TXA₂) are involved in atherosclerosis through LDL oxidation and platelet activation. Different aspects of the pathology can be described by 8-iso-PGF_2α and TXA₂ so it is important to determine both their concentrations to monitor the disease progression and/or therapy effects. We developed a simple and sensitive method based on liquid chromatography-tandem mass spectrometry, using electrospray ionization in negative-ion mode, for the simultaneous measurement of the concentration of 8-iso-PGF_2α and 11-dehydro thromboxane B₂ (11-DH-TXB₂), a TXA₂ metabolite. This method was applied to analyze urine samples collected overnight from 15 atherosclerotic patients, with documented carotid artery sclerosis (CAS), and from 20 controls. The detection limit was 0.097 pg/μL for 8-iso-PGF_2α and 0.375 pg/μL for 11-DH-TXB₂, with a linear range of 0.78-25 pg/μL; the inter- and intraday imprecision was <5% for both metabolites. These analytes were higher in CAS (P < 0.005 vs controls) and were positively correlated in patients but not in controls, even after adjustment for age and gender (r = 0.60; P = 0.032). This highly sensitive, precise, and rapid method allows for the simultaneous determination of 8-iso-PGF_2α and 11-DH-TXB₂ in human urine samples in order to evaluate oxidative stress and platelet aggregation.     

Quantitative analysis of biomarkers,drugs and toxins in biological samples by immunoaffinity chromatography coupled to mass spectrometry or tandem mass spectrometry: A focused review of recent applications

Immunoaffinity chromatography (IAC), mass spectrometry and especially tandem mass spectrometry (MS/MS) represent the most efficient and reliable analytical techniques for specific isolation, unequivocal identification and accurate quantification of numerous natural and synthetic substances in biological samples. This review article focuses on the combined use of these outstanding methodologies in basic and clinical research and in life sciences for the quantitative analysis of low- and high-molecular mass biomarkers, drugs and toxins in urine, plasma or serum samples, in tissue and other biologicals systems published in the last decade. The analytes discussed in some detail include the biomarkers of oxidative stress 15(S)-8-iso-prostaglandin F_2α {15(S)-8-iso-PGF_2α} and 3-nitrotyrosine, the major urinary metabolite of the lipid mediators cysteinyl leukotrienes, i.e., the leukotriene E₄ (LTE₄), melatonin, and the major collagen type II neoepitope peptide in human urine.     

Control system for detection of the illegal use of naturally occurring steroids in calves

Within the scope of the National Plan for Hormone Control in The Netherlands, a study was performed to develop a system for control of the illegal use of three naturally occurring hormones [oestradiol-17β (E₂-17β), testosterone (T), progesterone (P)] for fattening purposes in animal production. Using a specific high-performance liquid chromatographic—radioimmunoassay method, reference values were established for concentrations of E₂-17β, T and P and some of their metabolites in blood plasma and urine from untreated male and female veal calves. E₂-17β levels of both male and female calves were <0.01 μg/l in blood plasma and <0.2 μg/l in urine. For male veal calves levels of T and epitestosterone (epiT) in blood plasma and urine varied widely. The P levels were <0.1–0.3 μg/l in blood plasma and <0.6–10 μg/l in urine from both male and female calves. To investigate the effect of anabolic treatment on the hormone levels in plasma and excreta, male veal calves were injected, subcutaneously into the dewlap, with a solution containing 20 mg of E₂-17β benzoate and 200 mg of T propionate in 5 ml of arachis oil. Only the levels of E₂-17β and E₂-17α in blood plasma and excreta were elevated until about one week after injection, compared with the untreated control calves and the reference values. T and epiT levels were similar in plasma and excreta from both untreated and treated animals.     

Serum and urinary levels of reproductive hormones associated with the estrous cycle in white-tailed deer (Odocoileus virginianus)

Concentrations of estradiol-17β (E₂), progesterone (P_o), and luteinizing hormone (LH) in serum and estrone glucuronide (E₁G) and pregnanediol glucuronide (PdG) in urine were determined by direct radioimmunoassay in five yearling and three adult female white-tailed deer (Odocoileus virginianus) from 14 October to 20 December 1985. Elevated levels of LH were recorded before the first estrus of the breeding season and immediately preceding or during behavioral estrus. Urinary E₁G and PdG concentrations were poorly correlated with serum levels of E₂ and P_o, respectively. Serum P_o levels prior to the first estrus of the breeding season indicate that pubertal yearlings experience silent ovulations and suggest that silent ovulations may be important in the transition from adolescence to cycling adult in white-tailed deer. © 1992 Wiley-Liss, Inc.     

Analysis of A,B, E,and F prostaglandins as pentafluorobenzyl esters by electron-capture gas-liquid chromatography

A new and sensitive method is described for the simultaneous analysis of a mixture containing PGE₁, PGE₂, PGF_1α, and PGF_2α by electron-capture gas-liquid chromatography. During derivatization of the mixture, PGE₁ and PGE₂ were converted to PGB₁ and PGB₂, respectively, yielding a mixture of PGB₁, PGB₂, PGF_1α, and PGF_2α trimethylsilyl ether pentafluorobenzyl esters. Gas chromatographic resolution of all four derivatives is sufficient for quantitation of each prostaglandin. The A prostaglandins were analyzed by similar conversion to the respective B prostaglandin derivatives. Minimum detection limits for the B and F prostaglandin derivatives were 10 pg and 1 pg, respectively. Samples of rabbit kidney medulla were incubated and analyzed for A, B, E, and F prostaglandins. The results indicate that the method is capable of high recovery and reproducibility.     

Analysis of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine, the mercapturic acid derived from N,N-dimethylformamide

Human biotransformation of the industrial solvent N,N-dimethylformamide gives raise to N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) which has the longest half-life (about 23 h) among urinary metabolites of N,N-dimethylformamide. It could be used for monitoring industrial exposure over several workdays, by measuring it in urine samples collected at the end of the working week. This is consistent with the suggestions of the American Conference of Governmental Industrial Hygienists, which established a limit of 40 mg/l for the year 2000. An easy, cheap and user-friendly method has been developed for determination of urinary AMCC. Unlike currently available methods, it requires neither a time-consuming preparation phase nor gas chromatographic analysis with a nitrogen-phosphorus or mass detector. The method uses high-performance liquid chromatography (HPLC), with an UV detector at 436 nm. A 10-μl volume of urine is added to a carbonate–hydrogen carbonate buffer and mixed with a dabsyl chloride solution in acetonitrile. The reaction between AMCC and the reagent is performed at 70°C for 10 min. The ‘dabsylated’ product is stable for at least 12 h. After brief centrifugation, the solution is ready for HPLC analysis using a C₁₈ column (250×4.6 mm, 5 μm). The method is sensitive (detection limit 1.8 mg/l) and specific. It identified urinary AMCC in urine of 40 subjects not exposed to N,N-dimethylformamide with a median concentration of 3.9 mg/l. In urine samples from 20 workers exposed to N,N-dimethylformamide (5–40.8 mg/m³), AMCC concentrations ranged from 16 to 170 mg/l. Industrial toxicology laboratories with limited instrumentation will be able to use it in the biological monitoring of workers exposed to N,N-dimethylformamide.