Metabolism of cyadox in rat,chicken and pig liver microsomes and identification of metabolites by accurate mass measurements using electrospray ionization hybrid ion trap/time‐of‐flight mass spectrometry

Cyadox (CYX), (2‐formylquinoxaline)‐N¹,N⁴‐dioxide cyanoacetylhydrazone, is a growth promoter, which is more efficient and less toxic to animals. Few studies have been performed to reveal the metabolism of CYX in animals till now. In this study, the metabolic fate of CYX in the liver microsomes of animal was investigated firstly using high‐performance liquid chromatography combined with hybrid ion trap/time‐of‐flight mass spectrometry. CYX was incubated with rat, chicken and pig liver microsomes in the presence of a NADPH‐generating system. Multiple scans of metabolites in MS and MS² modes and accurate mass measurements were performed simultaneously through data‐dependent acquisition. Most measured mass errors were less than 10 ppm for both protonated molecules and fragment ions using external mass calibration. The structures of metabolites and their fragment ions were easily and reliably characterized based on the accurate MS² spectra and known structure of CYX. The relative biotransformation of CYX into characterized metabolites was estimated based on the UV absorption and the assumption that all metabolites had the same extinction coefficient as the parent compound at 305 nm. Totally, seven metabolites were identified as three reduced metabolites (cyadox 1‐monoxide (Cy1), cyadox 4‐monoxide (Cy2) and bisdesoxycyadox (Cy4)), three hydrolysis metabolites of the amide bond (N‐decyanoacetyl cyadox (Cy5), N‐decyanoacetyl cyadox 1‐monoxide (Cy6) and N‐decyanoacetyl bisdesoxycyadox (Cy7)) and a hydroxylation metabolite of Cy1 (Cy3). Cy1–Cy6 could be detected in rat, chicken and pig liver microsomes while metabolite Cy7 could only be observed in pig. The amounts of the metabolites in three species are different. For the formations of Cy1 and Cy3, the rank order was rat～chicken > pig. For Cy4 and Cy5, the order was pig > rat > chicken. Cy1 and Cy4 have been previously reported, whereas the other five metabolites were novel. The N→O group reduction and hydroxylation were the main metabolic pathways for CYX in the three species. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparative Metabolism of Mequindox in Liver Microsomes,Hepatocytes, and Intestinal Microflora of Chicken

Drug metabolism studies in vitro were carried out inexpensively and readily to serve as an adequate mechanism to characterize drug metabolites, elucidate their pathways, and make suggestions for further testing in vivo. In this work, the comparative metabolism of mequindox (MEQ) was investigated in vitro by incubation with chicken liver microsomes, hepatocytes, and intestinal microflora, followed by analysis using ultra-performance liquid chromatography coupled with electrospray ionization hybrid quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) for structure identification. There were 12 metabolites detected when MEQ was incubated with liver microsomes, 6 metabolites with the hepatocytes and 4 metabolites with intestinal microflora, respectively. The major metabolites in liver microsomes were bideoxymequindox and 2-isoethanol-N1-deoxymequindox, and that in hepatocytes were 2-isoethanol mequindox and 2-isoethanol-N1-deoxymequindox, but in intestinal incubations, N1-deoxymequindox and bideoxymequindox were the major metabolites. The results indicated that the metabolism of MEQ was active in vitro; meanwhile, revealed the main metabolic pathways of MEQ were N→O group reduction, carbonyl reduction and hydroxylation reaction. The information regarding in vitro metabolism of MEQ provided a better understanding of the role of the liver and intestinal tract in the disposition of MEQ.     

Identification of the major metabolites of quinocetone in swine urine using ultra‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Quinocetone (QCT), 3‐methyl‐2‐cinnamoylquinoxaline‐1,4‐dioxide, is a quinoxaline‐N,N‐dioxide used in veterinary medicine as a feed additive. QCT is broadly used in China to promote animal growth, but few studies have been performed to reveal the metabolism of QCT in animals until now. In the present study, the metabolites of QCT in swine urine were investigated using ultra‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight mass spectrometry (UPLC/ESI‐QTOF‐MS). Multiple scans of metabolites in MS and MS/MS modes and accurate mass measurements were performed simultaneously through data‐dependent acquisition. Most measured mass errors were less than ±5 mDa for both protonated molecules and product ions using external mass calibration. The structures of metabolites and their product ions were easily and reliably characterized based on the accurate MS² spectra and known structure of QCT. As expected, extensive metabolism was observed in swine urine. Thirty‐one metabolites were identified in swine urine, most of which were reported for the first time. The results reveal that the N‐O group reduction at position 1 and the hydroxylation reaction occurring at the methyl group, the side chain or on the benzene ring are the main metabolic pathways of quinocetone in swine urine. There was abundant production of 1‐desoxyquinocetone and hydroxylation metabolites of 1‐desoxyquinocetone. The proposed metabolic pathway of quinocetone in vivo can be expected to play a key role in food safety evaluations. Copyright © 2010 John Wiley & Sons, Ltd.     

Glycosidic linkage,N‐acetyl side‐chain,and other structural properties of methyl 2‐acetamido‐2‐deoxy‐β‐d‐glucopyranosyl‐(1→4)‐β‐d‐mannopyranoside monohydrate and related compounds

The crystal structure of methyl 2‐acetamido‐2‐deoxy‐β‐d ‐glycopyranosyl‐(1→4)‐β‐d ‐mannopyranoside monohydrate, C₁₅H₂₇NO₁₁·H₂O, was determined and its structural properties compared to those in a set of mono‐ and disaccharides bearing N‐acetyl side‐chains in βGlcNAc aldohexopyranosyl rings. Valence bond angles and torsion angles in these side chains are relatively uniform, but C—N (amide) and C—O (carbonyl) bond lengths depend on the state of hydrogen bonding to the carbonyl O atom and N—H hydrogen. Relative to N‐acetyl side chains devoid of hydrogen bonding, those in which the carbonyl O atom serves as a hydrogen‐bond acceptor display elongated C—O and shortened C—N bonds. This behavior is reproduced by density functional theory (DFT) calculations, indicating that the relative contributions of amide resonance forms to experimental C—N and C—O bond lengths depend on the solvation state, leading to expectations that activation barriers to amide cis–trans isomerization will depend on the polarity of the environment. DFT calculations also revealed useful predictive information on the dependencies of inter‐residue hydrogen bonding and some bond angles in or proximal to β‐(1→4) O‐glycosidic linkages on linkage torsion angles ? and ψ. Hypersurfaces correlating ? and ψ with the linkage C—O—C bond angle and total energy are sufficiently similar to render the former a proxy of the latter.     

Identification of carbadox metabolites formed by liver microsomes from rats, pigs and chickens using high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry

Carbadox (methyl-3-(2-quinoxalinylmethylene)-carbazate-N(1),N(4)-dioxide) is a chemotherapeutic growth promoter added to feed for starter pigs. In this work, the metabolism of carbadox in rat, pig and chicken liver microsomes has been studied firstly. The incubation mixtures were then processed and analyzed for metabolites with a sensitive and reliable method based on high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry (LC/MS-IT-TOF). With the help of chromatographic behavior and accurate mass measurements, it is possible to rapidly and reliably characterize the metabolites of carbadox. The structural elucidations of these metabolites were performed by comparing the changes in the accurate molecular masses and fragment ions generated from precursor ions with those of parent drug. The present results showed that the metabolism of carbadox in liver microsomes had qualitative species-difference. A total of seven metabolites were identified in rat liver microsomes. Five metabolites (Cb1-Cb3, Cb5, Cb7) were observed in pig and chicken liver microsomes. In addition, metabolite Cb6 was also detected in chicken liver microsomes. The peak areas of the metabolites in the three species are different. For the formations of Cb1, Cb2, Cb5 and Cb6, the rank order was rat>chicken>pig; Cb3; pig~chicken>rat. Cb1, Cb2 and Cb3 have been previously reported, whereas the other four metabolites were novel. The N→O group reduction and hydroxylation followed by N→O group reduction were the main metabolic pathways for carbadox in the three species.     

Structural elucidation of new urinary tamoxifen metabolites by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In this study, tamoxifen metabolic profiles were investigated carefully. Tamoxifen was administered to two healthy male volunteers and one female patient suffering from breast cancer. Urinary extracts were analyzed by liquid chromatography quadruple time‐of‐flight mass spectrometry using full scan and targeted MS/MS techniques with accurate mass measurement. Chromatographic peaks for potential metabolites were selected by using the theoretical [M + H]⁺ as precursor ion in full‐scan experiment and m/z 72, 58 or 44 as characteristic product ions for N,N‐dimethyl, N‐desmethyl and N,N‐didesmethyl metabolites in targeted MS/MS experiment, respectively. Tamoxifen and 37 metabolites were detected in extraction study samples. Chemical structures of seven unreported metabolites were elucidated particularly on the basis of fragmentation patterns observed for these metabolites. Several metabolic pathways containing mono‐ and di‐hydroxylation, methoxylation, N‐desmethylation, N,N‐didesmethylation, oxidation and combinations were suggested. All the metabolites were detected in the urine samples up to 1 week. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization and tentative identification of new flunitrazepam metabolites in authentic human urine specimens using liquid chromatography‐Q exactive‐HF hybrid quadrupole‐Orbitrap‐mass spectrometry (LC‐QE‐HF‐MS)

Flunitrazepam (FNZ) is a potent hypnotic, sedative, and amnestic drug used to treat severe insomnia. In our recent study, FNZ metabolic profiles were investigated carefully. Six authentic human urine samples were purified using solid phase extraction (SPE) without enzymatic hydrolysis, and urine extracts were then analyzed by liquid chromatography‐Q exactive‐HF hybrid quadrupole‐Orbitrap‐mass spectrometry (LC‐QE‐HF‐MS), using the full scan positive ion mode and targeted MS/MS (ddms2) technique to make accurate mass measurements. There were 25 metabolites, including 13 phase I and 12 phase II metabolites, which were detected and tentatively identified by LC‐QE‐HF‐MS. In addition, nine previously unreported phase II glucuronide conjugates and four phase I metabolites are reported here for the first time. Eight metabolic pathways, including N‐reduction and O‐reduction, N‐glucuronidation, O‐glucuronidation, mono‐hydroxylation and di‐hydroxylation, demethylation, acetylation, and combinations, were implicated in this work, and 2‐O‐reduction together with dihydroxylation were two novel metabolic pathways for FNZ that were identified tentatively. Although 7‐amino FNZ is widely considered to be the primary metabolite, a previously unreported metabolites (M12) can also serve as a potential biomarker for FNZ misuse.     

Identification of circulatory and excretory metabolites of meisoindigo in rat plasma,urine and feces by high‐performance liquid chromatography coupled with positive electrospray ionization tandem mass spectrometry

Meisoindigo has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia in China since the 1980s. However, information relevant to in vivo metabolism of meisoindigo is absent so far. In this study, in vivo circulatory metabolites of meisoindigo in rat plasma, as well as excretory metabolites in rat urine and feces, were identified by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Integration of multiple reaction monitoring with conventional metabolic profiling methodology was adopted to enable a more sensitive detection of in vivo metabolites. By comparing with the MS/MS spectra and retention times of the in vitro reduced metabolites, the major metabolites in rat plasma were proposed to form from 3,3′ double bond reduction, whereas the minor metabolites were formed from reduction followed by N‐demethylation, and reduction followed by phenyl mono‐oxidation. The major metabolites in the rat urine were proposed to form from reduction followed by phenyl mono‐oxidation, and its glucuronide conjugation and sulfate conjugation, whereas the minor metabolites were formed from 3,3′ double bond reduction, N‐demethylation, reduction followed by N‐demethylation, phenyl di‐oxidation, phenyl mono‐oxidation and its glucuronide conjugation and sulfate conjugation. The major metabolites in the rat feces were proposed to form from reduction followed by phenyl mono‐oxidation, whereas the minor metabolites were formed from reduction followed by N‐demethylation, and reduction followed by phenyl di‐oxidation. The phase I metabolic pathways showed a significant in vitro–in vivo correlation in rat. Copyright © 2010 John Wiley & Sons, Ltd.     

Metabolism of cyadox by the intestinal mucosa microsomes and gut flora of swine,and identification of metabolites by high‐performance liquid chromatography combined with ion trap/time‐of‐flight mass spectrometry

Cyadox (CYX), 2‐formylquinoxaline‐1,4‐dioxide cyanoacetylhydrazone, is an antimicrobial and growth‐promoting feed additive for food‐producing animals. To reveal biotransformation of CYX in swine intestine, CYX was incubated with swine intestinal microsomes and mucosa in the presence of an NADPH‐generating system and swine ileal flora and colonic flora, respectively. The metabolites of CYX were identified using high‐performance liquid chromatography combined with ion trap/time‐of‐flight mass spectrometry (LC/MS‐ITTOF). Structural elucidation of the metabolites was precisely performed by comparing their changes in molecular mass, full scan MS/MS spectra and accurate mass measurements with those of the parent drug. Finally, seven metabolites were identified as follows: three reduced metabolites (cyadox 1‐monoxide (Cy1), cyadox 4‐monoxide (Cy2) and bisdesoxycyadox (Cy4)); hydroxylation metabolite (3‐hydroxylcyadox 1‐monoxide (Cy3)); hydrolysis metabolite of the amide bond (N‐decyanoacetyl cyadox (Cy5)); a hydrogenation metabolite (11,12‐dihydro‐bisdesoxycyadox (Cy6)) and a side‐chain cleavage metabolite (2‐hydromethylquinoxaline (Cy7)). Only one metabolite (Cy1) was found in intestinal microsomes. Cy1, Cy2 and Cy4 were detected in intestinal mucosa, ileal and colonic flora. In addition, Cy3 and Cy5 were only obtained from ileal flora, and Cy6 and Cy7 alone were observed in colonic bacteria. The results indicated that N → O group reduction was the main metabolic pathway of CYX metabolism in swine ileal flora, intestinal microsomes and mucosa. New metabolic profiles of hydrogenation and cleavage on the side chain were found in colonic bacteria. Among the identified metabolites, two new metabolites (Cy6, Cy7) were detected for the first time. These studies will contribute to clarify comprehensively the metabolism of CYX in animals, and provide evidence to explain the pharmacology and toxicology effects of CYX in animals. Copyright © 2011 John Wiley & Sons, Ltd.     

Three sterically hindered 6‐amino‐5‐cyano‐2‐methyl‐4‐(1‐naphthyl)‐4H‐pyran‐3‐carboxylate derivatives

In the title compounds, 2‐methoxyethyl 6‐amino‐5‐cyano‐2‐methyl‐4‐(1‐naphthyl)‐4H‐pyran‐3‐carboxylate, C₂₁H₂₀N₂O₄, (II), isopropyl 6‐amino‐5‐cyano‐2‐methyl‐4‐(1‐naphthyl)‐4H‐pyran‐3‐carboxylate, C₂₁H₂₀N₂O₃, (III), and ethyl 6‐amino‐5‐cyano‐2‐methyl‐4‐(1‐naphthyl)‐4H‐pyran‐3‐carboxylate, C₂₀H₁₈N₂O₃, (IV), the heterocyclic pyran ring adopts a flattened boat conformation. In (II) and (III), the carbonyl group and a double bond of the heterocyclic ring are mutually anti, but in (IV) they are mutually syn. The ester O atoms in (II) and (III) and the carbonyl O atom in (IV) participate in intramolecular C—H...O contacts to form six‐membered rings. The dihedral angles between the naphthalene substituent and the closest four atoms of the heterocyclic ring are 73.3 (1), 71.0 (1) and 74.3 (1)° for (II)–(IV), respectively. In all three structures, only one H atom of the NH₂ group takes part in N—H...O [in (II) and (III)] or N—H...N [in (IV)] intermolecular hydrogen bonds, and chains [in (II) and (III)] or dimers [in (IV)] are formed. In (II), weak intermolecular C—H...O and C—H...N hydrogen bonds, and in (III) intermolecular C—H...O hydrogen bonds link the chains into ladders along the a axis.     

Characterization of imatinib metabolites in rat and human liver microsomes: differentiation of hydroxylation from N‐oxidation by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

In vitro metabolism of imatinib was investigated in rat and human liver microsomes. Atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) was applied in differentiating hydroxyl metabolites from N‐oxides of imatinib because N‐oxides are known to undergo deoxygenation during APCI. In addition, the major oxidative metabolite (M9, N‐oxidation on the piperazine ring) was observed to undergo in‐source fragmentation by elimination of formaldehyde. This fragment ion resulted from Meisenheimer rearrangement with migration of the N‐methyl group to the corresponding N‐methoxyl piperazine, followed by elimination of formaldehyde due to thermal energy activation at the vaporizer of APCI source. The presence of this fragment ion distinguished not only N‐oxide from isomeric hydroxylated metabolite, but also unambiguously indicated that oxidation occurred on the N‐4 of the piperazine ring where the methyl group was attached. Copyright © 2009 John Wiley & Sons, Ltd.     

ESI‐MS/MS analysis of protonated N‐methyl amino acids and their immonium ions

Methylation is one of the important posttranslational modifications of biological systems. At the metabolite level, the methylation process is expected to convert bioactive compounds such as amino acids, fatty acids, lipids, sugars, and other organic acids into their methylated forms. A few of the methylated amino acids are identified and have been proved as potential biomarkers for several metabolic disorders by using mass spectrometry–based metabolomics workstation. As it is possible to encounter all the N‐methyl forms of the proteinogenic amino acids in plant/biological systems, it is essential to have analytical data of all N‐methyl amino acids for their detection and identification. In earlier studies, we have reported the ESI‐MS/MS data of all methylated proteinogenic amino acids, except that of mono‐N‐methyl amino acids. In this study, the N‐methyl amino acids of all the amino acids ( 1 ‐ 21 ; including one isomeric pair) were synthesized and characterized by ESI‐MS/MS, LC/MS/MS, and HRMS. These data could be useful for detection and identification of N‐methyl amino acids in biological systems for future metabolomics studies. The MS/MS spectra of [M + H]⁺ ions of most N‐methyl amino acids showed respective immonium ions by the loss of (H₂O, CO). The other most common product ions detected were [MH‐(NH₂CH₃]⁺, [MH‐(RH)]⁺ (where R = side chain group) ions, and the selective structure indicative product ions due to side chain and N‐methyl group. The isomeric/isobaric N‐methyl amino acids could easily be differentiated by their distinct MS/MS spectra. Further, the MS/MS of immonium ions inferred side chain structure and methyl group on α‐nitrogen of the N‐methyl amino acids.     

N,N,N′,N′,N″‐Pentamethyl‐N″‐[(trifluorosilyl)methyl] phosphoric triamide. First example of the existence of intramolecular PO→Si coordination bond in the Si‐containing phosphoric triamides

The N,N,N′,N′,N″‐pentamethyl‐N″‐(trifluorosilylmethyl)phosphoric triamide O?P(NMe₂)₂N(Me) CH₂SiF₃ with intramolecular P?O→Si coordination was formed by the reaction of N,N,N′,N′,N″‐pentamethyl‐N″[(triethoxysilyl)methyl]phosphoric triamide with BF₃·Et₂O. Copyright © 2007 John Wiley & Sons, Ltd.     

In vitro metabolism studies of desoxy‐methyltestosterone (DMT) and its five analogues,and in vivo metabolism of desoxy‐vinyltestosterone (DVT) in horses

The positive findings of norbolethone in 2002 and tetrahydrogestrinone in 2003 in human athlete samples confirmed that designer steroids were indeed being abused in human sports. In 2005, an addition to the family of designer steroids called ‘Madol’ [also known as desoxy‐methyltestosterone ( DMT )] was seized by government officials at the US–Canadian border. Two years later, a positive finding of DMT was reported in a mixed martial arts athlete's sample. It is not uncommon that doping agents used in human sports would likewise be abused in equine sports. Designer steroids would, therefore, pose a similar threat to the horseracing and equestrian communities. This paper describes the in vitro metabolism studies of DMT and five of its structural analogues with different substituents at the 17α position (R ? H, ethyl, vinyl, ethynyl and ²H₃‐methyl). In addition, the in vivo metabolism of desoxy‐vinyltestosterone ( DVT ) in horses will be presented. The in vitro studies revealed that the metabolic pathways of DMT and its analogues occurred predominantly in the A‐ring by way of a combination of enone formation, hydroxylation and reduction. Additional biotransformation involving hydroxylation of the 17α‐alkyl group was also observed for DMT and some of its analogues. The oral administration experiment revealed that DVT was extensively metabolised and the parent drug was not detected in urine. Two in vivo metabolites, derived respectively from (1) hydroxylation of the A‐ring and (2) di‐hydroxylation together with A‐ring double‐bond reduction, could be detected in urine up to a maximum of 46 h after administration. Another in vivo metabolite, derived from hydroxylation of the A‐ring with additional double‐bond reduction and di‐hydroxylation of the 17α‐vinyl group, could be detected in urine up to a maximum of 70 h post‐administration. All in vivo metabolites were excreted mainly as glucuronides and were also detected in the in vitro studies. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro metabolic fate of alizapride: evidence for the formation of reactive metabolites based on liquid chromatography‐tandem mass spectrometry

The study of the formation of reactive metabolites during drug metabolism is one of the major areas of research in drug development since the link between reactive metabolites and drug adverse effects was well recognized. In particular, it has been shown that acrolein, a reactive carbonyl species sharing carbonylating and alkylating properties, binds covalently to nucleophilic sites in proteins, causing cellular damage. Alizapride, (±)‐6‐methoxy‐N‐{[1‐(prop‐2‐en‐1‐yl)‐pyrrolidin‐2‐yl]methyl}‐1H‐benzotriazole‐5‐carboxamide, is a N‐allyl containing dopamine antagonist with antiemetic properties for which no data concerning its metabolic fate are so far reported. The study of the in vitro metabolism of alizapride showed the formation of acrolein during the oxidative N‐deallylation. Moreover, the formation of an epoxide metabolite has been also described suggesting its role as a putative structural alert. The reactivity of the acrolein and the epoxide generated in alizapride metabolism was demonstrated by the formation of the corresponding adducts with nucleophilic thiols. Overall, ten metabolites have been identified and characterized by electrospray ionization tandem mass spectrometry analysis allowing to propose an in vitro metabolic scheme for alizapride. At the best of our knowledge, this is the second case of a drug involved in the generation of acrolein during its metabolism being the first represented by cyclophosphamide. Copyright © 2012 John Wiley & Sons, Ltd.     

Design of peptides with α,β‐de­hydro residues: pseudo‐tripeptide N‐benzyl­oxy­carbonyl–ΔLeu–l‐Ala–l‐Leu–OCH3

The title peptide N‐benzyl­oxy­carbonyl–ΔLeu–l ‐Ala–l ‐Leu–OCH₃ [methyl N‐(benzyl­oxy­carbonyl)‐α,β‐de­hydro­leucyl‐l ‐alanyl‐l ‐leucinate], C₂₄H₃₅N₃O₆, was synthesized in the solution phase. The peptide adopts a type II′β‐turn conformation which is stabilized by an intramolecular 4 1 N—H?O hydrogen bond. The crystal packing is stabilized by two intermolecular N—H?O hydrogen bonds.     

Caulerpin

The crystal structure of caulerpin (di­methyl 6,13‐di­hydro­dibenzo­[b,i]­phenazine‐5,12‐di­carboxyl­ate, C₂₄H₁₈N₂O₄), an indole alkaloid, reported in space group Cc with an acute β angle, has been redetermined in the correct space group, C2/c. The mol­ecule has twofold crystallographic symmetry and is composed of two essentially planar indole groups fused to an eight‐membered cyclo­octatetraene ring which adopts a boat conformation. The molecular dimensions are normal. The structure is stabilized by intermolecular and intramolecular interactions involving the indole N—H atom and carbonyl O atom [N?O 3.211 (4) and 2.836 (4) Å].     

Two‐injection workflow for a liquid chromatography/LTQ‐Orbitrap system to complete in vivo biotransformation characterization: demonstration with buspirone metabolite identification

The relatively high background matrix in in vivo samples typically poses difficulties in drug metabolite identification, and causes repeated analytical runs on unit resolution liquid chromatography/mass spectrometry (LC/MS) systems before the completion of biotransformation characterization. Ballpark parameter settings for the LTQ‐Orbitrap are reported herein that enable complete in vivo metabolite identification within two HPLC/MS injections on the hybrid LTQ‐Orbitrap data collection system. By setting the FT survey full scan at 60K resolution to trigger five dependent LTQ MS² scans, and proper parameters of Repeat Duration, Exclusion Duration and Repeat Count for the first run (exploratory), the Orbitrap achieved the optimal parallel data acquisition capability and collected maximum number of product ion scans. Biotransformation knowledge based prediction played the key role in exact mass ion extraction and multiple mass defect filtration when the initial data was processed. Meanwhile, product ion extraction and neutral loss extraction of the initial dependent data provided additional bonus in identifying metabolites. With updated parent mass list and the data‐dependent setting to let only the ions on the parent mass list trigger dependent scans, the second run (confirmatory) ensures that all precursor ions of identified metabolites trigger not only dependent product ion scans, but also at or close to the highest concentration of the eluted metabolite peaks. This workflow has been developed for metabolite identification of in vivo or ADME studies, of which the samples typically contain a high level of complex matrix. However, due to the proprietary nature of the in vivo studies, this workflow is presented herein with in vitro buspirone sample incubated with human liver microsomes (HLM). The major HLM‐mediated biotransformation on buspirone was identified as oxidation or hydroxylation since five mono‐ (+16 Da), seven di‐ (+32 Da) and at least three tri‐oxygenated (+48 Da) metabolites were identified. Besides the metabolites 1‐pyrimidinylpiperazine (1‐PP) and hydroxylated 1‐PP that formed by N‐dealkylation, a new metabolite M308 was identified as the result of a second N‐dealkylation of the pyrimidine unit. Two new metabolites containing the 8‐butyl‐8‐azaspiro[4,5]decane‐7,9‐dione partial structure, M240 and M254, were also identified that were formed apparently due to the first N‐dealkylation of the 1‐PP moiety. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolism of methyltestosterone in the greyhound

Gas chromatography/mass spectrometry and selective derivatisation techniques have been used to identify urinary metabolites of methyltestosterone following oral administration to the greyhound. Several metabolites were identified including reduced, mono‐, di‐ and trihydroxylated steroids. The major metabolites observed were 17α‐methyl‐5β‐androstane‐3α‐17β‐diol, 17α‐methyl‐5β‐androstane‐3α,16α,17β‐triol, and a further compound tentatively identified as 17α‐methyl‐5z‐androstane‐6z,17β‐triol. The most abundant of these was the 17α‐methyl‐5β‐androstane‐3α,16α,17β‐triol. This metabolite was identified by comparison with a reference standard synthesised using a Grignard procedure and characterised using trimethylsilyl (TMS) and acetonide‐TMS derivatisation techniques. There did not appear to be any evidence for 16β‐hydroxylation as a phase I metabolic transformation in the greyhound. However, significant quantities of 16α‐hydroxy metabolites were detected. Selective enzymatic hydrolysis procedures indicated that the major metabolites identified were excreted as glucuronic acid conjugates. Metabolic transformations observed in the greyhound have been compared with those of other mammalian species and are discussed here. Copyright © 2009 John Wiley & Sons, Ltd.     

Di‐ and tripeptide segments of zervamicin II‐2: Z‐Thr(OBn)‐Aib‐N(Me)Ph and Z‐Val‐Aib‐Hyp(OBn)‐OMe

The title compounds, O‐benzyl‐N‐(benzyl­oxy­carbonyl)­threonyl‐2,N‐dimethyl­alanin­anilide, C₃₀H₃₅N₃O₅, and methyl (4R)‐4‐benzyl­oxy‐N‐(benzyl­oxy­carbonyl)­valyl‐2‐(methyl­alanyl)prolinate, C₃₀H₃₉N₃O₇, were obtained from the `azirine coupling' of the corresponding protected amino acids with 2,2,N‐trimethyl‐2H‐azirin‐3‐amine and methyl (4R)‐4‐(benzyl­oxy)‐N‐(2,2‐dimethyl‐2H‐azirin‐2‐yl)prolinate, respectively. The Aib unit in each mol­ecule has the greatest turn‐ or helix‐inducing effect on the mol­ecular conformation. Inter­molecular N—H⋯O inter­actions link the mol­ecules of the tripeptide into sheets and those of the dipeptide into extended chains.