The methyl-5 alpha-dihydrotestosterones mesterolone and drostanolone; gas chromatographic/mass spectrometric characterization of the urinary metabolites.

Before including the detection of the methyl-5 alpha-dihydrotestosterones mesterolone (1 alpha-methyl-17 beta-hydroxy-5 alpha-androstan-3-one) and drostanolone (2 alpha-methyl-17 beta-hydroxy-5 alpha-androstan-3-one) in doping control procedures, their urinary metabolites were characterized by gas chromatography/mass spectrometry. Several metabolites were found after enzymatic hydrolysis and conversion of the respective metabolites to their trimethylsilyl-enol-trimethylsilyl ether derivatives. The major metabolites of mesterolone and drostanolone were identified as 1 alpha-methyl-androsterone and 2 alpha-methyl-androsterone, respectively. The parent compounds and the intermediate 3 alpha,17 beta-dihydroxysteroid metabolites were detected as well. The reduction into the corresponding 3 beta-hydroxysteroids was a minor metabolic pathway. All metabolites were found to be conjugated to glucuronic acid.     

Metabolic profiling and antioxidant activity during flower development in Agastache rugosa

Our previous study showed that flowers of Agastache rugosa had higher phenolic levels and higher antibacterial and antioxidant capacity compared to those of the leaves and stems. The aim of this study was to provide information on the variation in primary and secondary metabolites during flower development in A. rugosa by using high performance liquid chromatography (HPLC) and assays of total anthocyanin (TAC), flavonoid (TFC), and phenolic content (TPC), as well as gas chromatography time-of-flight mass spectrometry (GC-TOFMS) analysis. Assays of TPC, TAC, and TFC showed that the floral bud (stage I) contained higher TPC than did the partially open flower (stage II) and fully open flower (stage III). However, the TFC was the highest at stage II, and the highest TAC was observed at stage III. Furthermore, HPLC analysis revealed that the level of total phenylpropanoids, including rosmarinic acid, tilianin, acacetin, 4-hydroxybenzoic acid, caffeic acid, chlorogenic acid, trans-cinnamic acid, rutin, (-)-epicatechin, quercetin, and kaempferol, was higher in stages I and II, but the concentrations of rutin and rosmarinic acid were highest in stage III. A total of 43 compounds, including amino acids, organic acids, phenolic compounds, sugars, photorespiration-related compounds, and intermediates of the tricarboxylic acid cycle, were identified through GC-TOFMS analysis. Of these compounds, most amino acids decreased during flower development. In contrast, the increase in concentrations of glucose and sucrose were observed from stages I to III. In this study, health-beneficial compounds were identified and quantified in flowers of A. rugosa. Accordingly, our results suggests that A. rugosa flowers can potentially be used as biomaterials for pharmaceuticals, cosmetics, food, and related industries.Supplementary informationThe online version contains supplementary material available at (10.1007/s12298-021-00945-z).     

Interconversions of aglycone and host-selective toxin from Helminthosporium sacchari

Helminthosporium sacchari, a fungus that causes disease in sugarcane, produces oligosaccharide-sesquiterpene toxins (HS toxins A, B, and C) that are required for infection and disease development. Two free sesquiterpenes were isolated from mycelium but not from culture fluids of the fungus. One sesquiterpene was identified by HPLC and mass spectrometry as an aglycone of HS toxin C and could be obtained by enzymatic hydrolysis of this toxin. The other sesquiterpene appeared to be the 2-keto form of the first compound. The aglycone from toxin C hydrolysis was labelled with tritium by successive treatments with active manganese dioxide, sodium boro[³H]hydride, and lithium aluminium hydride. The labelled compound was fed to cultures of H. sacchari, radioactivity was incorporated into HS toxin C and into lower molecular weight homologues. The results suggest a metabolic route (aglycone → metabolite Y, → HS toxin → metabolite X) for the biosynthesis of HS toxin; metabolites X and Y are lower molecular weight homologues of the toxin.     

Evidence for selective hydrolysis of aliphatic copolyesters induced by lipase catalysis

High molar mass random poly(butylene succinate-co-butylene sebacate), P(BS-co-BSe), and poly(butylene succinate-co-butylene adipate), P(BS-co-BA), with different composition, were synthesized and subjected to enzymatic hydrolysis by Lipase from Mucor miehei or from Rhizopus arrhizus. The enzymatic hydrolysis of P(BS-co-BSe)s and P(BS-co-BA)s films produced a mixture of water-soluble monomers and co-oligomers that were separated and identified by on-line high performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). Optimization of the HPLC analysis allowed the separation of isobar co-oligomers, differing only for the co-monomers sequence. Oligomers with the same monomer composition and molar mass but different sequence were identified by HPLC/ESI-MS-MS on-line analysis. The results obtained show a preferential hydrolytic cleavage induced by the lipases used. In particular, these enzymes prefer cleaving sebacic ester bonds in P(BS-co-BSe) copolymers, whereas succinic ester bonds appear to be hydrolyzed faster than adipic ester bonds in P(BS-co-BA) copolyesters. 1H NMR analysis further substantiates these findings. The primary products generated by lipase hydrolysis of polyester films underwent further degradation at longer reaction times. The HPLC/ESI-MS analysis of these mixtures at various times provided the first evidence that lipase catalysis is active also in water solution, a hydrophobic effect induced by the aliphatic units of these polyesters.     

Gas chromatographic–mass spectrometric identification of main metabolites of stanozolol in cattle after oral and subcutaneous administration

An analytical method has been developed in order to control the illegal use of stanozolol as growth promoter in livestock. The procedure was based on enzymatic hydrolysis, purification on a Clean Screen DAU column and derivatization with heptafluorobutyric anhydride prior to GC–MS analysis. This method allowed us to study the metabolism of stanozolol in cattle after oral and subcutaneous administrations. Urinary metabolites were identified by mass spectrometry. Stanozolol and 16-hydroxystanozolol were detected after oral administration, while 16-hydroxystanozolol and 4,16-dihydroxystanozolol were found after subcutaneous administration.     

Oxygenation of linolenic and linoleic acid to novel vicinal dihydroxy acids by hepatic microsomes of the rabbit

[14C] Linolenic acid (18: omega 3) and [14C] linoleic acid (18:2 omega 6) were incubated with hepatic microsomes of the rabbit in the presence of NADPH (1 Mm) for 15 min at 37 degrees C. The products were extracted and purified by high performance liquid chromatography. The major metabolites of linolenic and linoleic acid were identified by capillary gas chromatography mass spectrometry as 15,16-dihydroxy-9,12-octadecadienoic acid, 12,13-dihydroxy-9,15-octadecadienoic acid and 9,10-dihydroxy-12,15-octadecadienoic acid and as 12,13-dihydroxy-9-octadecaenoic acid and 9,10-dihydroxy-12-octadecaenoic acid, respectively. The results were confirmed by comparison with mass spectra of the authentic compounds. These metabolites are presumably formed by cytochrome P-450 catalyzed epoxidation of the omega 3, omega 6 and omega 9 double bonds, followed by enzymatic hydrolysis to 1,2-diols. The ratio of omega 3, omega 6 and omega 9 oxygenated metabolites of linolenic acid was approximately 2:1:1 and the ratio of the omega 6 and omega 9 metabolites of linoleic acid was 2:1, indicating that the double bond closest the omega end is most easily oxygenated.     

Identification of several human urinary metabolites of 6-benzoyl benzoxazolinone by gas chromatography/mass spectrometry.

The biotransformation of 6-benzoyl benzoxazolinone (6-BB), a non-narcotic peripheral analgesic, was studied in eight healthy volunteers after oral administration of a single dose of 1 g. Urinary metabolites were extracted either with ethyl acetate at different pH values or by percolating at pH 5 through Amberlite XAD 2 ion-exchange resin. Eluates were concentrated under vacuum, purified by thin-layer chromatography and analysed by gas chromatography/mass spectrometry or direct insertion probe mass spectrometry. Metabolites were identified with reference to the mass spectra of various synthesized compounds assumed to be metabolites of 6-BB, as N-methylated or monohydroxylated compounds. Another metabolic pathway was cleavage of the benzoxazolinone heterocycle giving 2-amino-5-benzoyl phenol after hydrolysis and decarboxylation. N-methyl, N-acetyl and hydroxylated metabolites having an amino-5-benzoyl phenol structure were also found.     

Isolation,identification and determination of sulfadiazine and its hydroxy metabolites and conjugates from man and Rhesus monkey by high-performance liquid chromatography

The following metabolites of sulfadiazine (S) were isolated from monkey urine by preparative HPLC: 5-hydroxysulfadiazine (5OH), 4-hydroxysulfadiazine (4OH) and the glucuronide (5OHgluc) and sulfate conjugate of 5OH (5OHsulf). The compounds were identified by NMR, mass and infrared spectrometry and hydrolysis by β-glucuronidase. The analysis of S, the hydroxymetabolites (4OH, 5OH) and conjugates N₄-acetylsulfadiazine (N₄), 5OHgluc and 5OHsulf in human and monkey plasma and urine samples was performed using reversed-phase gradient HPLC with UV detection. In plasma, S and N₄ could be detected in high concentrations, whereas the other metabolites were present in only minute concentrations. In urine, S, the metabolites and conjugates were present. The limit of quantification of the compounds in plasma varies between 0.2 and 0.6 μg/ml (S 0.31, N₄ 0.40, 4OH 0.20, 5OH 0.37, 5OHgluc 0.33 and 5OHsulf 0.57 μg/ml). In urine it varies between 0.6 and 1.1 μg/ml (S 0.75, N₄ 0.80, 4OH 0.60, 5OH 0.80, 5OHgluc 0.80 and 5OHsulf 1.1 μg/ml). The method was applied to studies with healthy human subjects and Rhesus monkeys. The metabolites 5OH, 5OHgluc and 5OHsulf were present in Rhesus monkey and not in man. Preliminary results of studies of metabolism and pharmacokinetics in Rhesus monkey and man are presented.     

Rapid analysis of metabolic stability of dopamine receptor antagonists and detection of their metabolites by liquid chromatography/tandem mass spectrometry

In vitro metabolic stability of dopamine D(3)/D(4) receptor antagonists and identification of their metabolites by high-performance liquid chromatography (HPLC) coupled with ion-trap mass spectrometry (ITMS) were assessed in rat liver microsomes. The compounds were divided into three cassette groups for rapid quantitative analysis of multiple drugs and simultaneous detection of their metabolites. The samples from incubation with rat liver microsomes were pooled into designed cassette groups and analyzed by HPLC/electrospray ITMS in full-scan mode. The metabolic stability of the drugs was determined by comparing their signals after incubation for 0 and for 30min. The metabolic stability of the examined dopamine receptor antagonists was in the range of 9.9-84.4%. In addition, the present cassette analysis allowed the simultaneous detection of metabolites formed during the same incubation without having to reanalyze the samples. The metabolites were first characterized by nominal mass measurement of the corresponding protonated molecules. Subsequent multistage tandem mass spectrometry on the ion-trap instrument allowed characterization of the structure of the detected metabolites. N,O-dealkylation and ring hydroxylation reactions were identified as major metabolic reactions in piperazinylalkylisoxazole derivatives. These results suggested that the present approach is useful for the rapid evaluation of metabolic stability and structural characterization of metabolites within a short period in new drug discovery.     

Analytical strategies for the direct mass spectrometric analysis of steroid and corticosteroid phase II metabolites

The use of steroid hormones as growth promoters remains illegal in Europe. A classical approach used to control their utilization consists to measure the parent drug in target biological matrices. However, this strategy may fail when the parent drug is submitted to extensive metabolism reactions. For urine and tissue samples, chemical or enzymatic hydrolysis is usually applied in order to deconjugate glucuronide and sulfate phase II metabolites. But this treatment lead to the loss of information such as nature and relative proportions of the different conjugated forms, which can be useful, for example, to discriminate an endogenous production from an exogenous administration for natural hormones, or for other clinical or biochemical specific applications. For these purposes, direct measurement of conjugated metabolites using liquid chromatography-tandem mass spectrometry may represent a solution of choice. In this context, the mass spectrometric behavior of 14 steroid and corticosteroid phase II metabolites after electrospray ionization was investigated. Their fragmentation pathways in tandem mass spectrometry revealed some specificities within the different group of conjugates. A specific acquisition program (MRM mode) was developed for the unambiguous identification of the studied reference compounds. A more generic method (Parent Scan mode) was also developed for fishing approaches consisting to monitor several fragment ions typical of each conjugate class. A reverse phase HPLC procedure was also proposed for efficient retention and separation of the studied compounds. Finally, a protocol based on quaternary amine SPE was developed, permitting the separation of free, glucuronide, and sulfate fractions. Preliminary results on biological samples demonstrated the suitability of this analytical strategy for direct measurement of dexamethasone glucuronide and sulfate residues in bovine urine.     

High Performance Liquid Chromatography of Hydroperoxides Formed by Autoxidation of Vegetable Oils

Trilinoleoylglycerol (TL) was autoxidized at 37°C in the dark. Monohydroperoxides (MHP) obtained from the oxidized products were analyzed by high performance liquid chromatography (HPLC). Several peaks which appeared in the chromatogram were identified by infrared (IR), gas chromatography mass spectrometry (GC-MS) and enzymatic hydrolysis. Some positional and geometrical isomers of their hydroperoxy fatty acid components were separated using both absorption and reversed phase systems. Furthermore, 1-hydroperoxylinoleoyl-2,3-dilinoleoyl-glycerol and 1,3-dilinoleoyl-2-hydroperoxylinoleoylglycerol were partly separated by HPLC using an absorption system. MHP obtained from autoxidized corn oil, safflower oil and soybean oil were separated into some peaks by HPLC, although resolution into the individual isomers was incomplete. When oxidized oils were subjected to HPLC analysis directly, a linear relationship was observed between the peak areas of MHP and peroxide value in the range of 10 ~ 50 meq/kg.     

Narrow-bore sample trapping and chromatography combined with quadrupole/time-of-flight mass spectrometry for ultra-sensitive identification of in vivo and in vitro metabolites

The identification of in vitro and in vivo metabolites is vital to the discovery and development of new pharmaceutical therapies. Analytical strategies to identify metabolites at different stages of this process vary, but all involve the use of liquid chromatography separations combined with detection via mass spectrometry (HPLC/MS). Reported here is the use of narrow-bore column (0.5-1.0 mm i.d.) trapping of metabolites, followed by back-flushing onto a matching analytical column. Separated metabolites were then identified using quadrupole time-of-flight mass spectrometry (MS) and tandem MS. Metabolites in human plasma and from low-level in vitro incubations, that were not identified using standard HPLC/MS approaches, were characterized using the instrumental configuration described here.     

Mass spectral characterization of doxylamine and its rhesus monkey urinary metabolites

This study describes the use of mass spectrometry (MS), high-performance liquid chromatography (HPLC) and chemical derivatization techniques for the identification of doxylamine and five rhesus monkey urinary metabolites. The analyses were performed using chemical ionization mass spectrometry with either methane or ammonia as the reagent gas. The confirmation of the structures of two of these urinary metabolites was aided by the synthesis of doxylamine N-oxide and desmethyldoxylamine and by the use of methylation and acetylation derivatization techniques. Doxylamine N-oxide, desmethyldoxylamine, didesmethyldoxylamine, and two metabolites which resulted from the cleavage of the aliphatic tertiary nitrogen side chain to the subsequent 2-[1-phenyl-1-(2-pyridinyl)ethoxy]acetic acid or 2-[1-phenyl-1-(2-pyridinyl)ethoxy]methanol compounds were isolated and identified from rhesus monkey urine. Additional data concerning the mass spectral analysis of derivatization or reaction products from the three chloroformate reactions with doxylamine, and the synthesis and separation techniques which afforded mass spectral identification of the urinary metabolites are also presented.     

Detection of new urinary exemestane metabolites by gas chromatography coupled to mass spectrometry

Exemestane is an aromatase enzyme complex inhibitor. Its metabolism in humans is not fully described and there is only one known metabolite: 17β-hydroxyexemestane. In this work, excretion studies were performed with four volunteers aiming at the detection of new exemestane metabolites in human urine by gas chromatography coupled to mass spectrometry (GC-MS) after enzymatic hydrolysis and liquid-liquid extraction. Urine samples collected from four volunteers were analyzed separately. The targets of the study were mainly the 6-exomethylene oxidized metabolites. Two unreported metabolites were identified in both free and glucuconjugated urine fractions from all four volunteers, both of them were the result of the 6-exomethylene moiety oxidation: 6ξ-hydroxy-6ξ-hydroxymethylandrosta-1,4-diene-3,17-dione (metabolite 1) and 6ξ-hydroxyandrosta-1,4-diene-3,17-dione (metabolite 2). Furthermore, only in glucoconjugated fractions from all volunteers, one metabolite arising from the A-ring reduction was identified as well, 3ξ-hydroxy-5ξ-androst-1-ene-6-methylene-17-one (metabolite 3). The molecular formulae of all these metabolites were ascertained by the determination of exact masses using gas chromatography coupled to high resolution mass spectrometry (GC-HRMS). Moreover, all metabolites were confirmed using an alternative derivatization with methoxyamine and MSTFA/TMS-imidazole.     

Study of a virilizing adrenal cortical tumor. Analysis of urinary steroids by gas-liquid chromatography and mass spectrometry]

The C19 and C21 urinary steroids from a virilizing adrenal tumour with high levels of plasma 17alpha-progesterone and its urinary metabolites have been identified and quantitated by gas chromatography and mass spectrometry of sephadex fractions of the total urinary extract. Of the fifty five identified steroids thirteen were new compounds or known compounds not found before in such a case. The actiology of the apparent 21-steroid hydroxylase deficiency is discussed at the light of these analytical results and of the hormonogenesis enzymatic induction of the tumour biopsy.     

Measurement of the cortisol production rate in two sisters with 17 alpha-hydroxylase deficiency using [1,2,3,4-13C]cortisol and isotope dilution mass spectrometry

[1,2,3,4-13C]cortisol was i.v. administered to two sisters aged 11 yr (patient I) and 3 yr (patient II) who suffer from 17 alpha-hydroxylase deficiency. This is the first time that the cortisol production rate (CPR) in patients with 17 alpha-hydroxylase deficiency has been measured with a stable labelled tracer using the urinary method. The urine was collected for 3 days. High-performance liquid chromatography (HPLC) of approximately 100 ml urine extracts was carried out to isolate the small amount of cortisol metabolites excreted. The cortisol metabolites were oxidized to 11-oxo-aetiocholanolone. The isotope dilution in the methyl oxime tert-butyldimethylsilyl ether derivatives was measured by selected ion monitoring gas chromatography/mass spectrometry (GC/MS). The CPR calculated from tetrahydrocortisone (THE) and the cortolones was 765 and 536 nmol/day, respectively in patient I. The CPR in patient II was only calculated from THE and was 62 nmol/day. If radioactive labelled cortisol had been used, much larger quantities of urine would have been needed for isolation of sufficient mass of metabolites, even then purification may have been difficult. Steroid profiling of 1 ml urine samples by GC and identification by GC/MS revealed high concentrations of pregnenolone, progesterone, 11 beta-hydroxy progesterone and corticosterone metabolites. Tetrahydrocorticosterone and 5 alpha-tetrahydrocorticosterone were found in urine at elevated excretions of 2.5 and 5.7, 0.9 and 2.0 mumols/24 h, in patients I and II respectively. No cortisol metabolites were detected by routine GC or GC/MS as the low amounts excreted co-eluted with the relatively abundant corticosterone metabolites.     

Fragments from albumin and beta 2-microglobulin--constituents of the middle molecule fraction in hemofiltration]

Peptides were isolated from the middle molecule fraction of hemofiltrate obtained from uremic patients by adsorption of the organic compounds on RP 18. By stepwise elution an enriched peptide fraction was obtained. This procedure was followed by a combination of ion-exchange-chromatography, gel-chromatography and HPLC. The pure peptides obtained in this way were investigated by liquid secondary ion mass spectrometry (LSIMS)-if necessary after enzymatic digestion. The identified peptides turned out to be fragments of the human serum albumin and beta 2-microglobulin.     

Identification of new urinary metabolites of trimeprazine in rats by gas chromatography—mass spectrometry

The metabolites of trimeprazine were identified in urine of rats by gas chromatography—mass spectrometry. After the oral administration of trimeprazine, the urinary metabolites were extracted with diethyl ether before or after hydrolysis with β-glucuronidase. The identified metabolites were N-demethyltrimeprazine, 3-hydroxytrimeprazine, N-demethyl-3-hydroxytrimeprazine and trimeprazine sulphoxide.     

Optimized glucuronide hydrolysis for the detection of psilocin in human urine samples

In order to develop a sensitive and reliable analytical method for psilocin (PC) in urine samples, the hydrolysis conditions including the acid, alkaline and enzymatic hydrolyses have been investigated by monitoring not only PC but also psilocin glucuronide (PCG) by liquid chromatography tandem mass spectrometry (LC-MS-MS); PCG was initially identified in a "magic mushroom (MM)" user's urine by liquid chromatography mass spectrometry (LC-MS) and LC-MS-MS. The proposed conditions optimized for the hydrolysis are as follows: hydrolysis, enzymatic hydrolysis; enzyme, Escherichia coli beta-glucuronidase (5000 units/ml urine); incubation, pH 6 at 37 degrees C for 2h. The complete hydrolysis of PCG in urine was obtained under these conditions, while the enzymatic hydrolyses with three types of beta-glucuronidases originated from bovine liver (Type B-1), Helix pomatia (Type H-1) and Ampullaria provided uncompleted hydrolysis of PCG. Also, neither the acid nor alkaline hydrolysis was found to be applicable. According to the present method, 3.55 microg/ml of psilocin was detected in the "magic mushroom" user's urine after the enzymatic hydrolysis, though psilocin was not detected without hydrolysis.     

Fungal Transformation of the Tricyclic Antidepressant Amoxapine: Identification of N-Carbomethoxy Compounds Formed as Artifacts by Phosgene in Chloroform used for the Extraction of Metabolites

The biotransformation of the antidepressant drug amoxapine by Cunninghamella elegans formed three metabolites, 7-hydroxyamoxapine, N-formyl-7-hydroxyamoxapine, and N-formylamoxapine; two other compounds were only present when chloroform was used in the extraction process. All five of the compounds were separated by reversed-phase HPLC, then analyzed by ¹H NMR and mass spectrometry, and by ¹³C NMR when sample quantities permitted. The artifacts were identified as N-carbomethoxy-7-hydroxyamoxapine and N-carbomethoxyamoxapine. Phosgene is a decomposition product of chloroform that can form carbomethoxy compounds at the secondary nitrogen of a piperazine ring in an alcoholic solution. Since N-carbomethoxy compounds were not observed when ethyl acetate was used for extraction of the culture medium, they were considered artifacts and not metabolites. These findings suggest that chloroform should be tested for the formation of phosgene before using it to extract any compound with a piperazine ring or any other amine-containing structure.