Comparison of liquid chromatography-mass spectrometry interfaces for the analysis of polar metabolites of benz[a]pyrene

 The performance of two liquid chromatography-mass spectrometry (LC/MS) interfacing techniques, thermospray (TSP) and atmospheric pressure chemical ionization (APCI), for the analysis of benzo[a]pyrene (BaP) metabolites (hydroxy, epoxy and quinone derivatives) was compared. Interface and detection parameters such as source temperature, eluent composition or flow rate were optimized using negative ion mode. In TSP, the main ions are mostly [M]^-, [M−H₂O]^- or [M+CH₃COO]^-, whereas APCI gives mainly the [M]^- and [M−H]^- ions. Quantification was carried out by flow injection. Calibration graphs were linear in the range of 10 ng to 1000 ng in TSP and 0.1 ng to 10 ng in APCI. Detection limits were in the range of 1 ng to 20 ng in TSP and 0.002 ng to 0.2 ng in APCI. The presence of BaP-1,6-dione, BaP-3,6-dione, and BaP-6,12-dione was confirmed in environmental samples of air particulate matter. Received: 6 January 1997/Accepted: 18 April 1997     

Liquid chromatography-mass spectrometry with collision-induced dissociation of conjugated metabolites of benzol[a]pyrene

Benzo[a]pyrene (BP) metabolites conjugated with glutathione, cysteine-glycine, cysteine, N-acetylcysteine, and sulfuric and glucuronic acids have been studied by microcolumn liquid chromatography-electrospray mass spectrometry with collision-induced dissociation (CID) on a hybrid double focusing magnetic sector-orthogonal time-of-flight tandem mass spectrometer equipped with a focal plane array detector. Negative-ion electrospray mass spectra of the conjugated BP metabolites showed strong [M – H]^? ions. When the array detector was used, spectra were obtained from femtomoles of sample infused at mass resolutions of 5000 (full width at half maximum). Cone voltage fragmentation spectra show [M-H]^? ions and fragment ions indicative of the BP moiety and/or the conjugating group. Linked scan CID spectra at constant B/E were found to contain structurally informative product ions from infusion of as little as 1 pmol of sample. CID spectra were also recorded by using the double focusing sectors for precursor ion selection and the orthogonal time-of-flight analyzer for product ion mass separation. The method was applied to the analysis of conjugated BP metabolites in the urine of germ-free rats given a single intraperitoneal dose of BP.     

Rapid validated method for the analysis of benzo[a]pyrene in vegetable oils by using solid-phase microextraction-gas chromatography-mass spectrometry

A solid-phase microextraction method coupled with comprehensive gas chromatography and time-of-flight mass spectrometry for the determination of polycyclic aromatic hydrocarbons in vegetable oils has recently been reported. The present paper tested the possibility to use the solid-phase microextraction method coupled with one-dimensional gas chromatography-mass spectrometry of the only benzo[a]pyrene. Furthermore, an in-house validation for benzo[a]pyrene, used as a marker, as requested by the European regulation no. 208/2005, was carried out. Statistical tests were performed to elaborate the data. Linearity was satisfactory (r(2)=0.999), between about 0.5 and 15 microg/kg. Detection limit and quantification limit were 0.17 and 0.46 microg/kg, respectively. In-day and inter-day repeatability were less than 6% in both cases.     

Structure elucidation of drug metabolites using thermospray liquid chromatography-mass spectrometry

Thermospray liquid chromatography-mass spectrometry (LC-MS) has been used to provide structural information both from in vitro and in vivo experiments. This paper will describe the more salient aspects of the technique that have emerged. The ability of the interface to handle gradients was essential for its successful application to metabolism studies, owing to the wide range of compound polarity involved. The examples discussed in this paper include the use of LC-MS in the analysis of in vitro incubations of drugs with hepatocyte cell cultures and the direct analysis of plasma samples from in vivo studies in the dog.     

Determination of ketamine and metabolites in urine by liquid chromatography-mass spectrometry

We have developed an analytical method by using liquid chromatography-mass spectrometry (LC-MS) to determine ketamine and its metabolites in urine. The ionization efficiency between two ionization modes (ESI and APCI) of LC-MS was compared to each other. An easy and simple sample preparation of urine samples was made by passing samples through a 0.22 μm PVDF syringe filter. The results indicated that the ionization efficiency of positive APCI mode is better than positive ESI mode for determination of trace ketamines. A wide linearity range of the research is from 5 to 250 ng mL⁻¹ and the detection limits for ketamine, norketamine and dehydronorketamine were 0.95, 0.48 and 0.33 ng mL⁻¹, respectively. The proposed method was tested by analyzing ketamine and metabolites in the urines of volunteers. The concentrations of ketamine, norketamine and dehydronorketamine are ranged of 5.4-131.0, 12.5-74.1 and 22.8-278.9 ng mL⁻¹, respectively and the ketamines concentration profiles in human urine were also determined. The results demonstrate the suitability of liquid chromatography-mass spectrometry approach to analyze trace amount of ketamine and its metabolites in urine.     

Determination of dithiocarbamates and metabolites in plants by liquid chromatography-mass spectrometry

A quantitative matrix solid-phase dispersion and liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) method is outlined for the simultaneous analysis of dithiocarbamates (DTCs) and their degradation products in plants. Compounds analyzed are dazomet, disulfiram, thiram and the metabolites ethylenthiourea and propylenthiourea. The performance of two different sample preparation protocols, the proposed one and other based on solid-phase extraction, as well as, of both atmospheric pressure ionization sources, APCI and electrospray, were compared. The effect of several parameters on the extraction, separation and detection was studied. Dithiocarbamates and metabolites were dispersed with carbograph, eluted with a mixture of dichloromethane-methanol, and then, identified by monitoring the base peak of the spectra corresponding to [M + H]+. The method was validated for avocados, cherries, lemons, nuts, oat, oranges, peaches, rice and tomatoes. Average recoveries varied from 33 to 109%, and relative standard deviation were between 4 and 21% with limits of quantification ranged from 0.25 to 2.5 mg kg(-1), except for thiram and disulfiram, which were not recovered from fruits with high acid content. The procedure was applied to the determination of DTCs and their metabolites in fruits, vegetables and cereals taken from different markets of Valencia, Spain.     

Liquid chromatography-mass spectrometry and strategies for trace-level analysis of polar organic pollutants

Liquid chromatography–mass spectrometry using atmospheric pressure ionization (LC–API-MS) has drastically changed the analytical methods used to detect polar pollutants in water. The present status of application of this technique to organic water constituents is reviewed. The selection of the appropriate LC conditions, whether reversed-phase liquid chromatography, ion-pair chromatography, capillary electrophoresis or ion chromatography, and of the most sensitive ionization mode, electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), depends upon the polarity and acidity of the analytes. Strongly acidic compounds such as aromatic sulfonates, sulfonated dyes, haloacetic acids, linear alkylbenzene sulfonates, aliphatic sulfonates and sulfates and complexing agents, weakly acidic compounds such as carboxylates and phenols, neutral compound classes, namely alkylphenol ethoxylates, alcohol ethoxylates and polycyclic aromatic hydrocarbons and the basic toxins, quaternary ammonium compounds and organometallic compounds are considered. The selection of the mass spectrometer depends upon the analytical task: triple-quadrupole mass spectrometers are highly suited for sensitive quantitation and for qualitative analyses, ion traps are especially suited for structure elucidation, whereas time-of-flight mass spectrometers and quadrupole time-of-flight mass spectrometers with their higher mass resolution are ideal for the determination of molecular formulas of unknown compounds and for screening purposes. While large steps have already been made, future efforts with respect to water analysis may be directed at fine-tuning the methodical arsenal for increased sensitivity and selectivity and to extend LC–MS application to transformation products.     

Preliminary experiments for the analysis of tetrahydrotetrols of benz[a]anthracene and benzo[a]pyrene derived from their hemoglobin adducts using a coupled-column high-performance liquid chromatographic system

Improved technologies for the detection of polycyclic aromatic hydrocarbon adducts are required for human biomonitoring. Therefore, a coupled-column high-performance liquid chromatographic method, with system-integrated sample processing, has been developed and its applicability for determination of tetrahydrotetrols of polycyclic aromatic hydrocarbons in acid hydrolysates of human hemoglobin has been investigated. A novel column-switching technique applying ‘thermotransfer’ is used to separate tetrahydrotetrols of benzo[a]pyrene and benz[a]anthracene more efficiently. Derivatives of polycyclic aromatic hydrocarbons from blood hydrolysates are concentrated on a pre-column and then transferred to the analytical column by applying an electrical current to heat the solvent eluting the pre-column. This method allows for rapid and quantitative transfer of the analytes from the pre-column to the analytical column, after HPLC-integrated sample processing.     

古菌脂质的前处理方法比较及超高效液相色谱-高分辨质谱分析

古菌是一类兼具真菌及细菌细胞特征、大多生活在极端环境下的单细胞微生物。由于膜结构及代谢通路的特殊性,研究古菌脂质对深入探究极端环境下生命的正常活动具有重要意义。前处理方法在组学分析中会极大影响最终结果的准确度及可靠性。该文基于超高效液相色谱-高分辨质谱联用(ultra-performance liquid chromatography-high resolution mass spectrometry, UPLC-HRMS)技术,以嗜热嗜压古菌Pyrococcus yayanosii为模式生物,采用Bligh-Dyer加酸法、Folch法、甲基叔丁基醚(methyl tert-butyl ether, MTBE)法及固相萃取(SPE)法提取脂质成分,从提取效率、重复性、歧视性等角度进行方法评价,并最终确定SPE法和MTBE法的提取重复性和提取效率最好,适合高通量古菌全脂质提取。利用SPE提取、高分辨质谱分析对P.yayanosii的常规脂质进行了全面分析,共鉴定到了1 402种脂质。本研究旨在为古菌和其他极端微生物的非靶向脂质组学分析和脂质代谢研究提供方法参考。     

Tentative identification of new metabolites of epimedin C by liquid chromatography-mass spectrometry

Epimedin C is one of the major bioactive constituents of Herba Epimedii. The aim of this study is to characterize and elucidate the structure of metabolites in the rat after administration of epimedin C. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) scan in positive ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer. A total of 18 metabolites were characterized by the changes in their protonated molecular masses, their MS/MS spectrum and their retention times compared with those of the parent drug. The results reveal possible metabolite profiles of epimedin C in rats; the metabolic pathways including hydrolysis, hydroxylation, dehydrogenation, demethylation and conjugation with glucuronic acid and different sugars were observed. This study provides a practical approach for rapidly identifying complicated metabolites, a methodology that could be widely applied for the structural characterization of metabolites of other compounds.     

Structural elucidation of metabolites of ritonavir and indinavir by liquid chromatography-mass spectrometry

The structural elucidation of metabolites of ritonavir and indinavir, HIV-protease inhibitor drugs, by liquid chromatography-electrospray ionization mass spectrometry is described. Ritonavir and indinavir were biotransformed separately by incubation with transplant quality human liver microsomes. The incubation mixture was then analyzed by HPLC coupled to ion trap (ITMS) and triple quadrupole mass analyzers. The metabolites retained most of the structural features of the parent molecules. Baseline chromatographic resolution of isobaric species by gradient elution HPLC permitted rapid structural identification of these metabolites. Both drugs were biotransformed primarily by oxidative and hydrolytic pathways to numerous metabolites that retained many of the features of the parent molecules. Triple quadrupole and ion trap mass spectrometry were applied jointly to thoroughly detect and thoroughly characterize these metabolites. Furthermore, retention-time and data-dependent scanning assured acquisition of detailed MS-MS spectra for rapid detection of metabolic pathways of ritonavir and indinavir. Comparison of the ITMS and triple quadrupole data showed qualitative and quantitative differences in the mass spectral patterns, suggesting that these instruments should be used in parallel to ensure comprehensive metabolite detection and characterization by LC-MS.     

Characterization of in vivo metabolites of toad venom using liquid chromatography-mass spectrometry

The structures of in vivo metabolites of marinobufotoxin (marinobufagin 3-suberoylarginine ester), marinobufagin, or bufalin which are typical components of toad venom widely used as the traditional Chinese drug, Ch'an Su, are confirmed using authentic samples based on their liquid chromatography-mass spectrometric behavior. A rat is orally administered 2 mg of the previously mentioned components of toad venom, the serum is collected 30 min after the administration, extracted, and then characterized. Marinobufotoxin is hydrolyzed and further epimerized into 3-epimarinobufagin, but marinobufagin 3-hemisuberate is not detected. After the administration of marinobufagin, 3-epimarinobufagin is detected in both the male and female rats, but marinobufagin 3-sulfate is formed only in the female rats. Bufalin is metabolized to 3-epibufalin, which is found to undergo further conjugation resulting in its 3-glucuronide. Furthermore, 3-epibufalin 3-sulfate is formed only in the female rats.     

Synthesis of benz[e] indole and benz[g] indole carboxaldehydes

The Vilsmeier-Haack formylation reaction and several additional reactions have been employed to prepare a variety of benz[e]- and benz[g] indole-2- and 3-carboxaldehydes. Similar conditions have been utilized to prepare several 2,3-disubstituted benz[g] indole-5-carboxaldehydes.     

Elucidation of urinary metabolites of fluoxymesterone by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry

The suitability of liquid chromatography tandem mass spectrometry (LC-MS/MS) and gas chromatography mass spectrometry (GC-MS) for the elucidation of fluoxymesterone metabolism has been evaluated. Electrospray ionization (ESI) and collision induced dissociation (CID) fragmentation in LC-MS/MS and electron impact spectra (EI) in GC-MS have been studied for fluoxymesterone and two commercially available metabolites. MS(n) experiments and accurate mass measurements performed by an ion-trap analyser and a QTOF instrument respectively have been used for the elucidation of the fragmentation pathway. The neutral loss scan of 20 Da (loss of HF) in LC-MS/MS has been applied for the selective detection of fluoxymesterone metabolites. In a positive fluoxymesterone doping control sample, 9 different analytes have been detected including the parent compound. Seven of these metabolites were also confirmed by GC-MS including 5 previously unreported metabolites. On the basis of the ionization, the CID fragmentation, the accurate mass of the product ions and the EI spectra of these analytes, a tentative elucidation as well as a proposal for the metabolic pathway of fluoxymesterone has been suggested. The presence of these compounds has also been confirmed by the analysis of five other positive fluoxymesterone urine samples.     

Analysis of hydrophilic metabolites by high-performance liquid chromatography-mass spectrometry using a silica hydride-based stationary phase

A novel silica hydride-based stationary phase was used to evaluate the retention behavior in the aqueous normal-phase (ANP) mode of standards representing three classes of metabolites. The effects on retention behavior of amino acids, carbohydrates and small organic acids were examined by altering the column temperature, and by adding different additives to both the mobile phase and sample solvent. Gradient mode results revealed the repeatability of retention times to be very stable for these compound classes. At both 15 and 30 degrees C, excellent RSD values were obtained with less than 1% variation for over 50 injections of an amino acid mixture. The ability to separate the 19 nonderivatized amino acid standards, organic acids and carbohydrates was demonstrated as well as the potential for this material to separate polar metabolites in complex fluids such as urine.     

Urinary excretion profiles of toremifene metabolites by liquid chromatography-mass spectrometry. Towards targeted analysis to relevant metabolites in doping control

In the present study, toremifene urinary excretion studies were evaluated in order to examine main metabolic reactions and to select target metabolites in doping control analysis. Urine samples from three female subjects were collected every 3 h for at least 15 days after the oral administration of a single dose of Fareston® (60 mg). The elemental compositions of the compounds detected were determined by liquid chromatography-mass spectrometry using a time-of-flight system with accurate mass measurement. More detailed structure elucidation was obtained by monitoring the presence or absence of structure-specific ions, using product ion scan and neutral loss acquisition modes, whereas the metabolites urinary profiles were evaluated in selected reaction monitoring acquisition mode. The results showed that the main routes of phase-I modifications involved carboxylation of the chlorinated side chain, N-demethylation and hydroxylation in different positions. Fifteen metabolites were found in all subjects studied, most of them were detected for more than 10 days in the free, glucuronide and sulphate fractions, with a maximum of excretion generally after 9–22 and 34–47 h from drug administration. These metabolites can be divided in two groups: metabolites with the characteristic chlorine isotope pattern and metabolites without the characteristic chlorine isotope pattern. The most abundant and long-term compounds were the carboxylated metabolites followed by the hydroxylated metabolites. Their product ions originating after collision-induced dissociation were observed to occur prevalently in the dimethylaminoethoxy and in the chlorinated side chains. These structure-specific ions were used to design screening and confirmation procedures to positively identify toremifene administration in doping control analysis.

Figure Suggested main metabolic routes of toremifene, as postulated by excretion studies followed by both LC-MS/MS assays with different acquisition modes and LC-QTOF

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Supersonic jet fluorescence spectrometry of perylene and benzo[a]pyrene

A supersonic jet instrument for fluorescence spectrometry is described. It consists of a high-temperature free expansion nozzle for continuous sample introduction and a vacuum chamber equipped with a high-speed pumping system. Rotationally cooled spectra obtained with the supersonic jet are compared with gas-phase spectra measured at high temperature for perylene and benzo[a]pyrene molecules. Each component of the unresolved band structure in the high-temperature spectra was found to be composed of a rotational congestion of several vibrational bands. For a 1:1 mixture of perylene and benzo[a]pyrene, selective detection is possible by using supersonic jet spectrometry. The detection limit for perylene is 100 ng. The advantage of this technique over other low-temperature spectrometric methods based on Shpol'skii and matrix isolation effects are discussed.     

Micelle-sensitized constant-energy synchronous fluorescence spectrometry for the simultaneous determination of pyrene, benzo[a]pyrene and perylene.

The use of micellar media in constant-energy synchronous fluorescence spectrometry has been proposed. The influence of some aqueous micellar systems on the determination of pyrene, perylene and benzo[a]pyrene has been investigated. The presence of these micellar systems allows their determination in aqueous media, thus avoiding the use of an organic solvent, and greatly enhances the fluorescence signals. The combination of a constant-energy synchronous scanning technique and a micellar system provided a single spectrum for the simultaneous identification and quantitative determination of the three polycyclic aromatic hydrocarbons (PAHs). Further there was no energy transfer among them, making the measurement simple and fast. A constant-energy difference of 2800 cm(-1) was selected. The analytical characteristics of the proposed method in the presence of anionic micelles of sodium dodecylsulfate (SDS) were studied. The detection limits were at a level of ng ml(-1). Analysis of water samples from two different origins spiked with known amount of pyrene, perylene and benzo[a]pyrene also gave satisfactory results, and total average recoveries were greater than 97.1%.