The use of mass spectrometry in the study of chemically-reactive drug metabolises. Application of MS/MS and LC/MS to the analysis of glutathione- and related S-linked conjugates of N-methylformamide

The S-(N-methylcarbamoyl) derivatives of glutathione, cysteine and N-acetylcysteine, the S-linked conjugates derived from a reactive metabolite of N-methylformamide (NMF), were studied in mice dosed with an equimolar mixture of NMF and deuterium-labelled NMF. Following preparation of N-benzyloxycarbonyl derivatives in aqueous media, the title conjugates were isolated, purified as their methyl esters and subjected to analysis by fast atom bombardment mass spectrometry (FAB/MS), fast atom bombardment tandem mass spectrometry (FAB/MS/MS) or thermospray liquid chromatography/mass spectrometry (TSP LC/MS). Characteristic isotope clusters in the FAB or TSP mass spectra facilitated recognition of drug metabolites, while constant neutral loss (89 u) and daughter ion scanning tandem mass spectrometry (MS/MS) experiments provided unique structural information on the conjugates of interest. It is concluded that the combined use of stable isotopes, aqueousphase derivatization and contemporary mass spectrometric techniques represents a powerful approach for the analysis of glutathione adducts and related S-linked conjugates of chemically-reactive drug metabolites.     

Studies on the metabolism of propafenone. 3rd Comm.: Isolation of the conjugated metabolites in the dog and identification using fast atom bombardment mass spectrometry

The metabolism and urinary and biliary excretion of propafenone (2-(2'-hydroxy-3'-propylamino-propoxy)-omega-phenyl-propiophenone hydrochloride) were studied in the dog. Approximately 20% of the dose was excreted in 24 h with the urine and about 65% with the bile. Propafenone was extensively metabolized. Less than 4% of the dose was recovered unchanged in urine and bile. The metabolites were mainly excreted as conjugates. Free metabolites accounted for less than 20% of the dose. Separation methods were developed to isolate and purify the conjugated metabolites. Fractionation on an Al2O3-column yielded a sulphate and a glucuronide fraction, further separation and purification was done by TLC and HPLC. Positive and negative ion fast atom bombardment mass spectra (FAB/MS) were obtained of the purified glucuronides. The structures of two hydroxylated propafenone derivatives and two O-methylated catechol-like derivatives were definitely proven by FAB/MS, the glucuronic acid moiety being conjugated to the hydroxyl function in the different aromatic rings. Two isomeric propafenone glucuronides were found in the bile, probably diastereomeric forms of the O-glucuronide. Thus FAB/MS proved to be a complementary method to electron impact ionization mass spectrometry (EI/MS) for studying drug metabolism. The structures of free and conjugated metabolites can be defined from the combination of both mass spectrometric techniques.     

In vitro formation of glutathione conjugates of the dimethylester of bilirubin.

Rat hepatic microsomes catalyzed the formation of two distinct glutathione conjugates of bilirubin dimethylester (DMB). The two conjugates were identical to those isolated from the bile of Gunn rats infused with DMB. The microsomal reaction was dependent on NADPH, oxygen and glutathione and was inhibited by nitrogen and the cytochrome P450 inhibitors metyrapone, 1-benzyl-imidazole, and alpha-naphthoflavone. Conjugate formation was inducible with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but not phenobarbital pretreatment. The rate of formation of conjugates was not affected by washings of the microsomal pellet or by the presence of superoxide dismutase and/or catalase. Cation fast atom bombardment mass spectrometry (FAB/MS) of the conjugates indicated a molecular ion of 937 atomic mass units (amu). Fragmentation revealed a loss of 307 amu, consistent with glutathione, and a residual mass of 629 amu suggesting a hydroxylated derivative of DMB (612 amu). Cation FAB/MS/MS of conjugates formed in vitro under an atmosphere of oxygen-16 and oxygen-18 demonstrated the incorporation of molecular oxygen by a difference of 2 amu in the respective molecular ions. Our results suggest that DMB is oxidized by the cytochrome P450 IA gene family to an epoxide intermediate which is then subsequently conjugated with glutathione.     

Identification of phase-I and phase-II metabolites of fluphenazine in rat bile. Intact glucuronide and sulfate conjugates.

In rat bile following ip administration of fluphenazine (FLU) dihydrochloride (20 mg/kg body weight), phase-I metabolites 7-hydroxyfluphenazine (7-HOFLU) and FLU sulfoxide (FLUSO), together with unmetabolized FLU, were isolated and identified by HPLC and fast atom bombardment spectrometry (FAB/MS) and also by comparison with authentic compounds. Two intact glucuronide conjugates of FLU were isolated and identified as phase-II metabolites: 7-hydroxyfluphenazine ring glucuronide (glucuronic acid linked to the aromatic hydroxyl group of 7-hydroxyfluphenazine), and FLU glucuronide (glucuronide linked to the aliphatic group of the side chain of FLU) by HPLC and FAB/MS in comparison with authentic compounds. Further confirmed by FAB/MS were several sulfate conjugates of FLU that were isolated and identified indirectly as phase-II sulfate metabolites: FLU sulfate, 7-hydroxyfluphenazine sulfate and/or 7-hydroxyfluphenazine ring sulfate, and FLU sulfoxide sulfate by HPLC and FAB/MS; their aglycones were identified after sulfatase hydrolysis as FLU, 7-HOFLU and FLUSO. A further phase-II metabolite, for which no authentic standard was available, was tentatively identified as a monoglucuronide of dihydroxy derivative of FLU. To our knowledge, this report provides the first direct evidence of the presence of intact phase-II metabolites of FLU in rat bile.     

电喷雾离子阱质谱法直接测定几种药物的葡萄糖苷酸型代谢物

为研究药物代谢产物的质谱规律,用电喷雾离子阱质谱法对溶液中乙氧苯柳胺、SFZ-47羧基衍生物、5-羟基普罗帕酮及普罗帕酮的β-D-葡萄糖苷酸型代谢物的结构进行了测定。结果表明,它们的(-)ESI-MS均生成[M-H]^-准分子离子,(-)ESI-MS²和(-)ESI-MS³则分别生成m/z175和m/z113碎片离子。提示这些共同特征可用于LC/MS法直接分析药物的葡萄糖苷酸型代谢物。     

Structural characterization of cisplatin analogues by fast atom bombardment (FAB) and laser microprobe mass spectrometry (LAMMA)

The present study is concerned with the investigation of the potentials and limitations of fast atom bombardment (FAB) and laser microprobe mass spectrometry (LAMMA) for the structural characterization of a series of cisplatin analogues. The limiting factors for obtaining good quality FAB spectra are the solubility and the stability of the organometallic platinum complexes in the FAB matrix. In the case of a suitable matrix being found, molecular weight information is derived from the (M + H)⁺ and/or (M — H)⁻ ions. Drawbacks of the application of FAB are (i) the low signal intensities of the molecular ion-like species as compared to the matrix signals and (ii) the scarcity of fragmentation necessary for structure determination. Combination of FAB with tandem mass spectrometry was used to overcome these problems. LAMMA provides a valuable alternative for the direct mass spectral analysis of cisplatin analogues. For some compounds, LAMMA results in useful mass spectra, whereas FAB fails. The abundant fragmentation yields structural information which is complementary for positive and negative ions. The laser power density applied to the sample is of critical importance for the quality of the spectra.     

Biliary excretion and intestinal metabolism in the intermediary metabolism of pentachlorothioanisole

1. Biliary metabolites from rats dosed with pentachlorothioanisole (PCTA) were characterized by fast atom bombardment mass spectrometry and electron impact mass spectrometry. 2. Most of the biliary metabolites from PCTA were mercapturic acid pathway metabolites of methylsulphinyltetrachlorobenzene (51% of the dose); the remaining characterized biliary metabolites (20%) were mainly methylsulphinyltetrachlorothiophenols excreted as unknown conjugates. 3. Pathways are proposed for the intermediary metabolism of PCTA to bis-(methylthio)tetrachlorobenzene (bis-MTTCB) involving glutathione conjugation, biliary excretion, intestinal metabolism, and enterohepatic circulation.     

Identification of glutathione conjugates and mercapturic acids of 1,2-dibromopropane in female BALB/c mice by liquid chromatography-electrospray ionization tandem mass spectrometry

Based on recent results that 1,2-dibromopropane (1,2-DBP) causes hepatotoxicity and immunotoxicity in female BALB/c mice as well as a reduction of hepatic glutathione levels, the possible formation of glutathione conjugates and mercapturic acids of 1,2-DBP was investigated in vivo in the present studies. The following four metabolites were identified in the liver at 12?h after treatment with 1,2-DBP, by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS): M1, 2-hydroxypropylglutathione; M2, 2-oxopropylglutathione; M3, N-acetyl-S-(2-hydroxypropyl)-L-cysteine; and M4, N-acetyl-S-(2-oxopropyl)-L-cysteine. Ions of individual conjugates were observed at m/z 366, 364, 222 and 220, respectively. Characteristic product ions at m/z 237, 217, 204 and 202 for the identification of M1, M2, M3 and M4 were observed, respectively. In the sera isolated from the same animals, only mercapturic acids (M3 and M4) were observed by LC-ESI/MS. When female BALB/c mice were treated orally with 1,2-DBP at doses of 150, 300 and 600?mg?kg^?1 once for 12?h, the production of glutathione conjugates and mercapturic acids in liver was apparently dose dependent, as were the concentrations of them in sera. When the production of metabolites from 1,2-DBP was investigated in liver following oral treatment with 600?mg?kg^?1 1,2-DBP for 6, 12, 24 and 48?h, metabolite concentrations were greatest at the first time point (6?h). The results explain the authors’ previous studies that oral treatment with 1,2-DBP reduces the hepatic content of glutathione.     

Formation, characterization, and toxicity of the glutathione and cysteine conjugates of toxic heptapeptide microcystins.

Microcystins LR, YR, and RR, cyclic heptapeptide hepatotoxins produced by cyanobacteria, were synthetically converted into glutathione (GSH) and cysteine (Cys) conjugates. Fast atom bombardment mass spectra showed [M + H]+ ions corresponding to GSH and Cys conjugates of microcystins LR, YR, and RR for the obtained compounds. 1H NMR spectral analyses revealed that two singlet signals of olefinic protons of N-methyldehydroalanine (Mdha) in microcystins disappeared in the conjugates, confirming that thiols of GSH and Cys added nucleophilically to the alpha, beta-unsaturated carbonyl of the Mdha moiety. On examination of the 50% lethal dose (LD50) with intravenous injection using mice, both GSH and Cys conjugates showed reduction in toxicity compared with microcystins, but their toxicity still remained. Microcystin LR and its GSH conjugate were separated and identified in a standard mixture by using a frit-fast atom bombardment liquid chromatography/mass spectrometry (Frit-FAB LC/MS) method. Obtained conjugates in the present study would be important compounds as the standard samples for study of metabolism of microcystins, and the Frit-FAB LC/MS method would be applicable to mass spectrometric identification of metabolites of microcystins.     

高效液相色谱-电喷雾离子阱串联质谱分析水苏碱及其大鼠体内代谢物

目的鉴定水苏碱在大鼠体内的代谢物。方法应用高效液相色谱-电喷雾离子阱串联质谱(HPLC-ESI/MSⁿ)技术研究水苏碱的一级质谱电离规律、二级质谱裂解规律及其色谱保留，以此作为水苏碱大鼠体内代谢物分析鉴定的依据；再将健康大鼠空腹灌胃25 mg·kg^-1水苏碱，收集0～24 h的尿样，经C₁₈小柱固相萃取分离纯化后，应用HPLC-ESI/MS分析尿样中水苏碱代谢物。结果在大鼠尿样中发现了母药及其N-去甲基、氧化脱氢、环氧化等6种I相代谢产物及两种环氧化物的甘氨酸轭合II相代谢产物。结论HPLC-ESI/MS法灵敏度高，快速，定性能力强，适合于水苏碱大鼠体内代谢物的分析。     

Biliary excretion and intestinal metabolism in the intermediary metabolism of pentachlorothioanisole

1. Biliary metabolites from rats dosed with pentachlorothioanisole (PCTA) were characterized by fast atom bombardment mass spectrometry and electron impact mass spectrometry.

2. Most of the biliary metabolites from PCTA were mercapturic acid pathway metabolites of methylsulphinyltetrachlorobenzene (51% of the dose); the remaining characterized biliary metabolites (20%) were mainly methylsulphinyltetrachlorothio-phenols excreted as unknown conjugates.

3. Pathways are proposed for the intermediary metabolism of PCTA to bis-(methylthio)tetrachlorobenzene (bis-MTTCB) involving glutathione conjugation, biliary excretion, intestinal metabolism, and enterohepatic circulation.     

Digestion by pancreatic juice of a beta-casomorphin-containing fragment of buffalo beta-casein

Degradation by pig pancreatic juice of a beta-casomorphin-containing fragment (tryptic peptide corresponding to residues 49–68 of buffalo beta-casein) was investigated. The FAB/MS (fast atom bombardment mass spectrometry) technique was used to identify the fragments produced by the concerted action of pancreatic proteases. Pancreatic juice, under our experimental conditions, is not able to release beta-casomorphins or morphiceptin from the tryptic peptide sequence. Furthermore, the present report shows that the rapid hydrolysis of a peptide bond by a single protease can prevent the cleavage of peptide bonds by a different protease. Therefore the formation of some peptides in the gastrointestinal tract can depend on the protease ratio.     

ACYLPOLYAMINES: MASS SPECTROMETRIC ANALYTICAL METHODS FOR Araneidae SPIDER ACYLPOLYAMINES

A new class of compounds, acylpolyamine has been isolated from spider venom constituents. Recent advances in highly sensitive mass spectrometric techniques have been applied successfully to characterize these acylpolyamines even with the use of a single venom gland. This has been achieved, in part, by improvements in fast atom bombardment mass spectrometry (FAB), continuous flow (FRIT) FAB-MS combined with reversed-phase high performance liquid chromatography (HPLC), matrix assisted laser desorption/ionization (MALDI) and high energy collision induced dissociation (CID) tandem MS/MS. Crude venom analysis without chromatographic separation can be realized directly by MALDI-MS. A charge-remote fragmentation method has provided abundant structure-related product ions and have reduced the quantity of required venom for the structure analysis of acylpolyamines. These mass spectrometric methods were proved to be useful for the analysis of complex constituents of spiders and other arthropod venom glands.     

Fast atom bombardment mass spectrometry of ceruletide and [Tyr 4] ceruletide

Fast atom bombardment (FAB) mass spectrometry has revealed a very useful technique in obtaining the mass spectrum of ceruletide (formerly caerulein), a decapeptide with a tyrosyl-O-sulphate residue, and of its desulphated analog. Molecular weight value for ceruletide is obtained only in the negative ion mode, whereas for [Tyr⁴] ceruletide this information is obtained both in negative and in positive ion detection. Positive ion mass spectra show more extended fragmentation which allows the complete amino acids sequence to be checked. The lability of the sulphate group has been confirmed, examining other sulphated peptides by field desorption mass spectrometry.     

质谱解析多糖结构的方法学进展

在多糖结构分析的诸多方法中,质谱技术被认为是一个重要的不可缺少的手段,其中快原子轰击、电喷雾离子化、基质辅助激光解吸、串联质谱等质谱技术已在多糖结构的分析中有许多成功的应用。     

Formation and reversibility of S-linked conjugates of N-(1-methyl-3,3-diphenylpropyl)isocyanate, an in vivo metabolite of N-(1-methyl-3,3-diphenylpropyl)formamaide, in rats

The metabolic disposition of N-(1-methyl-3,3-diphenylpropyl) formamide was studied in rats. The water-soluble metabolites, N-acetyl-S-[N-(1-methyl-3,3-diphenylpropylcarbamoyl)]cysteine and S-[N-(1-methyl-3,3-diphenylpropylcarbamoyl)]glutathione, were identified in urine and bile, respectively, of rats doses with the secondary formamide. The structures of these metabolites were confirmed by comparison with synthetic standards and by using liquid chromatography mass spectrometry and fast atom bombardment mass spectrometry. Synthetic standards of these metabolites were obtained by reacting the N-(1-methyl-3,3-diphenylpropyl)isocyanate with glutathione or N-acetylcysteine in methanolic solutions. The isocyanate was obtained in high yield by reacting 1-methyl-3,3-diphenylpropylamine with trichloromethyl chloroformate. The S-linked conjugates released the isocyanate in mild alkali, but were stable under acidic conditions. The released isocyanate was characterized by comparison with the synthetic standard using GC/MS and HPLC. A mechanism is proposed for the base-catalyzed elimination of the isocyanate from the thiol conjugates.     

Identification of two glucuronide metabolites of doxylamine via thermospray/mass spectrometry and thermospray/mass spectrometry/mass spectrometry

Analysis of a high-pressure liquid chromatography fraction containing two urinary glucuronide metabolites of doxylamine by thermospray mass spectrometry (TSP/MS) provided [MH]+ ions for each metabolite. TSP/MS/MS of the [MH]+ ions provided a fragment ion characteristic of these metabolites. The results demonstrate the utility of TSP/MS analysis for biologically derived glucuronide metabolites.     

Structural characterization of bacitracin components by Frit-fast atom bombardment (FAB) liquid chromatography/mass spectrometry (LC/MS).

The structural characterization of minor components of bacitracin (BC) complex was carried out using a technique of liquid chromatography/mass spectrometry (LC/MS). Satisfactory total ion current chromatogram of BC complex and excellent mass spectra of many components were given by Frit-fast atom bombardment (FAB) LC/MS analytical system, and the structures of 13 minor components could be proposed. The 13 minor components were classified into two groups, bacitracin A (BC-A) related components and bacitracin F (BC-F) related components depending on their common N-terminal moieties. The structures of BC-A related components and BC-F related components were the same as those of BC-A and BC-F, respectively, except that one to three of isoleucine and leucine residues are replaced by valines. The BC-F related components were degradation products of BC-A related components through the same degradation process as that of BC-A.     

Stereoselectivity of naphthalene epoxidation by mouse, rat, and hamster pulmonary, hepatic, and renal microsomal enzymes

Previous studies have demonstrated the formation of three glutathione conjugates during the hepatic and pulmonary microsomal metabolism of naphthalene in the presence of reduced glutathione and cytosolic enzymes containing the glutathione transferases. These glutathione conjugates now have been identified by negative ion fast atom bombardment mass spectrometry, by proton NMR spectroscopy, and by chemical synthesis from the (1S,2R)- and (1R, 2S)-naphthalene 1,2-oxide enantiomers as isomeric hydroxyglutathionyldihydronaphthalene derivatives. All three glutathione adducts yielded prominent mass spectral ions at m/z 450 (M-H)-, 432 (dehydration product), and 306 (glutathionyl moiety) which were consistent with the monoglutathionyl derivatives of hydroxydihydronaphthalene. Signals in the proton NMR spectra at 3.60 and 4.95 ppm (adduct 1) and 3.60 and 4.95 ppm (adduct 2) indicated that these conjugates were diastereomers of 1-hydroxy-2-glutathionyl-1,2-dihydronaphthalene. Corresponding signals for H1 and H2 at 4.22 and 4.45 ppm for adduct 3 showed that this isomer was generated from attack of glutathione at the 1 position of the naphthalene 1,2-oxide. Incubation of synthetic (1S, 2R)-naphthalene 1,2-oxide with glutathione in the presence of glutathione transferases resulted in the formation of adducts 1 and 3 in approximately equal proportions; under identical conditions, glutathione conjugate 2 was formed from (1R, 2S)-naphthalene 1,2-oxide. Incubation of naphthalene, glutathione, and glutathione transferases with pulmonary, hepatic, or renal microsomal preparations from mouse, rat, and hamster resulted in the formation of all three glutathione conjugates. Substantial differences in the rates of formation of the individual conjugates were observed.(ABSTRACT TRUNCATED AT 250 WORDS)