Methyloxime-trimethylsilyl Ether Derivatives Research Articles

PP.09.20

Objective: To assess if 24 h urinary steroid metabolite profiles are related to vascular parameters such as arterial stiffness and blood pressure (BP) Design and method: 43 eligible participants, 25–81 years, with eGFR > 45 mL/min and no serious illness. Urine was collected over 24 h (12 h day and night samples). Peripheral BP, central pulse wave velocity (aPWV) (by Arteriograph) and cardio ankle vascular index (CAVI) (by VaSera) measures were performed on the collection day. Steroids were extracted from urine aliquots and conjugates hydrolysed by Helix pomatia digestive juice. Free steroids were re-extracted and methyl oxime-trimethylsilyl ether derivatives prepared for GC-MS analysis. Pearson correlation and multiple regression analysis were performed. Results: The urinary steroid metabolites detailed in the figure (figure on the following page) were quantified. Systolic BP and aPWV correlated positively with the ratio of cortisol to cortisone metabolites [aTHF, THF, αcol and βcol: THE, αcone and βcone, (colmets: conemets)]; r = 0.32, 0.37 respectively, p < 0.05 for day samples and r = 0.33, 0.34 respectively, p < 0.05 for night samples. Both BP and aPWV also correlated negatively in the day samples with the ratio αcol, βcol, αcone and βcone: aTHF, THF and THE (20OH: 20oxo); r = -0.33, -0.32 respectively, p < 0.05. Night samples only showed this correlation with diastolic BP r = 0.31, p < 0.05. No correlations were seen with CAVI. Regression analysis with these metabolite ratios (including age and body mass index) did not show they were associated with BP or CAVI (including BP). While aPWV was borderline associated with colmets: conemets, p = 0.077, 20OH: 20oxo was significantly associated (p = 0.026). BP and BMI remained independent predictors. Conclusions: 24 h urinary 20OH:20oxo ratio was independently related to aPWV in those with or at risk of T2D and colmets:conemets had borderline associations with aPWV. There were no associations with the urinary steroids and BP.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 4. Characterization, using GC–MS and GC–MS/MS, of 11oxo-pregnanes and 11oxo-pregnenes

In 21-hydroxylase deficiency, urinary metabolites of 21-deoxycortisol, mainly derived from its 11oxo form 21-deoxycortisone, are indicators of intra-adrenal overproduction of 17-hydroxyprogesterone. In affected neonates these metabolites are numerous and most have not been previously described. This work forms the concluding part of a comprehensive study of urinary steroids, aiming to enhance the diagnosis of this disorder and to further elucidate steroid metabolism in neonates. Cortisol metabolites found in untreated patients, similarly almost exclusively present in their 11oxo form in neonates, have been included for completeness.Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC–MS and GC–MS/MS spectra were used together to determine the structure of hitherto undescribed 11oxo-pregnane(enes).Few GC–MS features were associated with the presence of the non-derivatizeable 11oxo group in pregnane(ene)s. GC–MS/MS contributed only to the characterization of structures outside the C-ring, as described in the preceding parts of this study. Parallels were found between the metabolism of 21-deoxycortisone and cortisone. The major metabolic pathway was that of classical 3α,5β-reduction with a prominent further hydroxylation, predominantly at C6. Oxidation of the 6-hydroxyl was also common.We conclude that further oxygenated metabolites of 21-deoxycortisone have potential as more reliable markers of 21-hydroxylase deficiency in the early neonatal period, because their levels are higher during that period of life than for the classical marker 11oxo-pregnanetriol.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 3. Characterization, using GC–MS and GC–MS/MS, of androstanes and androstenes

Urine from neonates with 21-hydroxylase deficiency contains a large range of androstane(ene)s, many of which have not been previously described. We present their characterization as the third part of a comprehensive study of urinary steroids, aiming to enhance the diagnosis of this disorder and to further elucidate steroid metabolism in neonates.Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC–MS and GC–MS/MS spectra were used together to determine the structure of hitherto undescribed androstane(ene)s.GC–MS/MS was pivotal for the structural characterization of 2-hydroxylated androstenediones but GC–MS was generally more informative for androstane(ene)s, in contrast to 17-hydroxylated pregnane(ene)s. Parallels were found between the GC–MS and GC–MS/MS characteristics of structurally similar androstenediones and progesterones without a substituent on the D-ring, but not with those of 17-hydroxylated progesterones. Assignment of 5α(β) orientation, based on GC–MS characteristics, was possible for 11-oxo-androstanes.The major endogenous 3β-hydroxy-5-enes in 21-hydroxylase deficiency did not differ from those in unaffected neonates. The key qualitative and quantitative differences encompassed 5α(β)-androstanes and 3-oxo-androst-4-enes. Major positions of hydroxylation in these were C2, C6, C11, C16 and C18. Additional oxo-groups were common at C6, C11 and C16.We conclude that the presence of multiple further oxygenated metabolites of androstenedione in urine from neonates with 21-hydroxylase deficiency and their pattern indicate a predominance of the classical pathway of androgen synthesis and reflect an increased demand for clearance. The positions of oxygenation in androstane(ene)s are dependent on the configuration at C3–C5.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 2. Characterization, using GC–MS and GC–MS/MS, of pregnanes and pregnenes with an oxo- group on the A- or B-ring

Urine from neonates with 21-hydroxylase deficiency contains a large range of metabolites of 17-hydroxyprogesterone, 21-deoxycortisol and androgens but few have been previously described. We present the second part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal steroid metabolism.Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC–MS and GC–MS/MS spectra were used together to determine the structure of the A- and B-rings containing an oxo group.Fragmentations indicating presence of 3-, 6-, and 7-oxo groups and also 1β-, 2α-, 4β-, and 6β-hydroxyls are presented and discussed for the first time. Interpretation was aided by comparison with spectra of available relevant standards, of oxidation products of standards and urinary metabolites and of deuterated derivatives. Endogenous 1-enes and 2(3)-ene artifacts of non-hydrolyzed 3α-sulfates are also reported. D-ring and side chain structure was determined according to our previously published criteria. Likely metabolic relationships were also explored.We conclude that GC–MS combined with GC–MS/MS allows identification of the A- and B-ring structure of pregnane and pregnenes in the presence of an oxo group on one of these rings. Major oxygenations are 1β, 15β, 16α and 21-hydroxy and 6- and 7-oxo groups. Minor positions of hydroxylation are those at 2α, 4β and 6β. Three major metabolic streams exist in affected neonates in addition to the classical 3α-hydroxy-5β-pregnane pathway, i.e. these of the 3-oxo-4-enes as well as 3α- and 3β-hydroxy-5α-anes.

Urine metabolomics analysis for adrenal incidentaloma activity detection and biomarker discovery

This study describes the development of a method suitable for the analysis of nineteen major urinary steroid metabolites in human urine. The analytes of interest were isolated from urine using solid phase extraction, subjected to enzymatic hydrolysis and again extracted applying solid phase extraction. After derivatization, methyloxime-trimethylsilyl ether derivatives of steroid hormones were identified by gas chromatography–mass spectrometry (GC/MS) and quantified by gas chromatography with flame ionization detector (GC/FID). The quantification method was validated for linearity, trueness, precision and selectivity. The limits of detection were between 6.2 and 7.2 ng/mL and limits of quantification were between 12.3 and 14.8 ng/mL. The established method was applied to analyze 28 urine samples from patients diagnosed with non-functioning adrenal incidentalomas (AIs) and 30 healthy subjects. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were employed to visualize the differences between metabolic profiles of patients and the control group and to determine possible markers of AIs activity. Both multivariate methods separated seven patients from the rest of the examined individuals. Five urinary metabolites including α-cortol, tetrahydrocorticosterone, tetrahydrocortisol, allo-tetrahydrocortisol and etiocholanolone were identified as potential biomarkers of pathological adrenal function. The altered metabolites reflected pathological metabolism mainly of cortisol and cortisone. This research proved that metabolomics is a suitable tool for disease research.

A new marker for early diagnosis of 21-hydroxylase deficiency: 3β,16α,17α-trihydroxy-5α-pregnane-7,20-dione

In neonates with 21-hydroxylase deficiency the specific marker 11-oxo-pregnanetriol is at low levels in the first days of life and this drives the search for alternatives. We describe the structural characterisation of a new early marker, 3β,16α,17α-trihydroxy-5α-pregnane-7,20-dione.Urine samples from 87 untreated and 11 recently treated newborns with 21-hydroxylase deficiency (42 males and 56 females) between birth and 40 days of age and control samples from 7 healthy neonates (4 males, 3 females) were compared. Steroids were analyzed as methyloxime-trimethylsilyl ether derivatives by GC–MS and GC–MS/MS, after extraction and enzymatic conjugate hydrolysis. Microchemical methods and deuterated derivatives were used.The new steroid was identified by comparison with 3β,16α,17α-trihydroxy-preg-5-en-20-one and 3β-hydroxy-5α-pregnane-7,20-dione standards. It was present for the first 4 weeks after birth (with a maximum around day 4) and showed a marked inter-individual variability. No effect of treatment was evident and levels were much higher than for 11-oxo-pregnanetriol in the first days of life. Only traces were found in controls.The likely involvement of oxysterol 7α-hydroxylase (CYP7B1) from the ‘acidic’ pathway of bile acid synthesis and 11β-hydroxysteroid dehydrogenase-1 in the generation of the 7-oxo group is discussed.We conclude that this steroid is a useful early marker of 21-hydroxylase deficiency.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency: Characterization, using GC–MS and GC–MS/MS, of the D-ring and side chain structure of pregnanes and pregnenes

Steroid metabolites in urine from neonates with 21-hydroxylase deficiency are predominantly polyhydroxylated 17-hydroxyprogesterone and androgen metabolites, and most have incompletely defined structure. This study forms part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal types of steroid metabolism. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC–MS and GC–MS/MS spectra, obtained with constant excitation conditions, were used together to determine the structure of the D-ring and the side chain of 20-oxo and 20-hydroxy pregnane(ene)s without oxo groups on the A-, B-, and C-ring. All possible combinations of D-ring and side chain configuration were considered. Most fragmentations could be interpreted as partial or complete D-ring cleavages with loss of the side chain, aided by comparison with spectra of deuterated derivatives and of borohydride reduced metabolites. Possible rearrangement ions are also discussed. More than 140 endogenous metabolites were characterized. GC–MS/MS was especially beneficial for characterization of compounds with 16,17-dihydroxy-20-oxo structure, interpreted as markers of intra-uterine enzyme induction. It also assisted the differentiation of 16-hydroxy-20-oxo metabolites, present in urine of non-affected neonates, from the diagnostic 17-hydroxy-20-oxosteroids and enabled the detection of 15,17-dihydroxy-20-oxo compounds in low concentrations. The presence of 17,21-dihydroxylated pregnane(ene)s despite the deficit in CYP21A2 is discussed. We conclude that GC–MS combined with GC–MS/MS allows reliable identification of the structure of the D-ring and side chain of pregnane(ene)s without prior isolation, even when in low concentrations in urine.

Identification of Neuroactive Steroids and Their Precursors and Metabolites in Adult Male Rat Brain

Steroids in the brain arise both from local synthesis and from peripheral sources and have a variety of effects on neuronal function. However, there is little direct chemical evidence for the range of steroids present in brain or of the pathways for their synthesis and inactivation. This information is a prerequisite for understanding the regulation and function of brain steroids. After extraction from adult male rat brain, we have fractionated free steroids and their sulfate esters and then converted them to heptafluorobutyrate or methyloxime-trimethylsilyl ether derivatives for unequivocal identification and assay by gas chromatography analysis and selected ion monitoring mass spectrometry. In the free steroid fraction, corticosterone, 3alpha,5alpha-tetrahydrodeoxycorticosterone, testosterone, and dehydroepiandrosterone were found in the absence of detectable precursors usually found in endocrine glands, indicating peripheral sources and/or alternative synthetic pathways in brain. Conversely, the potent neuroactive steroid 3alpha,5alpha-tetrahydroprogesterone (allopregnanolone) was found in the presence of its precursors pregnenolone, progesterone, and 5alpha-dihydroprogesterone. Furthermore, the presence of 3beta-, 11beta-, 17alpha-, and 20alpha-hydroxylated metabolites of 3alpha,5alpha-tetrahydroprogesterone implicated possible inactivation pathways for this steroid. The 20alpha-reduced metabolites could also be found for pregnenolone, progesterone, and 5alpha-dihydroprogesterone, introducing a possible regulatory diversion from the production of 3alpha,5alpha-tetrahydroprogesterone. In the steroid sulfate fraction, dehydroepiandrostrone sulfate was identified but not pregnenolone sulfate. Although pharmacologically active, identification of the latter appears to be an earlier methodological artifact, and the compound is thus of doubtful physiological significance in the adult brain. Our results provide a basis for elucidating the origins and regulation of brain steroids.

Routine screening and quantitation of urinary corticosteroids using bench-top gas chromatography-mass-selective detection.

A method was developed for the routine screening, confirmation and quantitation of corticosteroids in human urine using bench top capillary gas chromatography (GC)-mass-selective detection. The free and conjugated corticosteroid fractions were isolated by liquid-liquid partition. After evaporation to dryness under vacuum the corticosteroid residues were derivatized to form the methyloxime trimethylsilyl ether derivatives. Both GC retention data and characteristic spectral data based on authentic reference standards were used for the identification and quantitation of cortisol, cortisone, tetrahydrocortisol and tetrahydrocortisone in the ppb (ng/ml) concentration range. The method is simpler and more efficient than the other GC-mass spectrometric (MS) techniques. It is also more sensitive than the liquid chromatographic-MS method.

Degradation of steroid derivatives at a gas chromatograph mass spectrometer interface

Methyloxime trimethylsilyl ether derivatives are frequently employed in gas chromatographic and mass spectrometric analysis of steroids. Under certain instrumental conditions, unusual mass spectra were recorded from some corticosteroid methyloxime trimethylsilyl ether derivatives, reflecting degradation by contact of the steroids with active surfaces in the gas chromatograph mass spectrometer interface. This occurred prior to electron impact ionization. Fragmentations of the degradation products of methyloxime trimethylsilyl ether derivatives of tetrahydrocortisol and tetrahydrodeoxycorticosterone are described, as representatives of C21 steroids with dihydroxyacetone and α-ketol side chains. Elemental compositions of ions were confirmed by high resolution mass spectrometric analysis. Derivatives of steroids with glycerol side chains, e.g. cortolone, showed no evidence of degradation. The problem was overcome by ensuring inertness of the glass restrictor interface and by preventing, as far as posssible, contact of sample with metal surfaces in the source housing.

Derivatization of estrogen conjugates for analysis by capillary gas chromatography.

By using 20 meter wall-coated open tubular glass capillary columns of high stability, analysis of methyl ester, methyloxime trimethylsilyl ether derivatives of estrogen glucuronides had been achieved. Relative retention times of five glucuronide conjugates on OV-1 stationary phase are reported. Estrogen sulfates conjugated at the 3-position were shown to be quantitatively hydrolyzed and derivatized in a single trimethylsilylation step, and this method of direct derivatization was compared to two solvolysis methods. These analytical methods could be further developed to allow rapid and quantitative analysis of estrogens in biological fluids, and may prove particularly useful for analysis of labile compounds.

Mass spectrometric analysis for tetrahydroaldosterone

Trimethylsilyl ether and methyloxime-trimethylsilyl ether derivatives of 3α,11β,21-trihydroxy-5β-pregnan-20-on-18-al (tetrahydroaldosterone) and its etiolactone were analysed by gas Chromatography. The structures were established from the fragmentation pattern during mass spectrometry. A quantitative method for measuring urinary tetrahydroaldosterone is described based on selected-ion-monitoring gas chromatography-mass spectrometry (GC-MS). Urine samples were hydrolysed by β-glucuronidase, 3β- allo-tetrahydroaldosterone was added as internal standard, the steroids were extracted on Amberlite XAD-2 resin then purified by Sephadex LH-20 chromatography. Methyloxime-trimethylsilyl ethers were prepared and the ions of m e 638 (M) and 607 (M-31) simultaneously monitored by GC-MS. Both the analyte and internal standard were resolved on OV-1 columns and the peak heights were determined. The ratio of peak height of analyte to internal standard was constant up to 40 ng per injection and the lower limit of sensitivity giving acceptable signal-to-noise ratio was around 200 pg.

Identification and measurement of 18-hydroxycorticosterone metabolites by gas chromatography-mass spectrometry.

Abstract Combined gas chromatography-mass spectrometry was used to identify methyloxime-trimethylsilyl ether derivatives of 18-hydroxycorticosterone metabolites in a urinary extract from an infant with pseudo-hypoaldosteronism. Mass spectra are presented of derivatives of two metabolites (3α,18,21-trihydroxy-5β-pregnane-11, 20-dione and 3α,11β,18,21-tetrahydroxy-5β-pregnan-20-one) and a mechanism is proposed for a fragmentation characteristic of these 18-hydroxylated C-21 steroids. A method is described for the quantitative determination of 3α,18,21-trihydroxy-5β-pregnane-11,20-dione in urine. 3β,17,21-Trihydroxy-5α-pregnane-11,20-dione was used as internal standard and the ions of mass 609 and 578 were selectively monitored by the mass spectrometer.