Mass spectrometric analysis of cyclofenil and its metabolites in human urine

Using gas chromatography/electron impact-mass spectrometry (GC/EI-MS) and high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS), the structures of cyclofenil metabolites in human urine have been assigned. The hydroxyl metabolites liberated from the glucuronide conjugates after acid hydrolysis were characterized as the trimethylsilyl (O-TMS) derivatives using GC/MS. The conjugate glucuronide forms were detected without hydrolysis by HPLC/MS. Cyclofenil was not observed in urine. Tentative structures for the two metabolites are proposed.     

New designer drug 2,5-dimethoxy-4-ethylthio-beta-phenethylamine (2C-T-2): studies on its metabolism and toxicological detection in rat urine using gas chromatography/mass spectrometry

Studies are described on the metabolism and the toxicological analysis of the phenethylamine-derived designer drug 2,5-dimethoxy-4-ethylthio-beta-phenethylamine (2C-T-2) in rat urine using gas chromatography/mass spectrometry (GC/MS) after enzymatic cleavage of conjugates, liquid-liquid extraction and derivatization. The structures of 14 metabolites were assigned tentatively by detailed interpretation of their mass spectra. Identification of these metabolites indicated that 2C-T-2 was metabolized by sulfoxidation followed by N-acetylation and either hydroxylation of the S-ethyl side chain or demethylation of one methoxy group, O-demethylation of the parent compound followed by N-acetylation and sulfoxidation, deamination followed by reduction to the corresponding alcohol followed by partial glucuronidation and/or sulfation or by oxidation to the corresponding acid followed either by partial glucuronidation or by degradation to the corresponding benzoic acid derivative followed by partial glucuronidation. Furthermore, 2C-T-2 was metabolized by N-acetylation of the parent compound followed either by O-demethylation and sulfoxidation or by S-dealkylation, S-methylation and sulfoxidation. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction microwave-assisted acetylation allowed the detection of an intake of a dose of 2C-T-2 in rat urine, which corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-T-2 in human urine.     

New designer drug 1-(3-trifluoromethylphenyl) piperazine (TFMPP): gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry studies on its phase I and II metabolism and on its toxicological detection in rat urine

Studies are described on the phase I and II metabolism and the toxicological analysis of the piperazine-derived designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP) in rat urine using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS). The identified metabolites indicated that TFMPP was extensively metabolized, mainly by hydroxylation of the aromatic ring and by degradation of the piperazine moiety to N-(3-trifluoromethylphenyl)ethylenediamine, N-(hydroxy-3-trifluoromethylphenyl)ethylenediamine, 3-trifluoromethylaniline, and hydroxy-3-trifluoromethylaniline. Phase II reactions included glucuronidation, sulfatation and acetylation of phase I metabolites. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of TFMPP and its above-mentioned metabolites in rat urine after single administration of a dose calculated from the doses commonly taken by drug users. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of TFMPP in human urine.     

Simultaneous determination of dimethylarsinic acid and monomethylarsonic acid after derivatization with thioglycol methylate by gas chromatography/mass spectrometry

A gas chromatography/mass spectrometry (GC/MS) method has been developed to determine two methylated arsenic species in human urine samples. The yield of derivatization for dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA) using thioglycol methylate (TGM) was measured. The detection limit for the derivatized DMA and MMA using the GC/MS method are 0.95 and 0.8 ng cm-3, respectively. This simple and rapid method has good precision and accuracy. Fragmentation routes of derivatized MMA and DMA are suggested on accurate mass measurements.     

Comparative metabolite profiling of carboxylic acids in rat urine by CE‐ESI MS/MS through positively pre‐charged and 2H‐coded derivatization

A new approach to the selective comparative metabolite profiling of carboxylic acids in rat urine was established using CE‐MS and a method for positively pre‐charged and ²H‐coded derivatization. Novel derivatizing reagents, N‐alkyl‐4‐aminomethyl‐pyridinum iodide (alkyl=butyl, butyl‐d9 or hexyl), containing quaternary amine and stable‐isotope atoms (deuterium), were introduced for the derivatization of carboxylic acids. CE separation in positive polarity showed high reproducibility (0.99–1.32% RSD of migration time) and eliminated problems with capillary coating known in CE‐MS anion analyses. Essentially complete ionization and increased hydrophobicity after the derivatization also enhanced MS detection sensitivity (e.g. formic acid was detected at 0.5 pg). Simultaneous derivatization of one sample using two structurally similar reagents, N‐butyl‐4‐aminomethyl‐pyridinum iodide (BAMP) and N‐hexyl‐4‐aminomethyl‐pyridinum iodide, provided additional information for recognizing a carboxylic acid in an unknown sample. Moreover, characteristic fragmentation acquired by online CE‐MS/MS allowed for identification and categorization of carboxylic acids. Applying this method on rat urine, we found 59 ions matching the characteristic patterns of carboxylic acids. From these 59, 32 ions were positively identified and confirmed with standards. For comparative analysis, 24 standard carboxylic acids were derivatized by chemically identical but isotopically distinct BAMP and N‐butyl‐d9‐4‐aminomethyl‐pyridinium iodide, and their derivatization limits and linearity ranges were determined. Comparative analysis was also performed on two individual urine samples derivatized with BAMP and N‐butyl‐d9‐4‐aminomethyl‐pyridinium iodide. The metabolite profiling variation between these two samples was clearly visualized.     

New designer drug 1-(3,4-methylenedioxybenzyl) piperazine (MDBP): studies on its metabolism and toxicological detection in rat urine using gas chromatography/mass spectrometry

Studies are described on the metabolism and toxicological analysis of the piperazine-derived designer drug 1-(3,4-methylenedioxybenzyl)piperazine (MDBP) in rat urine using gas chromatography/mass spectrometry (GC/MS). The identified metabolites indicated that MDBP was metabolized by demethylenation and subsequent methylation to N-(4-hydroxy-3-methoxybenzyl)piperazine followed by partial glucuronidation or sulfation. Additionally, degradation of the piperazine moiety to N-(3,4-methylenedioxybenzyl)ethylenediamine and 3,4-methylenedioxybenzylamine and N-dealkylation to piperazine were observed. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid/liquid extraction and microwave-assisted acetylation allowed the detection of MDBP and its above-mentioned metabolites in rat urine after single administration of a dose calculated from the doses commonly taken by drug users. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of MDBP by analysis of human urine.     

固相萃取-气相色谱-质谱法检测尿液中乙基葡萄糖醛酸苷

建立了固相萃取(SPE)-气相色谱-质谱(GC-MS)检测尿液样本中乙基葡萄糖醛酸苷(EtG)的方法.1 mL尿液样本经100μL 3 mol/L盐酸去蛋白后,通过SPE法提取上清液中的目标物质及内标,提取物经衍生化后,采用GC-MS检测,选择离子模式(SIM)扫描,内标法定量分析.该方法在0.1～3.2 mg/L范围...     

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

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

Quantitative determination of chelators and their degradation products in mixed hazardous wastes from Tank 241-SY-101 using derivatization GC/MS

Considerable attention has been focused on chelators such as ethylenediaminetetraacetic acid (EDTA) and N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA), which form water-soluble complexes with most heavy metals. Most radionuclides are included in this class of constituents. As a result, chelator complexes have become very important environmentally because of their tendency to enhance the mobility of heavy metals through the soil and potentially contaminate groundwater. In addition, there is a correlation between chelator concentration and crust formation/gas release. The chelators are a class of compounds whose low volatility and high polarity preclude analysis by gas chromatography/mass spectrometry (GC/MS) without prior derivatization. Waste samples from a double-shell storage tank at Hanford were derivatized with BF₃/methanol and analyzed using GC/MS. Results indicate the presence of EDTA, HEDTA, nitrilotriacetic (NTA), and citric acid. Nitrosoiminodiacetic acid was identified and determined to be an artifact of the derivatization procedure; it is assumed to arise from nitrosation of iminodiacetic acid in the waste sample.     

Identification of conjugation positions of urinary glucuronidated vitamin D3 metabolites by LC/ESI–MS/MS after conversion to MS/MS‐fragmentable derivatives

A liquid chromatography/electrospray ionization–tandem mass spectrometry‐based method was developed for the identification of the conjugation positions of the monoglucuronides of 25‐hydroxyvitamin D₃ [25(OH)D₃] and 24,25‐dihydroxyvitamin D₃ [24,25(OH)₂D₃] in human urine. The method employed derivatization with 4‐(4‐dimethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione to convert the glucuronides into fragmentable derivatives, which provided useful product ions for identifying the conjugation positions during the MS/MS. The derivatization also enhanced the assay sensitivity and specificity for urine sample analysis. The positional isomeric monoglucuronides, 25(OH)D₃‐3‐ and ‐25‐glucuronides, or 24,25(OH)₂D₃‐3‐, ‐24‐ and ‐25‐glucuronides, were completely separated from each other under the optimized LC conditions. Using this method, the conjugation positions were successfully determined to be the C3 and C24 positions for the glucuronidated 25(OH)D₃ and 24,25(OH)₂D₃, respectively. The 3‐glucuronide was not present for 24,25(OH)₂D₃, unlike 25(OH)D₃, thus we found that selective glucuronidation occurs at the C24‐hydroxy group for 24,25(OH)₂D₃.     

Simultaneous enantiomeric determination of MDMA and its phase I and phase II metabolites in urine by liquid chromatography–tandem mass spectrometry with chiral derivatization

A liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) procedure was developed for the simultaneous determination of enantiomers of the prevalent designer drug 3,4-methylenedioxymethamphetamine (MDMA) and its phase I and phase II metabolites in urine with chiral derivatization. The analytes in urine were directly derivatized with chiral Marfey’s reagent, N ^α- (5-fluoro-2,4-dinitrophenyl)-d-leucinamide, without extraction. The diastereomers of the N ^α-(2,4-dinitrophenyl)-d-leucinamide derivatives generated were determined by LC-MS/MS. Satisfactory chromatographic separation was achieved for the enantiomers of MDMA and its metabolites 3,4-methylenedioxyamphetamine, 4-hydroxy-3-methoxymethamphetamine (HMMA), HMMA glucuronide, and HMMA sulfate on a semimicro octadecylsilane column using linear gradient elution. With use of multiple reaction monitoring mode, the limits of detection of these analytes ranged from 0.01 to 0.03?μg/mL. Linear calibration curves were obtained for all enantiomers from 0.1 to 20?μg/mL in urine. The method showed sufficient reproducibility and quantitative ability. This is the first report of a simple LC-MS/MS-based analytical procedure with direct chiral derivatization in aqueous media that allows simultaneous enantiomeric determination of drugs and their metabolites, including glucuronide and sulfate derivatives.     

Determination of anabolic steroids by gas chromatography/negative-ion chemical ionization mass spectrometry and gas chromatography/negative-ion chemical ionization tandem mass spectrometry with heptafluorobutyric anhydride derivatization

A gas chromatography/mass spectrometry (GC/MS) method is described which uses negative ion chemical ionization (NCI) and tandem mass spectrometry (MS/MS) for the determination of eight anabolic steroids in human urine. Eight anabolic steroids were derivatized by heptafluorobutyric anhydride (HFBA), and were determined using GC/NCI-MS and GC/NCI-MS/MS. The linear correlation coefficients for calibration in NCI-MS/MS were in the range 0.9880-0.9988. This method of derivatization with HFBA for use with GC/NCI was useful in determinations of 19-norandrosterone, boldenone, 19-noretiocholanolone, 2-methylandrosterone, nandrolone, 1-methyleneandrosterone, 1-methylandrosterone, 4-dihydroboldenone and mesterolone. The detection limits of this procedure were 5-20 ppb at a signal-to-noise (S/N) ratio of 3.     

Analysis of some metabolites of T-2 toxin, diacetoxyscirpenol and deoxynivalenol by thermospray high-performance liquid chromatography-mass spectrometry

Detection and identification of mycotoxin metabolites is a very challenging task. In order to achieve adequate sensitivity and specificity an analytical technique must overcome serious matrix interferences. Gas chromatography-mass spectrometry (GC-MS) which has the sensitivity and specificity to detect and identify mycotoxin metabolites requires hydrolysis of conjugated metabolites as well as derivatization. Thermospray high-performance liquid chromatography-mass spectrometry (HPLC-MS) offers the sensitivity, specificity, and structural information to detect and identify some mycotoxin metabolites in fecal and urine samples without derivatization. The mycotoxins evaluated in this study include deoxynivalenol (DON), T-2 toxin, and diacetoxyscirpenol. The de-epoxy and hydroxy metabolites of each toxin and the glucuronide conjugate of DON were isolated, extracted, and analyzed to detect their occurrence in animals. The thermospray mass spectra of the toxins showed an [M + H]+ ion and numerous structurally significant fragment ions in the positive ion detection mode. Negative ion detection exhibited primarily [M + acetate]- cluster ions with less fragmentation than observed by positive ion detection. The operation of the interface in the filament-on mode greatly increased the sensitivity in both positive and negative ion detection mode. Detection limits of 50-500 pg injected on column are obtained for these toxins and their metabolites using multiple ion detection. The urine and fecal extracts from rats, hens, and cows did not interfere with the HPLC-MS analysis for the specific metabolites or the glucuronide conjugate.     

High-temperature ultra-performance liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry applied to ibuprofen metabolites in human urine

The application of sub-2 microm porous particle liquid chromatography (LC) operated at elevated temperatures, coupled with time-of-flight mass spectrometry (MS), to the separation and identification of metabolites of ibuprofen present in human urine following oral administrations is illustrated. The LC/MS system generated a high-resolution analytical separation that, with an analysis time of 20 min, provided a peak capacity in the order of ca. 350. Using this system a total of nine glucuronides of the drug and its metabolites were detected, including a number of isomeric acyl glucuronides of ibuprofen itself, a side-chain-oxidized carboxylic acid acyl glucuronide and a number of acyl glucuronides of various hydroxylated metabolites. The identities of the metabolites were confirmed by their accurate mass values and the presence of the common fragment ions from ibuprofen.     

New designer drug N-(1-phenylcyclohexyl)-3-ethoxypropanamine (PCEPA): studies on its metabolism and toxicological detection in rat urine using gas chromatographic/mass spectrometric techniques

Studies are described on the metabolism and toxicological detection of the phencyclidine-derived designer drug N-(1-phenylcyclohexyl)-3-ethoxypropanamine (PCEPA) in rat urine using gas chromatographic/mass spectrometric techniques. The identified metabolites indicated that PCEPA was metabolized by N-dealkylation, O-deethylation partially followed by oxidation of the resulting alcohol to the corresponding carboxylic acid, hydroxylation of the cyclohexyl ring at different positions of PCEPA, N-dealkyl PCEPA, O-deethyl PCEPA, and of the corresponding carboxylic acids. Finally, aromatic hydroxylation of PCEPA, the corresponding carboxylic acids, and O-deethyl PCEPA, the latter partially followed by oxidation to the corresponding carboxylic acid and hydroxylation of the cyclohexyl ring could be observed. All metabolites were partially excreted in the conjugated form. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction, and microwave-assisted acetylation allowed the detection in rat urine of an intake of a common drug users' dose of PCEPA. Assuming a similar metabolism in humans, the STA in human urine should be suitable as proof of intake of PCEPA.     

A general approach for the quantitative analysis of bisphosphonates in human serum and urine by high-performance liquid chromatography/tandem mass spectrometry

Bisphosphonates are extremely hydrophilic and structurally similar to many endogenous phosphorylated compounds, making their selective extraction from serum or urine very challenging. Many bisphosphonates lack strong chromophores for sensitive UV or fluorescence detection. We report here the first general approach to enable sensitive and selective quantitation of N-containing bisphosphonates by liquid chromatography/tandem mass spectrometry (LC/MS/MS) following derivatization with diazomethane. The novelty of the strategy lies in performing the derivatization on silica-based anion-exchange sorbents as an integrated step in the sample purification by solid-phase extraction (SPE). The 'on-cartridge' reaction with diazomethane not only led to higher efficiency of derivatization, but also enabled a more discriminatory recovery of the drug's derivatives. The derivatized bisphosphonates demonstrated improved chromatographic separation and increased sensitivity of the detection. The general applicability of the approach was demonstrated by validation of bioanalytical methods for risedronate and alendronate in human serum and urine. Sensitivity was achieved at the pg/mL level with merely 100-200 microL of sample.     

Identification of chemical constituents and rat metabolites of Kangxianling granule by HPLC‐Q‐TOF‐MS/MS

A high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass tandem mass spectrometry method was established to characterize the chemical constituents of Kangxianling granule (KXL), a traditional Chinese medicine formula, and the metabolic profile in rat urine and plasma after oral administration of KXL. A total of 27 compounds in KXL extract and 13 prototype compounds with 12 metabolites in rat urine and plasma were identified. Among the 27 detected compounds, 15 were identified by comparing the retention time and MS data with that of reference compounds and the other 12 compounds were tentatively assigned based on the MS data and reference literature. The main prototype components absorbed in rat were amygdalin, salvianolic acid B, tanshinones and anthraquinones. Hydroxylation, glucuronidation and sulfation were the principal metabolic pathways in rat. The results revealed that the 25 compounds identified in rat urine and plasma were the potential active ingredients of KXL, which provides helpful chemical information for further study of the pharmacology mechanism of KXL. Copyright © 2015 John Wiley & Sons, Ltd.     

Novel phosphoryl derivatization method for peptide sequencing by electrospray ionization mass spectrometry

A one-step phosphoryl derivatization method has been used in a peptide sequencing procedure for electrospray ionization tandem mass spectrometry (ESI-MS/MS). The sodiated derivatized peptides exhibit very simple dissociation patterns, in which two kinds of fragment ions, [b(n) + OH + Na]+ and [a(n) + Na]+, are formed. Since the amino acid residues are lost sequentially from the C-terminus, peptide sequences can be identified easily. The fragmentation efficiency of peptides increased as a result of the phosphorylation, and also provided peaks of useful intensity at lower m/z. A peptide with lysine at the C-terminus was derivatized and analyzed by ESI-MS/MS. Similar mass spectra, from which the sequence could be read out, were obtained. This is a novel derivatization method yielding neutral derivatives that should be suitable for peptide sequencing by LC/ESI-MS/MS.     

Postcolumn derivatization liquid chromatography/mass spectrometry for detection of chemical-weapons-related compounds

Postcolumn derivatization for liquid chromatography/mass spectrometry (LC/MS) analysis was characterized for detection of some compounds related to chemical-weapons (CW) agents using an Atmospheric Pressure Chemical Ionization (APCI) source. The derivatizing reagents were added directly to the LC eluent flow, and the derivatization reactions occurred in the APCI source under typical operating conditions. The compound S-[2-(diisopropylamino)ethyl] methylphosphonothioic acid was methylated using the derivatizing reagent trimethylphenyl ammonium hydroxide (TMPAH). Methylphosphonic acid was doubly derivatized to form dimethyl methylphosphonate, although the signal for the derivatization product was very sensitive to the amount of TMPAH. Arsenic compounds related to the CW agent lewisite, including chlorovinyl arsonous acid and arsenic (III) oxide, were derivatized using 2-mercaptopyridine. The thiol group reacted readily with the arsenic (III) center and provided a significant improvement in sensitivity relative to the underivatized signal using APCI or electrospray ionization. Triethanolamine and ethyl diethanolamine were derivatized with benzoyl chloride, a commonly used LC derivatizing reagent for alcohols, to modify their mass spectra. Postcolumn derivatization using an APCI source gives an alternative for detecting some difficult-to-ionize compounds. It has the limitations that sensitivity was not always improved even though the major mass spectral peaks can be shifted; it is necessary to carefully select the reagent; and some reagents introduced strong interference peaks at specific masses in the spectrum and may suppress the ionization of some derivatized analyte ions. The reagent also produced contamination in the source, which had to be cleaned daily.     

Highly sensitive and positively charged precolumn derivatization reagent for amines and amino acids in liquid chromatography/electrospray ionization tandem mass spectrometry

We have developed a highly sensitive and positively charged precolumn derivatization reagent, (5‐N‐succinimidoxy‐5‐oxopentyl)triphenylphosphonium bromide (SPTPP), for amines and amino acids in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS). The handling of the derivatization reaction is quite simple and the reagent reacts with the analytes rapidly and with high efficiency. The derivatized analytes were observed to form regular and intense product ions upon MS/MS analysis; thus, highly sensitive and selective detection was possible in the selected reaction monitoring (SRM) mode. The limits of detection of the SPTPP‐derivatized analytes were less than sub‐femtomole levels. The sensitivities of the derivatized analytes increased about 500‐fold compared to those of underivatized analytes. Since the hydrophobicities of the samples increased after their derivatization, the resolution of the analytes improved dramatically when a reversed‐phase system was used. The relative standard deviations of intra‐day and inter‐day variations were below 10.6% and 13.3%, respectively. The accuracy ranged between 86.6–113% and 83.4–113%, respectively. Furthermore, the developed reagent was used for the analysis of the neurotransmitter 4‐aminobutanoic acid (GABA) and oxidative stress markers such as oxidized, nitrated, and halogenated tyrosines in rat serum. Copyright © 2010 John Wiley & Sons, Ltd.