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.     

Effects of stereoisomerism on the electron impact fragmentation of some compounds with an eburnane skeleton1

The mass spectra of five sets of stereoisomers possessing an eburnane basic skeleton (vincamine, vincaminic acid ethyl ester, deoxyvincamine, deoxyvincaminic acid ethyl ester, apovincaminic acid ethyl ester and all their possible epimers) have been compared. All of the epimers, among them the 3H-β isomers, not studied formerly, have characteristically different electron impact spectra. For vincamine and its epimers similar differences were also found in the argon charge exchange ionization, mass-analysed ion kinetic energy and collisionally activated mass-analysed ion kinetic energy spectra. The main effects of stereoisomers on the abundance ratios clearly indicated the occurrence of some highly stereospecific or stereoselective decomposition processes. Conclusions concerning the mechanism of these reactions, based on the normal and metastable ion spectra as well as on some energetic data for the epimers, have also been drawn. For several fragment ions the observed unexpected abundance differences seem to be rationalized in terms of the competing effects of other decomposition processes of higher stereoselectivity.     

Characterization of oxysterols by electrospray ionization tandem mass spectrometry after one-step derivatization with dimethylglycine

We report a novel approach to derivatize the primary, secondary, and tertiary hydroxy group(s) of oxysterols with N,N-dimethylglycine (DMG) in the presence of both 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 4-(N,N-dimethylamino)pyridine to yield their corresponding mono- or di-DMG esters. Eight oxysterols including 7-oxocholesterol, 5alpha,6alpha- and 5beta,6beta-epoxycholesterols, as well as 7alpha-, 7beta-, 24(S)-, 25-, and 27-hydroxycholesterols, were studied. Electrospray ionization tandem mass spectrometric characterization of these singly or doubly protonated derivatives demonstrates the presence of an informative fragmentation pattern for each oxysterol derivative. Potential dissociation pathways for the production of these unique fragmentation patterns are proposed and discussed. Collectively, these informative and unique fragmentation patterns allow rapid and direct discrimination of the identities of 7alpha-, 7beta-, 24(S)-, 25-, and 27-hydroxycholesterol isomers, as well as 5alpha,6alpha- and 5beta,6beta-epoxycholesterol isomers, thereby potentially providing a foundation for quantitative analysis of oxysterols in biological samples in combination with a chromatographic separation.     

Mass spectrometric analysis of androstan-17β-ol-3-one and androstadiene-17β-ol-3-one isomers

Mass spectrometric identification and characterization of steroids using electrospray ionization and tandem mass spectrometry has advantages in drug testing and doping control analysis attributable to limitations of gas chromatography followed by electron ionization mass spectrometry. Steroids with an androstadiene-17beta-ol-3-one nucleus and double bonds located either at C-1 and C-4, C-4 and C-9, or C-4 and C-6 were used to determine characteristic fragmentation pathways. Diagnostic dissociation routes are proposed using deuterium labeling, MS3 experiments, and analyses of structurally closely related compounds. Steroids such as boldenone (androst-1,4-diene-17beta-ol-3-one) produced characteristic product ions at m/z 121, 135, and 147. Compounds with double bonds at C-4 and C-9 generated abundant product ions at m/z 145 and 147. Conjugated double bonds at C-4 and C-6 gave rise to an intense and characteristic signal at m/z 133. Stereochemical differentiation between 5alpha- and 5beta-isomers of androstan-17beta-ol-3-ones was possible because of significant differences in relative abundance of product ions generated by elimination of acetone from alpha,beta-saturated 3-keto steroids.     

Stereospecific ion-molecule reactions of anabolic-type steroid tertiary alcohols with proton-donating cations

Isobutane and methane chemical ionization (CI) mass spectra of C-17a-epimeric, 17a-substituted 3-methoxyestra-1,3,5(10),8-tetraen-17a-ols and at C-17-epimeric 17-substituted 3-methoxyestra-1,3,5(10)-trien-17-ols, as well as of some their derivatives, have been studied. In each epimeric pair, the peak intensity ratio [MH-H(2)O](+) / [MH](+) is greater for stereoisomers having an axial (or quasi-axial) hydroxyl group. The same regularity in the peak intensity ratio [MH-CH(3)COOH](+) / [MH](+) is valid for acetates in the D-homo series. The observed quantitative differences in CI mass spectra of epimers are explained by steric hindrance of the attack of the proton-donating cation caused by the angular 18-methyl group. No differences in the ease of elimination of the silanol molecule were observed in CI mass spectra of epimeric silyl ethers.     

Electron ionization mass spectral fragmentation of C19 isoprenoid aldehydes and carboxylic acid methyl and trimethylsilyl esters

The electron ionization (EI) mass spectra of saturated and alpha,beta-unsaturated C(19) isoprenoid aldehydes and carboxylic acid methyl and trimethylsilyl esters are reported. Different pathways are proposed in order to explain the main fragmentations observed. The conjugated double bond migrates more or less readily before gamma-hydrogen rearrangement according to the structure of the considered compound. Configurations of the double bond of alpha,beta-unsaturated C(19) isoprenoid aldehydes and fatty acid methyl and trimethylsilyl esters can be easily determined thanks to the peaks at m/z 97, 127 and 185, respectively, which are much more abundant in the mass spectra of the Z isomers owing to the formation of a cyclic ion. In the case of trimethylsilyl esters, subsequent fragmentation of the cyclic ion at m/z 185 affords two other diagnostic ions at m/z 95 and 169.     

Mass spectrometric study of persistent acid metabolites of nonylphenol ethoxylate surfactants

A mass spectrometric study was carried out on two nonylphenoxycarboxylic acids, NP1EC and NP2EC (where 1 and 2 indicate the number of ethoxylate units attached to the nonylphenoxy moiety), that are persistent metabolites of widely used nonionic surfactant nonylphenol ethoxylates. In a gas chromatographic/mass spectrometric (GC/MS) study of the methyl esters of NP1EC and NP2EC, two series of fragment ions were observed in electron ionization (EI) mass spectra; m/z (179 + 14n, n = 0-7) and m/z (105 + 14n, n = 0-4) for NP1ECMe and m/z (223 + 14n, n = 0-7) and m/z (107 + 14n, n = 0-5) for NP2ECMe. Similarity indices were used to compare quantitatively the mass spectra of isomers. The mass spectra of two isomers were found to be similar whereas those of the remaining isomers were readily distinguishable from each other. The abundant fragment ions of the two NPECMes were investigated further by GC/MS/MS; product ions resulting from cleavage in the alkyl moiety, cleavage in the ECMe moiety and cleavage in both moieties were detected. Possible structures of the nonyl groups in the two esters were inferred. GC/chemical ionization (CI) mass spectra of the NPECMes with isobutane as reagent gas showed characteristic hydride ion-abstracted fragment ions shifted by 1 Da from those in the corresponding EI mass spectra. The sensitivity of a selected ion monitoring quantitation method for the NPECMes is enhanced under CI conditions compared with that under EI conditions. With electrospray ionization MS/MS, [M - H](-) ions of NP1EC (m/z 277) and NP2EC (m/z 321) were observed and, upon collision-induced dissociation of [M - H](-) of each of the two acids, fragment ions of m/z 219 corresponding to deprotonated nonylphenol, were observed in each case. Based on this observation, a rapid, simple and reliable selected product ion quantitation method is proposed for NP1EC and NP2EC.     

Determination of three major triterpenoids in epicuticular wax of cabbage (Brassica oleracea L.) by high-performance liquid chromatography with UV and mass spectrometric detection

Lupeol, together with alpha- and beta-amyrins in smaller quantities, has been found for the first time in the epicuticular wax of white cabbage (Brassica oleracea L. convar. capitata (L.) Alef. var. alba DC) leaf surface extract. The three triterpenoids were identified by a new high-performance liquid chromatographic (HPLC) method with UV and mass spectrometric (MS) detection using atmospheric pressure chemical ionization (APCI). All three isomeric compounds gave a parent ion peak at m/z 409 [M+H-18](+) and the relative intensities of some characteristic fragment ion peaks in tandem mass spectrometric (MS-MS) spectra of this parent ion enabled differentiation between the isomers. An additional peak at m/z 439 [M+H](+), which could be oleanonic or ursonic aldehyde, was detected by HPLC-APCI-MS. Saponification of cabbage leaf surface extract with 20% NaOH in methanol at 65 degrees C for 2h had no influence on lupeol, or alpha- or beta-amyrins, but lead to the formation of three additional compounds, which were not identified.     

Distinguishing by principal component analysis of o-xylene, m-xylene, p-xylene and ethylbenzene using electron ionization mass spectrometry

The possibility of distinguishing between o-, m-, p-xylene and ethylbenzene on the basis of only their 70 eV electron ionization (EI) mass spectra has been investigated. These four isomers were distinguished by principal component analysis (PCA) of respective EI mass spectra (recorded under identical conditions). Considered mass spectra contained either eleven or five m/z values, which had intensity greater than 5% or 10% of base peak, respectively.     

Linkage position and residue identification of disaccharides by tandem mass spectrometry and linear discriminant analysis

The discrimination of isomeric disaccharides with different linkage types and different monosaccharide residues--glucose (Glc), galactose (Gal), and mannose (Man) at the non-reducing end--was investigated with tandem mass spectrometry (MS/MS) and linear discriminant analysis (LDA). Conventional matrix-assisted laser desorption/ionization (MALDI)-MS has strong interference peaks from matrix ions in the low mass region (<500 Da). This greatly limits the application of MALDI-MS for the analysis of small molecules such as saccharides. We solved this problem by using LDI with acidic fullerene matrix, which gives a very clean background in the low-mass region. Disaccharides with different linkage types give different tandem mass spectral profiles from various cross-ring fragmentation pathways. Disaccharides with the same linkage type but with three different kinds of monosaccharide residues bear the same fragmentation profiles. However, the relative ratios of the fragment ion intensities were found to be distinctly different among the three disaccharide isomers. By employing statistical tools such as LDA to classify the tandem mass spectra, disaccharide isomers with either different linkages or different monosaccharide residues were successfully classified.     

Electrospray collision-induced dissociation of testosterone and testosterone hydroxy analogs

Complications with the gas chromatographic analysis of steroids prompted the use of alternative techniques for their identification. High-performance liquid chromatography/mass spectrometry with atmospheric pressure ionization allowed the collection of data for structural identification of these compounds. The objective of this study was to investigate the up-front collision-induced dissociation (UFCID) electrospray ionization (ESI) mass spectra of testosterone and monohydroxylated testosterones. The positive ion UFCID ESI mass spectrum of testosterone showed three significant ions at m/z 97, 109 and 123. The relative abundance of these ions in the UFCID ESI mass spectra of monohydroxylated testosterones varied with the position of the hydroxy group. Statistical data allowed the prediction of hydroxy group position on testosterone by evaluation of the relative abundance of the m/z 97, 109, 121 and 123 ions. Data from the ESI mass spectral analysis of testosterone in a deuterated solvent and from the analysis of cholestenone and 4-androstene-3 beta, 17 beta-diol indicated that the initial ionization of testosterone occurred at the 3-one position. CID parent ion monitoring analyses of the m/z 97, 109 and 123 ions indicated that each resulted from different fragmentation mechanisms and originated directly from the [M + H]+ parent ion. The elemental composition of these fragment ions is proposed based on evidence gathered from the CID analysis of the pseudo-molecular ions of [1,2-2H2]-, [2,2,4,6,6-2H5]-, [6,7-2H2]-, [7-2H]-, [19,19,19-2H3]- and [3,4-13C2]testosterone. The structure and a possible mechanism of formation of the m/z 109 and 123 ions is presented. The results of this study advance the understanding of the mechanisms of collision-induced fragmentation of ions.     

The distinction of underivatized monosaccharides using electrospray ionization ion trap mass spectrometry

A convenient method for distinguishing underivatized isomeric monosaccharides has been established using electrospray ionization ion trap mass spectrometry (ESI-ITMS). Mass spectra of hexoses (glucose, galactose, and mannose), N-acetylhexosamines (N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine) and hexosamines (glucosamine, galactosamine, and mannosamine) dissolved in solvent containing 1 mM ammonium acetate were obtained in the positive ion mode. Glucose was distinguished from galactose and mannose in the MS(2) spectrum of the [M+NH(4)](+) ion at m/z 198. The MS(3) spectra generated from [M+NH(4)-H(2)O-NH(3)](+) at m/z 163 showed that galactose and mannose could be distinguished by the ratio of peak intensities at m/z 145 and 127, while the three N-acetylhexosamine and hexosamine stereochemical isomers could be identified by the relative abundance ratios of product ions observed in MS(3) spectra. The investigation of MS and MS(2) spectra from complexes of these monosaccharides with Na(+) and Pb(2+) failed to distinguish these monosaccharide isomers. Therefore, multiple stage mass analysis by ESI-ITMS using either [M+NH(4)](+) or [M+H](+) was useful to distinguish between the isomers of monosaccharides.     

GC-MS and GC-IRD studies on the ring isomers of N-methyl-2-methoxyphenyl-3-butanamines (MPBA) related to 3,4-MDMA

The mass spectra of the controlled substance 3,4-MDMA and its regioisomer 2,3-MDMA are characterized by an imine fragment base peak at m/z 58 and additional fragments at m/z 135/136 for the methylenedioxybenzyl cation and radical cation, respectively. Three positional ring methoxy isomers of N-methyl-2-(methoxyphenyl)-3-butanamine (MPBA) have an isobaric relationship to 2,3- and 3,4-MDMA. All five compounds have the same molecular weight and produce similar EI mass spectra. This lack of mass spectral specificity for the isomers in addition to the possibility of chromatographic co-elution could result in misidentification. The lack of reference materials for the potential imposter molecules constitutes a significant analytical challenge. Perfluoroacylation of the amine group reduced the nitrogen basicity and provided individual fragmentation pathways for discrimination among these compounds based on unique fragment ions and the relative abundance of common ions. Studies using gas chromatography with infrared detection provided additional structure-IR spectra relationships. The underivatized amines and the perfluoroacylated derivatives (PFPA and HFBA) were resolved by capillary gas chromatography on a 100% dimethylpolysiloxane stationary phase. The perfluoroacylated derivatives showed better resolution on a cyclodextrin modified stationary phase.     

Mass spectra of tetrahydroisoquinoline-fused 1,3,2-O,N,P- and 1,2,3-O,S,N-heterocycles: influence of ring size and fusion, of present heteroatoms, substituent effects and of the stereochemistry on fragmentation

The electron ionization (EI) mass spectra of a variety of stereoisomeric tricyclic 1,3,2-oxazaphosphino[4,3-a]isoquinolines (1-4), 1,2,3-oxathiazino[4,3-a]isoquinoline-4-oxides (5-7) and the -4,4-dioxides (8-10) of oxazaphospholo- and oxathiazolo[4,3-a]- (11, 12, 15 and 16) and -[3,4-b]isoquinolines (13, 14 and 17) were recorded. Ring size and fusion, the different heteroatoms (P and S) and substituents on the ring systems strongly influence the mass spectra. In addition, mass spectra of the stereoisomers of compounds 1, 2 and 13, 14 revealed stereochemically relevant differences which are not observed for the other pairs of isomers.     

N-methyl-N-alkylsilyltrifluoroacetamide-I2 as a new derivatization reagent for anabolic steroid control.

Analytical methods for the control of growth promoters have to be specific and sensitive. At low concentration levels, it is difficult to identify some molecules unambiguously even with the improved performance of analytical methods. GC-MS analysis of 17 beta-trenbolone and its major metabolite, 17 alpha-trenbolone, is a good example. A new derivatization agent has been developed which is based on silylation of the 3- and 17-oxygenated functions and nucleophilic substitution in the 4-position. The structure of the derivatized products was demonstrated using a simple model, cyclohex-2-en-1-one, by NMR and MS spectrometry. In contrast to data found in the literature, this derivative permitted specific mass spectra for trenbolone, sensitive signals for high mass ions and reproducible gas chromatograms to be obtained. The addition of an N(CH3)COCF3 radical to the steroid nucles allowed highly specific detection in GC-high resolution MS even following extraction from complex matrices; sensitive responses were also observed in the negative chemical ionization mode. Moreover, there are significant differences in the electron ionization mass spectra of the two stereoisomers, 17 alpha- and 17 beta-trenbolone. These preliminary results and those obtained for androsta-1,4-dien-3-one and pregna-4,6-dien-3-one indicate useful advances for the determination of steroids and potential applications for metabolism studies on such compounds.     

Electrospray and atmospheric pressure chemical ionization tandem mass spectrometric behavior of eight anabolic steroid glucuronides

Mass spectrometric and tandem mass spectrometric behavior of eight anabolic steroid glucuronides were examined using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) in negative and positive ion mode. The objective was to elucidate the most suitable ionization method to produce intense structure specific product ions and to examine the possibilities of distinguishing between isomeric steroid glucuronides. The analytes were glucuronide conjugates of testosterone (TG), epitestosterone (ETG), nandrolone (NG), androsterone (AG), 5alpha-estran-3alpha-ol-17-one (5alpha-NG), 5beta-estran-3alpha-ol-17-one (5beta-NG), 17alpha-methyl-5alpha-androstane-3alpha,17beta-diol (5alpha-MTG), and 17alpha-methyl-5beta-androstane-3alpha,17beta-diol (5beta-MTG), the last four being new compounds synthesized with enzyme-assisted method in our laboratory. High proton affinity of the 4-ene-3-one system in the steroid structure favored the formation of protonated molecule [M + H]+ in positive ion mode mass spectrometry (MS), whereas the steroid glucuronides with lower proton affinities were detected mainly as ammonium adducts [M + NH4]+. The only ion produced in negative ion mode mass spectrometry was a very intense and stable deprotonated molecule [M - H]- . Positive ion ESI and APCI MS/MS spectra showed abundant and structure specific product ions [M + H - Glu]+, [M + H - Glu - H2O]+, and [M + H - Glu - 2H2O]+ of protonated molecules and corresponding ions of the ammonium adduct ions. The ratio of the relative abundances of these ions and the stability of the precursor ion provided distinction of 5alpha-NG and 5beta-NG isomers and TG and ETG isomers. Corresponding diagnostic ions were only minor peaks in negative ion MS/MS spectra. It was shown that positive ion ESI MS/MS is the most promising method for further development of LC-MS methods for anabolic steroid glucuronides.     

Mass spectra of 1,2-alkylenedioxy-substituted and related benzofurazans and benzofuroxans: differentiation between linear and angular isomers.

The electron ionization (EI) mass spectra of [1,3]-dioxolo-, 6, 7-dihydro-[1,4]-dioxino-, 7,8-dihydro-6H-[1,4]dioxepino-, 6,7,8, 9-tetrahydro[1,4]dioxocino-, 7,8,9,10-tetrahydro-6H-[1,4]dioxonino- and 6,7,8,9,10,11-hexahydro[1,4]dioxecino[2,3-f]-2,1, 3-benzoxadiazoles (benzofurazans) and the corresponding 1-oxides (benzofuroxans), all linear isomers, are reported. Also reported are the EI mass spectra of the corresponding angular isomers, [1, 3]dioxolo-, 7,8-dihydro[1,4]dioxino-, 8,9-dihydro-7H-[1,4]dioxepino-, 7,8,9,10-tetrahydro[1,4]dioxocino-, 8,9,10,11-tetrahydro-7H-[1, 4]dioxonino-, 7,8,9,10,11,12-hexahydro[1,4]dioxecino[2,3-e]-2,1, 3-benzoxadiazoles and the corresponding 1-oxides. The relative abundance of the fragment ion of m/z 80 is characteristically enhanced in the spectra of the angular derivatives compared to the spectra of the linear counterparts. Collisionally activated decomposition studies show that it is structurally identical whether derived from the benzofurazans or the benzofuroxans. The relative abundance of the m/z 80 ion serves to distinguish between linear and angular isomeric pairs. In addition, the fragment ion at m/z 80 also serves to differentiate between the pairs of isomers, analogous to linear and angular, of six acyclic methoxy- and methoxymethyl-substituted benzofurazans and their corresponding benzofuroxans.     

Electron and chemical ionization mass spectrometry in stereochemical differentiation of some 1,3-amino alcohols

Mass spectral fragmentations of two cyclopentane, eight cyclohexane and four norbornane/one 1,3-amino alcohols were studied under electron ionization (EI) by low-resolution, high-resolution, metastable ion analysis and collision-induced dissociation (CID) techniques. All stereoisomeric compounds gave rise to identical 70 eV EI mass spectra. However, the spectra of positional isomers clearly differed. The main fragmentation pathway for the saturated compounds began as an α-cleavage reaction with respect to the nitrogen atom. For the norbornene compounds a retro-Diels—Alder reaction was favoured. Relative to the aminomethyl-substituted compounds the fragmentation patterns for the compounds having the amino group connected directly to the ring were more complicated. The chemical ionization (CI) mass spectra were recorded using ammonia, isobutane, methane, dichloromethane and acetone as reagent gas. From the norbornane/One compounds the di-exo isomers decomposed more easily than the di-endo isomers with most of the reagent gases used. Differences between stereoisomers were observed directly only under methane CI. The decomposition products of the [M + H]⁺ ions generated under ammonia and isobutane CI were studies by recording their CID mass spectra. These spectra allowed the differentiation of the stereoisomers, at least to some extent.     

Structure Analysis and Ion Abundance in CID- MS- MS Spectra of Isomeric Oligosaccharides Using Quadrupole Time-of- flight Mass Spectrometry: Distinguishing between Isomeric Oligosaccharides

N - Linked oligosaccharide were analyzed by using electrospray ionization (ESI) quadrupole time - of- fight mass spectrometry(Q - Tof MS).The isomers showed the same MS and collisioninduced desociation(CID) MS - MS spectra in the m/z values because the sequence of the sugar residues was the same.But the relative ion abundance of the specific fragment ion was greatly different between the isomers.So, the isomeric oligosacchariedes were distinguished by using the ion abundance in their CID -MS - MS spectra.Discussing the ion abundance in accurate level, quantitative analysis of the mixtures of isomers were also performed.     

Mass spectrometry of steroid glucuronide conjugates. II-Electron impact fragmentation of 3-keto-4-en- and 3-keto-5alpha-steroid-17-O-beta glucuronides and 5alpha-steroid-3alpha,17beta-diol 3- and 17-glucuronides

The steroid glucuronide conjugates of 16,16,17-d(3)-testosterone, epitestosterone, nandrolone (19-nortestosterone), 16,16,17-d(3)-nortestosterone, methyltestosterone, metenolone, mesterolone, 5alpha-androstane-3alpha,17beta-diol, 2,2,3,4,4-d(5)-5alpha-androstane-3alpha,17beta-diol, 19-nor-5alpha-androstane-3alpha,17beta-diol, 2,2,4,4-d(4)-19-nor-5alpha-androstane-3alpha,17beta-diol and 1alpha-methyl-5alpha-androstane-3alpha/beta,17beta-diol were synthesized by means of the Koenigs-Knorr reaction. Selective 3- or 17-O-conjugation of bis-hydroxylated steroids was performed either by glucuronidation of the corresponding steroid ketole and subsequent reduction of the keto group or via a four-step synthesis starting from a mono-hydroxylated steroid including (a) protection of the hydroxy group, (b) reduction of the keto group, (c) conjugation reaction and (d) removal of protecting groups. The mass spectra and fragmentation patterns of all glucuronide conjugates were compared with those of the commercially available testosterone glucuronide and their characterization was performed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy. For mass spectrometry the substances were derivatized to methyl esters followed by trimethylsilylation of hydroxy groups and to pertrimethylsilylated products using labelled and unlabelled trimethylsilylating agents. The resulting electron ionization mass spectra obtained by GC/MS quadrupole and ion trap instruments, full scan and selected reaction monitoring experiments are discussed, common and individual fragment ions are described and their origins are proposed.