Mass spectrometry of steroid glucuronide conjugates. I. Electron impact fragmentation of 5alpha-/5beta-androstan-3alpha-ol-17-one glucuronides, 5alpha-estran-3alpha-ol-17-one glucuronide and deuterium-labelled analogues

Owing to the developments of analytical instruments and interfaces (e.g. coupling high-performance liquid chromatography to mass spectrometry), there has been increased interest in new reference materials, for example in doping analysis with steroid glucuronide conjugates. The synthesized reference material has to pass several characterization steps including the use of gas chromatography/mass spectrometry (GC/MS) for its structure confirmation. In the present study, the fragmentation and mass spectrometric behaviour of several steroid glucuronide conjugates of endogenous and anabolic steroids after derivatization to pertrimethylsilylated products and to methyl ester pertrimethylsilylated products were investigated using GC/MS ion trap and GC/MS quadrupole instruments. The mass spectra of the derivatives of androsterone glucuronide, d5-androsterone glucuronide, epiandrosterone glucuronide, etiocholanolone glucuronide, 11beta-hydroxy etiocholanolone glucuronide, 19-norandrosterone glucuronide, d4-19-norandrosterone glucuronide and 1alpha-methyl-5alpha-androstan-3alpha-ol-17-one glucuronide are presented and the origin of typical fragment ions of the glycosidic and steroidal moieties is proposed, based on different derivatization techniques including derivatization with d18-bistrimethylsilylacetamide, methyl ester and trimethylsilyl ester derivatization and selected reaction monitoring. Typical fragmentation patterns which are related to the steroid structure are discussed.     

Chemical conversion of corticosteroids to 3 alpha,5 alpha-tetrahydro derivatives. Synthesis of 5 alpha-cortol 3-glucuronides and 5 alpha-cortolone 3-glucuronides.

The synthesis of the 3-glucuronides of 5 alpha-cortol-20 alpha, 5 alpha-cortolone-20 alpha and their 20 beta-epimers is described. The 5 alpha-cortol 20,21-diacetates (12, 17) and 5 alpha-cortolone 20,21-diacetates (14, 19) were the key intermediates. Sodium borohydride reduction of the carbonyl group at C-20 in 5 alpha-tetrahydrocortisol 3-tert-butyldimethylsilyl ether 17,21-acetonide (8) gave the 20 alpha-hydroxy-acetonide (9). Selective removal of the acetonide ring was successful when the 20 alpha-acetoxy-17 alpha,21-acetonide (10) was treated with 50% acetic acid. Subsequent acetylation with acetic anhydride in pyridine, followed by removal of the protecting group at C-3 in the silyl ether-acetate (11) gave the desired 20 alpha-intermediate (12). The 11-ketone (14) was prepared from 11 by oxidation with pyridinium chlorochromate, followed by desilylation. The 20 beta-acetates (17, 19) were synthesized from 21-acetoxy-3 alpha,11 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one 3-tert-butyldimethylsilyl ether (15). Introduction of the glucuronyl residue at C-3 was carried out by means of the Koenigs-Knorr reaction.     

Synthesis of 3-keto-4-diazo-5-α-dihydrosteroids as potential irreversible inhibitors of steroid 5-α-reductase

3-Keto-4-diazo-5-α-dihydrosteroids are prepared using the Hendrickson diazo transfer reaction on the corresponding β-diketones, which are available from 3-ketoΔ-4-steroids $\text{via}$ Li/NH₃ reduction and acylation of the kinetic enolate.     

Chemical conversion of corticosteroids to 3 alpha,5 alpha-tetrahydro derivatives. Synthesis of allotetrahydrocortisol glucuronides and allotetrahydrocortisone glucuronides

The synthesis of the 3- and 21-glucuronides of allotetrahydrocortisol (allo-THF) and allotetrahydrocortisone (allo-THE) is described. 5 alpha-Dihydrocortisol (5) was prepared by selective hydrogenation of 21-acetoxy-3, 11 beta, 17 alpha-trihydroxy-3,5-pregnadien-20-one 3-ethyl ether (3), followed by acid hydrolysis and saponification. When 5 alpha-dihydrocortisol 21-tetrahydropyranyl ether (6) was treated with potassium tri-sec-butylborohydride in tetrahydrofuran under mild conditions, regioselective and stereoselective reduction at C-3 took place to give allo-THF 21-tetrahydropyranyl either (7). This compound was converted into the 3- and 21-monoacetates of allo-THF and allo-THE, key intermediates. Introduction of the glucuronyl residue at C-3 or C-21 was carried out by means of the Koenigs-Knorr reaction. Prior to saponification yielding the 3-glucuronides (20,23), the alkali-sensitive ketol side chain at C-17 was protected as 20-semicarbazones.     

Studies on neurosteroids XXIV. Determination of neuroactive androgens, androsterone and 5alpha-androstane-3alpha,17beta-diol, in rat brain and serum using liquid chromatography-tandem mass spectrometry

The development and validation of liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) methods that enable the quantification of neuroactive androgens, androsterone (5alpha-androstan-3alpha-ol-17-one, 3alpha,5alpha-A) and 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-Adiol), in the rat brain and serum are presented. The androgens were extracted with methanol-acetic acid, purified using solid-phase extraction cartridges, derivatized with an ESI-active reagent, isonicotinoyl azide (INA), and then subjected to LC-ESI-MS/MS. The quantifications were based on selected reaction monitoring mode using the characteristic transitions of the INA derivatives. The methods allowed the reproducible and accurate quantification of the brain and serum neuroactive androgens using a 100 mg or 100 microL sample; the intra- and inter-assay relative standard deviations were below 3.6%, and the percentage accuracy values were 97.1-103.7% for both androgens. The animal study using the methods suggests that most of 3alpha,5alpha-Adiol found in the brain is derived from the periphery, while 3alpha,5alpha-A is not only transported from the periphery into the brain, but also synthesized in the brain by the oxidation of 3alpha,5alpha-Adiol. The androgens in the rats intraperitoneally administered finasteride, a 5alpha-reductatse inhibitor, were also measured; this treatment significantly reduced the brain 3alpha,5alpha-A and 3alpha,5alpha-Adiol levels and increased only the brain level of androstenedione, the precursor of 3alpha,5alpha-A.     

The electron impact fragmentation of 3- and 4-styrylpyridazine

The electron impact mass spectrometric fragmentation of trans-3- and trans-4-styrylpyridazine is reported in detail, including a comparison with other aza-stilbenes. With regard to a distinction between the two isomeric styrylpyridazines, the intensity ratio of the M⁺ and [M-1]⁺ ions, the general degree of fragmentation and the elimination pathways of nitrogen proved to be most characteristic.     

Mass spectral fragmentation pattern of 3-methyl- and 3-phenyl-4-(N-methylarylhydrazono)isoxazol-5-ones

3-Methyl-4-(N-methylarylhydrazono)isoxazol-5-ones and 3-phenyl-4-(N-methylarylhydrazono)isoxazol-5-ones undergo considerable fragmentation on electron impact involving rupture of the isoxazolone ring and bonds in the N-methylarylhydrazono side chain.     

Steroid total synthesis. IV.(+)-13 -ethyl-17 -ethynyl-17 -hydroxy-gon-4-en-3-one

The title compound (Norgestrel), a potent progestational agent, has been prepared by a new total synthesis. The scheme is based on earlier work with BCD-tricyclic intermediates, condensation of 2-ethyl-cyclopentane-1,3-dione with 2-[2-diethylamino-ethyl]-6-[4-t-butoxy-pentyl]-tetrahydropyran-2-ol being the key step. The synthesis features the addition of acetylene to a Δ⁵⁽¹⁰⁾-4-oxa-17-keto intermediate, followed by a novel transformation to the final product.     

Mass spectral fragmentation pattern of 3-methyl-4-arylaminomethyleneisoxazol-5-ones

The mass spectral fragmentation patterns of 3-methyl-4-arylaminomethyleneisoxazol-5-ones obtained by electron impact have been elucidated. The base peaks are due to the molecular ions. The main fragmentation routes involve loss of H, OH, H₂O, CO₂ and COOH from the molecular ions as well as rupture of the exocyclic CH-NH bond.     

Studies in organic mass spectrometry. IV. Electron impact induced fragmentation of 2-substituted 3-(5-isoxazolyl)-4(3H)-quinazolinones of pharmaceutical interest

The fragmentation under electron impact of thirteen 2-substituted-3-(5-isoxazolyl)-4(3H)-quinazolinones has been investigated with the aid of metastable ion detection and high resolution measurements. Molecular ions are always abundant and the main primary fragmentation route involves acetonitrile elimination through isoxazole ring opening. The other common processes, particularly those leading to the abundant [R-C₈H₄N₂]⁺ ion ( b or b' ), as well as those due to the nature of the 2-substituent are reported and discussed.     

Electron ionization mass spectral fragmentation of cholestane-3beta,4alpha,5alpha-triol and cholestane-3beta,5alpha,6alpha/beta-triol bis- and tris-trimethylsilyl derivatives

The electron ionization (EI) mass spectral fragmentation of the bis- and tris-trimethylsilyl derivatives of cholestane-3beta,4alpha,5alpha-triol, cholestane-3beta,5alpha,6beta-triol and cholestane-3beta,5alpha,6alpha-triol was investigated. The EI mass spectrum of the 3beta,4alpha-bis-trimethylsilyl derivative of cholestane-3beta,4alpha,5alpha-triol exhibits interesting fragment ions at m/z 142 and 332 resulting from the initial loss of TMSOH between the carbons 2 and 3 and subsequent retro-Diels-Alder (RDA) cleavage of the ring A. Trimethylsilyl transfer between the 4alpha- and the 5alpha-hydroxy groups acts significantly before RDA cleavage affording an ion at m/z 404. Complete silylation of cholestane-3beta,4alpha,5alpha-triol strongly stabilizes the molecule, affording an abundant molecular ion at m/z 636 and decreasing the abundance of the RDA cleavage. Loss of water (from the non-silylated 5alpha-hydroxy group) plays a very important role during the decomposition of the molecular ion of 3beta,6alpha/beta-bis-trimethylsilyl derivatives of cholestane-3beta,5alpha,6alpha/beta-triols. These derivatives appear to be very useful in assigning the configuration of the carbon 6. This assignment is based on the abundance of the fragment ions at m/z 321, 367 and 403, which are more prominent in the EI mass spectrum of the beta-isomer. In contrast, EI mass spectra of the tris-trimethylsilyl derivatives of cholestane-3beta,5alpha,6beta-triol and cholestane-3beta,5alpha,6alpha-triol differ only slightly and appear to be poorly informative.     

Photochemical reactions. V (1). Photo-oxidation of 17 beta-acetoxy-4-aza-androst-5-en-3-one.

The photo-oxidation of the title compound ( 1 ) is described. The isolated products are the result of a [2 + 2] oxygen addition to the olefinic double bond of the enamide.     

Electron impact mass spectrometry of 3-methyl-4-benzylideneamine-5-styrylisoxazoles. II

The mass spectra of a series of substituted 3-methyl-4-benzylideneamine-5-styrylisoxazoles are reported and discussed. The spectra of these isoxazoles show a characteristic fragmentation pattern different from the 5-styryl-4-nitroisoxazoles. Perhaps this is due to the presence of the Schiff base at position 4.     

Mass spectrometry of macroheterocycles. 2. Characteristics of the fragmentation of azacrown ethers under electron impact

The fragmentation of aza-crown and diaza-crown ethers under electron impact was studied. It is shown that for the former the primary pathways of fragmentation of the molecular ions involve the intracyclic migration of a hydrogen atom and the elimination of C₂H₃O and CH₂OH particles. A characteristic feature of the diaza-crown ethers is the ejection of a divinylaminyl radical.For Communication 1 see [1].Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 2, pp. 242–246, February, 1992.     

The fragmentation of 5- and 3-substituted thiophene-2-carboxamides under electron impact

The 70 eV electron impact mass spectra of twelve 5- and 3-substituted thiophene-2-carboxamides are discussed with the aid of exact mass measurements and labelling experiments. All mass spectra exhibit pronounced molecular ions. Some isomeric 5- and 3-substituted title compounds can be differentiated by mass spectrometry. The fragmentation is influenced by a strong ‘ortho-effect’ which activates the NH₃ elimination. In the other cases the most important fragmentation is NH₂˙ loss, followed by CO elimination.