Simultaneous determination of endogenous and stable isotope-labelled 6beta-hydroxycortisols in human urine by stable isotope dilution mass spectrometry

This study describes a capillary GC-MS method for the simultaneous determination of endogenous 6beta-hydroxycortisol (6beta-OHF) and its stable isotope-labelled analogue, 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6beta-OHF-2H(5)), in human urine. 6beta-Hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (cortisol-13C(4),(2)H(5)) was used as an analytical internal standard. The methoxime trimethylsilyl ether (MO-TMS) derivatization was employed for the GC-MS analysis of 6beta-OHF. Quantitation was carried out by selected-ion monitoring (SIM) of the characteristic fragment ion ([M-31](+.)) of the MO-TMS derivative of 6beta-OHF. The sensitivity limit of the present GC-MS-SIM method was found to be 25 pg per injection for 6beta-OHF (S/N ratio=5.6). The within-day reproducibility in the amounts of unlabelled and labelled 6beta-OHFs determined were in good agreement with the actual amounts added, the relative errors being less than 5.30%. The inter-assay RSDs were less than 4.95% for unlabelled and labelled 6beta-OHFs.     

Simultaneous determination of tetrahydrocortisol and tetrahydrocortisone in human plasma and urine by stable isotope dilution mass spectrometry

A capillary gas chromatographic–mass spectrometric method for the simultaneous determination of tetrahydrocortisol (THF, 3α,11β,17α,21-tetrahydroxy-5β-pregnane-20-one), allo-tetrahydrocortisol (allo-THF, 3α,11β,17α,21-tetrahydroxy-5α-pregnane-20-one) and tetrahydrocortisone (THE, 3α,17α,21-trihydroxy-5β-pregnane-11,20-dione) in human plasma and urine is described. [1,2,3,4,5-²H₅]THF (THF-d₅), allo-[1,2,3,4,5-²H₅]THF (allo-THF-d₅) and [1,2,3,4,5-²H₅]THE (THE-d₅) were used as internal standards. A double derivatization (bismethylenedioxypentafluoropropionate, BMD-PFP) made possible the separation of the three tetrahydrocorticoids with good gas chromatographic behavior. Quantitation was carried out by selected-ion monitoring of the characteristic fragment ions ([M−30]⁺) of the BMD-PFP derivatives of THF, allo-THF and THE. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring low concentrations of THF, allo-THF and THE in human plasma and urine.     

Galactonate determination in urine by stable isotope dilution gas chromatography-mass spectrometry

A stable isotope dilution assay was developed for the sensitive determination of D-galactonic acid. D-[U-13C(6)]galactono-1,4-lactone was prepared as internal standard. Unlabelled and U-13C-labelled D-galactonic acid species were converted to the N-(1-butyl)galactonamide pentaacetate derivatives and assessed by gas chromatography-mass spectrometry (GC-MS). Positive chemical ionisation and monitoring of the [MH-60](+)-ions in the galactonate chromatographic peak at m/z 402 and m/z 408 were used for quantification. The procedure was applied to study the variability of D-galactonate excretion in healthy subjects and galactosemic patients and to monitor the D-galactonate-D-galactitol ratio in human urine.     

Simultaneous determination of endogenous and 13C-labelled thyroid hormones in plasma by stable isotope dilution mass spectrometry

This study describes a capillary gas chromatography-mass spectrometry (GC-MS) method for the simultaneous determination of endogenous thyroid hormone (thyroxine, T4) and its 13C-labelled analogue (13C6-thyroxine) in plasma. 13C9-thyroxine was used as analytical internal standard. A double derivatization (CH3OH/HCl and HFBA) inducing good GC mobility was used for the GC-MS analysis of the thyroid hormones. Quantification was carried out by selected ion monitoring (SIM) of specific ions of the fragment ions (m/z 970/976/979). The detection limit of the present GC-MS-SIM method was found to be 100 pg per injection for thyroxine (S/N=3.0). A first implementation in in vivo tests of 13C6-T4 like metabolic tracer was carried out under veterinary control on one cat and one rabbit. The thyroxine follow-up was done by GC-MS and based on double isotopic dilution with two different regio-selective 13C-labelled molecules of the same hormone. The present paper discusses the possibilities and limitations of this methodology. The in vivo experiment demonstrated that the use of stable isotopes and mass spectrometry provide a reliable methodology for hormonal monitoring.     

Simultaneous determination of selenomethionine enantiomers in biological fluids by stable isotope dilution gas chromatography-mass spectrometry

A method for the stereoselective determination of D- and L-enantiomers of selenomethionine in mouse plasma was developed using gas chromatography-mass spectrometry with selected-ion monitoring (GC-MS-SIM). DL-[(2)H(3,)(82)Se]selenomethionine was used as analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. Selenomethionine enantiomers in mouse plasma were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N-acylation with optically active (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. Quantification was performed by SIM of the molecular-related ions of the diastereomers on the chemical ionization mode. The intra- and inter-day precision for D- and L-selenomethionine spiked to mouse plasma gave good reproducibility with relative standard deviation of 3% and 3% for D-selenomethionine and 6% and 3% for L-selenomethionine, respectively. The estimated amounts were in good agreement with the actual amounts spiked, the intra- and inter-day relative error being 5% and 2% for D-selenomethionine and 2% and 1% for L-selenomethionine, respectively. The present method is sensitive enough to determine pharmacokinetics of selenomethionine enantiomers.     

Simultaneous determination of 6-oxo-prostaglandin F1 alpha and 2,3-dinor-6-oxo-prostaglandin F1 alpha in biological fluids by stable isotope dilution and negative ion chemical ionization mass spectrometry

A stable isotope dilution assay for the simultaneous determination of two metabolites of prostacyclin (1), 6-oxo-prostaglandin F1 alpha (2a) and 2,3-dinor-6-oxo-prostaglandin F1 alpha (3a), in human seminal fluid and human urine is described. A new chemical total synthesis of deuterated internal standard, 18,18,19,19-(2H4)-2,3-dinor-6-oxo-PGF1 alpha (3b), is presented and enables specific and sensitive quantification based on negative ion chemical ionization mass spectrometry. 2a and 3a were analysed as their methoxime pentafluorobenzyl ester tris(trimethylsilyl) ether derivatives in the selected ion monitoring mode registrating the [M-181]- fragments with a detection limit for both prostanoids of 10 pg per injection. The two metabolites occur in human seminal fluid in very low concentrations (2a: 2.8 ng ml-1; 3a: 1.7 ng ml-1) and cannot contribute significantly to the urinary metabolite levels which are in the range of 108-265 ng/24 h for 3a and 124-574 ng/24 h for 2a.     

Simultaneous determination of stable isotopically labelled l-histidine and urocanic acid in human plasma by stable isotope dilution mass spectrometry

A capillary gas chromatographic—mass spectrometric method for the simultaneous determination of stable isotopically labelled l-histidine (l-[3,3-²H₂,1′,3′-¹⁵N₂]histidine, l-His-[M + 4]) and urocanic acid ([3-²H,1′,3′-¹⁵N₂]urocanic acid, UA-[M + 3]) in human plasma was developed using dl-[2,3,3,5′-²H₄,2′-¹³C,1′,3′-¹⁵N₂]histidine (dl-His-[M + 7]) and [2,3,5′-²H₃,2′-¹³C,1′,3′-¹⁵N₂]urocanic acid (UA-[M + 6]) as internal standards. l-Histidine and urocanic acid were derivatized to ^αN-(trifluoroacetyl)-^imN-(ethoxycarbonyl)-l-histidine n-butyl ester and ^imN-(ethoxycarbonyl)urocanic acid n-butyl ester. Quantification was carried out by selected ion monitoring of the molecular ions of the respective derivatives of l-His-[M + 4], dl-His-[M + 7], UA-[M + 3] and UA-[M + 6]. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring plasma concentrations of l-His-[M + 4] and UA-[M + 3] following administration of trace amounts of l-His-[M + 4] to humans.     

Quantitative determination of the β-adrenoceptor stimulant formoterol in urine by gas chromatography mass spectrometry

A method for the quantitative determination of the β-stimulant formoterol in urine, using a gas chromatograph—mass spectrometer, is described. Formoterol can be analyzed after the addition of a deuterium-labelled internal standard and conversion to a mixed bispentafluoropropionyl-methyl derivative for selected ion monitoring. The detection limit was 5 ng/ml.Urinalysis after the oral administration of formoterol fumarate, using a combined enzymic hydrolysis method, revealed that the drug was conjugated with glucuronic acid in rats, dogs and humans.     

Measurement of thromboxane B2 in human urine by isotope dilution and negative ion chemical ionization mass spectrometry

A highly sensitive and specific assay for the quantification of thromboxane B2 (TXB2)(1) in human urine is described. The method is based on the use of low-blank (1H less than or equal to 0.2%) tetradeuterated internal standard 2 (18, 18, 19, 19-2H4-thromboxane B2), whose chemical synthesis is reported. After purification and high-performance liquid chromatography (HPLC) samples are derivatized to give an open-chain derivative of thromboxane B2, the methoxime pentafluorobenzyl ester tris(trimethylsilyl) ether (TXB2-MO-PFB-TMS3), most suitable for negative ion chemical ionization mass spectrometry. In the selected ion monitoring mode limits of detection per injection for pure standards and biological samples of 10 pg and 30 pg, respectively, are established. Normal urinary excretion of 1 in humans is 37-112 ng/24 h (n = 12).     

Quantification of melatonin in human saliva by liquid chromatography-tandem mass spectrometry using stable isotope dilution

A method for the determination of melatonin in human saliva has been developed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS). Saliva was collected in plastic tubes. 7-D-Melatonin was added as internal standard and the samples were cleaned and concentrated by solid-phase extraction. The limit of detection was 1.05 pg x ml(-1) and the limit of quantification was 3.0 pg x ml(-1). The accuracy of the method was +/-14% at 5.60 pg x ml(-1) and +/-9% at 19.6 pg x ml(-1). The precision was +/-13% at 6.18 pg x ml(-1) and +/-11% at 31.2 pg x ml(-1), respectively. Our HPLC-MS-MS method shows a high sensitivity and specificity for melatonin and more reliable results compared with a radioimmunoassay. The chromatographic method has been used to determine the circadian rhythm of melatonin among three nurses working the night shift and a patient suffering from an inability to fall asleep at night.     

Quantification of monofluoroacetate and monochloroacetate in human urine by isotope dilution liquid chromatography tandem mass spectrometry

The rodenticide monofluoroacetate (MFA) and monochloroacetate (MCA), a chemical intermediate from several chemical syntheses, have been identified as potential agents of chemical terrorism due to their high toxicity. In preparation for response to poisonings and mass exposures, we have developed a quantification method using isotopic dilution to determine MFA and MCA in urine from 50 to 5000 ng/mL. Both analytes were extracted from urine using solid-phase extraction; extraction recoveries were 62% (MFA) and 76% (MCA). The extracts were then separated with isocratic high-performance liquid chromatography and identified using electrospray ionization tandem mass spectrometry, with detection limits of 0.9 and 7.0 ng/mL for MFA and MCA, respectively. Selectivity was established for both analytes with unique chromatographic retention times which were correlated with isotopically labeled internal standards and the use of two mass spectral transitions for each compound. The intra-day variability was less than 5% for both analytes and the inter-day variability was 7% for MFA and 6% for MCA.     

Determination of 9α,11β-prostaglandin F2 in human urine and plasma by gas chromatography—mass spectrometry

Sensitive and specific assay methods for 9α,11β-prostaglandin F₂ (9α,11β-PGF₂) by gas chromatography—mass spectrometry with electron impact ionization are described. The mass spectrometric assay for 9α,11β-PGF₂ was based on the use of the methyl ester—dimethylisopropylsilyl ether derivative, and pentadeuterated PGF_2α as a convenient internal standard. The calibration graph for 9α,11β-PGF₂ was linear from 5 pg to 100 ng for both the standard and spiked biological samples. The limit of detection was 50 pg/ml for urine and 25 pg/ml for plasma (signal-to-noise RATIO = 2.3). The method was applied to the determination of 9α,11β-PGF₂ in urine and plasma samples from patients with bronchial asthma.     

Stable isotope dilution high-performance liquid chromatography-electrospray ionization mass spectrometry method for endogenous 2- and 4-hydroxyestrones in human urine

A sensitive, precise and accurate stable isotope dilution high-performance liquid chromatography-electrospray ionization mass spectrometry method has been developed for measuring endogenous 2- and 4-hydroxyestrones, the main catechol estrogens in human urine. Compared to the published methods using gas chromatography-mass spectrometry, this approach simplifies sample preparation and increases the throughput of analysis. The unique part of our method is the use of a simple and rapid derivatization step that forms a hydrazone at the C-17 carbonyl group of catechol estrogens. This derivatization step has greatly enhanced method sensitivity as well as HPLC separability of 2- and 4-hydroxyestrones. Standard curves were linear over a 100-fold calibration range with correlation coefficients for the linear regression curves typically greater than 0.996. The lower limit of quantitation for each catechol estrogen is 1 ng per 10-ml urine sample, with an accuracy of 97-99% and overall precision, including the hydrolysis, extraction and derivatization steps, of 1-3% for samples prepared concurrently and 2-11% for samples prepared in several batches. This method is adequate for measuring the low endogenous levels of catechol estrogens in urine from postmenopausal women.     

Determination of cytokinins by mass spectrometry based on stable isotope dilution.

A method for quantitative determination of individual cytokinin species has been developed, based on gas chromatography-mass spectrometry and selected ion monitoring. Deuterated internal standards were prepared for analysis of N⁶-isopentenyladenosine, N⁶-isopentenyl-2-methylthioadenosine, zeatin riboside, and 2-methylthiozeatin riboside and were tested over the range of 1 to 20 ng of endogenous cytokinin per injection, relative to 100 ng of labeled standard. An isolation procedure for extracts of cabbage hearts as a model plant source has been developed that gives maximum recovery and minimum interference for gas chromatographic-mass spectrometric measurements. The present method differs from the commonly used bioassay by its selectivity for individual cytokinin components and shortened analysis time, including extractions, of 3 days vs several weeks.     

Estrogen 2-, 4-, 6- or 16-hydroxylation by human follicles shown by gas chromatography-mass spectrometry associated with stable isotope dilution

Follicular fluid, obtained by aspiration of human Graafian follicles in cycles stimulated by clomiphene and hMG + hCG, was analyzed for estrogen content. Carefully controlled extraction and efficient preliminary chromatographic separations were set up. Deuterium labelled analogues were used as internal standards for the quantitative determination by gas chromatography-mass spectrometry and some new derivatives were included in the identification procedure. The identified estrogens and their mean concentrations (ng/ml) are: 2-hydroxy-estrone (0.14), 4-hydroxy-estrone (0.12), 2-hydroxy-estradiol (0.36), 4-hydroxy-estradiol (0.34), 6α-hydroxy-estradiol (13.2), 6β-hydroxy-estradiol (6.40), 2-methoxy-estrone (0.83), 2-methoxy-estradiol (10.5), 16-oxo-estradiol (0.41), estriol (10.2), estradiol-17β (1365), estradiol 17α (1.91), estrone (211). Metabolism of estradiol by 6-hydroxylation seems to be predominant in the human ovary. The other data suggest that 2-hydroxylation, with subsequent O-methylation, and 16-hydroxylation may be by equivalent pathways, since the sum of the 2-methoxy-estrone and 2-methoxy estradiol concentrations is rather similar to the estriol concentration. Hence, the latter three compounds and the 6-hydroxy-estradiols may be end-products of follicular estrogen metabolism. Catechol estrogen formation by 2-hydroxylation and 4-hydroxylation is shown to be of equal importance in the ovary. These results confirm the presence in the human follicle of various competing estrogen hydroxylases and catechol-O-methyltransferase.     

Stable isotope dilution analysis of human urinary metabolites of 17α-methyltestosterone

A method based on gas chromatography–mass spectrometry–selected-ion monitoring was developed to measure the main metabolites of 17α-methyltestosterone, 17α-methyl-5α-androstan-3α,17β-diol and 17α-methyl-5β-androstan-3α,17β-diol, in human urine. 17α-Methyl-[²H₃]-5α-androstan-3α,17β-diol and 17α-methyl-[²H₃]-5β-androstan-3α,17β-diol were used as internal standards. The methods involved purification using a Sep-Pak C₁₈ cartridge, hydrolysis by β-glucuronidase from Ampullaria and derivatization with N-methyl-N-trimethylsilyl-trifluoroacetamide/dithioerythriol/ammonium iodide. Quantitation was achieved by selected-ion monitoring of the characteristic fragment ions ([(M+H)−2×TMSOH]⁺) of the di-TMS derivatives on the chemical ionization mode. The method provides a specific, sensitive and reliable technique to determine the urine levels of 17α-methyl-5α-androstan-3α,17β-diol and 17α-methyl-5β-androstan-3α,17β-diol, and can be applied to pharmacokinetic studies of 17α-methyltestosterone.     

Simultaneous determination of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk by isotope dilution ultraperformance liquid chromatography-tandem mass spectrometry

A simple and sensitive analytical method was developed for the simultaneous determination of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk by isotope dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). Samples were directly purified through HLB cartridge. The organic phase was dried under nitrogen and residues were redissolved in mobile phase. Samples were analyzed by UPLC–MS/MS on an Acquity UPLC^® BEH C₁₈ column with gradient elution. The samples were quantified using clenbuterol-D₉, chloramphenicol-D₅ and diethylstilbestrol-D₈ as internal standards. The proposed method was validated according to the European Union regulation 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), trueness, precision, linearity and stability. The method is demonstrated to be suitable for the determination of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk. The total time required for the analysis of one sample was about 50 min.     

Quantitation of vitamin B6 in biological samples by isotope dilution mass spectrometry

Methods have been developed for the simultaneous quantitative analysis of vitamin B6 forms in biological samples by isotope dilution mass spectrometry using deuterated forms of pyridoxine, pyridoxal, pyridoxamine, and pyridoxic acid. The biological fluid or tissue sample was homogenized and then treated with a cocktail containing appropriate amounts of each deuterated vitamer, as well as the deuterated, phosphorylated vitamer forms. The individual vitamers were isolated from the homogenate by a complex high-performance liquid chromatographic procedure that provided separate fractions for each of the six vitamers found in biological samples. Aldehydic B6 vitamers were reduced to the alcohol form prior to acetylation and analysis by gas chromatography/mass spectrometry (GC/MS). The three resulting vitamers were analyzed by electron ionization GC/MS using a silicone capillary column. The methods have been applied to analysis of vitamin B6 in liver, milk, urine, and feces at levels as low as 0.02 nmol/ml.     

Measurement of trimethylamine-N-oxide by stable isotope dilution liquid chromatography tandem mass spectrometry

Trimethylamine-N-oxide (TMAO) levels in blood predict future risk for major adverse cardiac events including myocardial infarction, stroke, and death. Thus, the rapid determination of circulating TMAO concentration is of clinical interest. Here we report a method to measure TMAO in biological matrices by stable isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS) with lower and upper limits of quantification of 0.05 and >200 μM, respectively. Spike and recovery studies demonstrate an accuracy at low (0.5 μM), mid (5 μM), and high (100 μM) levels of 98.2, 97.3, and 101.6%, respectively. Additional assay performance metrics include intraday and interday coefficients of variance of <6.4 and <9.9%, respectively, across the range of TMAO levels. Stability studies reveal that TMAO in plasma is stable both during storage at −80 °C for 5 years and to multiple freeze thaw cycles. Fasting plasma normal range studies among apparently healthy subjects (n = 349) show a range of 0.73–126 μM, median (interquartile range) levels of 3.45 (2.25–5.79) μM, and increasing values with age. The LC/MS/MS-based assay reported should be of value for further studies evaluating TMAO as a risk marker and for examining the effect of dietary, pharmacologic, and environmental factors on TMAO levels.     

Simultaneous determination of mevalonate and 7α-hydroxycholesterol in human plasma by gas chromatography—mass spectrometry as indices of cholesterol and bile acid biosynthesis

A very sensitive and specific method for the simultaneous determination of mevalonate and 7α-hydroxycholesterol in human plasma is described. The assay is based on isotope dilution mass spectrometry: the extracts from plasma were treated with benzylamine followed by dimethylethylsilylimidazole, then the resulting dimethylethylsilyl ether derivatives of mevalonylbenzylamide and 7α-hydroxycholesterol were determined by gas chromatography—mass spectrometry using high-resolution selected-ion monitoring. Simple regression analysis revealed significant correlations between the plasma level of mevalonate and the hepatic activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34) (r = 0.83, P < 0.01) and between the plasma level of free 7α-hydroxycholesterol and the hepatic activity of cholesterol 7α-hydroxylase (EC 1.14.13.7) (r = 0.76, P < 0.05).