Identification of new phase II metabolites of xanthohumol in rat in vivo biotransformation of hop extracts using high-performance liquid chromatography electrospray ionization tandem mass spectrometry

Polyphenolic compounds occurring in hop extracts and their phases I and II metabolites formed during in vivo rat biotransformation have been analyzed using HPLC/MS/MS with electrospray ionization (ESI). Two main groups of polyphenolics are present in the hops, i.e., xanthohumol related compounds and so called α- and β-bitter acids (humulones and lupulones). In our study, hybrid quadrupole-time-of-flight (QqTOF) analyzer is used for the identification of both natural phenolics and their metabolites due to the possibility of accurate mass measurements in full scan and tandem mass spectra supported by MSⁿ data obtained with the ion trap analyzer. Both ESI polarity modes are used for the determination of molecular weights based on [M+H]⁺ and [M−H]⁻ ions in the full scan spectra and the structural information in subsequent tandem mass spectra. The emphasis is given on the elemental composition determination of individual metabolites based on accurate masses typically better than 5 ppm even with the external calibration. Advanced software tools are used for the metabolite identification using the comparison of the blank chromatogram with the real incubation sample together with the software prediction and detection of possible metabolites. Chromatograms of rat incubations are also compared with chromatograms of pure rat feed, rat feed enriched with hop extracts and the placebo experiment. More than ten compounds originating from the hops are identified in rat feces, two of them belong to phase I metabolites and five compounds are phase II metabolites.     

High-performance liquid chromatography–tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites

Applications of tandem mass spectrometry (MS/MS) techniques coupled with high-performance liquid chromatography (HPLC) in the identification and determination of phase I and phase II drug metabolites are reviewed with an emphasis on recent papers published predominantly within the last 6 years (2002–2007) reporting the employment of atmospheric pressure ionization techniques as the most promising approach for a sensitive detection, positive identification and quantitation of metabolites in complex biological matrices. This review is devoted to in vitro and in vivo drug biotransformation in humans and animals. The first step preceding an HPLC-MS bioanalysis consists in the choice of suitable sample preparation procedures (biomatrix sampling, homogenization, internal standard addition, deproteination, centrifugation, extraction). The subsequent step is the right optimization of chromatographic conditions providing the required separation selectivity, analysis time and also good compatibility with the MS detection. This is usually not accessible without the employment of the parent drug and synthesized or isolated chemical standards of expected phase I and sometimes also phase II metabolites. The incorporation of additional detectors (photodiode-array UV, fluorescence, polarimetric and others) between the HPLC and MS instruments can result in valuable analytical information supplementing MS results. The relation among the structural changes caused by metabolic reactions and corresponding shifts in the retention behavior in reversed-phase systems is discussed as supporting information for identification of the metabolite. The first and basic step in the interpretation of mass spectra is always the molecular weight (MW) determination based on the presence of protonated molecules [M+H]⁺ and sometimes adducts with ammonium or alkali-metal ions, observed in the positive-ion full-scan mass spectra. The MW determination can be confirmed by the [M-H]^- ion for metabolites providing a signal in negative-ion mass spectra. MS/MS is a worthy tool for further structural characterization because of the occurrence of characteristic fragment ions, either MS ⁿ analysis for studying the fragmentation patterns using trap-based analyzers or high mass accuracy measurements for elemental composition determination using time of flight based or Fourier transform mass analyzers. The correlation between typical functional groups found in phase I and phase II drug metabolites and corresponding neutral losses is generalized and illustrated for selected examples. The choice of a suitable ionization technique and polarity mode in relation to the metabolite structure is discussed as well.     

Low resolution and high resolution MS for studies on the metabolism and toxicological detection of the new psychoactive substance methoxypiperamide (MeOP)

In 2013, the new psychoactive substance methoxypiperamide (MeOP) was first reported to the European Monitoring Centre for Drug and Drug Addiction. Its structural similarity to already controlled piperazine designer drugs might have contributed to the decision to offer MeOP for online purchase. The aims of this work were to identify the phase I/II metabolites of MeOP in rat urine and the human cytochrome P450 (CYP) isoenzymes responsible for the initial metabolic steps. Finally, the detectability of MeOP in rat urine by gas chromatography–mass spectrometry (GC‐MS) and liquid chromatography coupled with multistage mass spectrometry (LC‐MSⁿ) standard urine screening approaches (SUSAs) was evaluated. After sample preparation by cleavage of conjugates followed by extraction for elucidating phase I metabolites, the analytes were separated and identified by GC‐MS as well as liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). For detection of phase II metabolites, the analytes were separated and identified after urine precipitation followed by LC‐HR‐MS/MS. The following metabolic steps could be postulated: hydrolysis of the amide, N‐oxide formation, N‐ and/or O‐demethylation, oxidation of the piperazine ring to the corresponding keto‐piperazine, piperazine ring opening followed by oxidation of a methylene group to the corresponding imide, and hydroxylation of the phenyl group. Furthermore, N‐acetylation, glucuronidation and sulfation were observed. Using human CYPs, CYP1A2, CYP2C19, CYP2D6, and/or CYP3A4 were found to catalyze N‐oxide formation and N‐, O‐demethylation and/or oxidation. Mostly MeOP and N‐oxide‐MeOP but to a minor degree also other metabolites could be detected in the GC‐MS and LC‐MSⁿ SUSAs. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of ginsenoside compound K metabolites in rat urine and feces by ultra‐performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Ginsenoside compound K (CK) is an active metabolite of ginsenoside and has been shown to have ameliorative property in various diseases. However, the detailed in vivo metabolism of this compound has rarely been reported. In the present study, a method using liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry together with multiple data processing techniques, including extracted ion chromatogram, multiple mass defect filter and MS/MS scanning, was developed to detect and characterize the metabolites of CK in rat urine and feces. After oral administration of CK at a dose of 50 mg/kg, urine and feces were collected for a period of time and subjected to a series of pretreatment. A total of 12 metabolites were tentatively or conclusively identified, comprising 11 phase I metabolites and a phase II metabolite. Metabolic pathways of CK has been proposed, including oxidation, deglycosylation, deglycosylation with sequential oxidation and dehydrogenation and deglycosylation with sequential glucuronidation. Relative quantitative analyses suggested that deglycosylation was the main metabolic pathway. The result could offer insights for better understanding of the mechanism of its pharmacological activities.     

Determination of triclosan metabolites by using in‐source fragmentation from high‐performance liquid chromatography/negative atmospheric pressure chemical ionization ion trap mass spectrometry

Triclosan is a widely used broad‐spectrum antibacterial agent that acts by specifically inhibiting enoyl–acyl carrier protein reductase. An in vitro metabolic study of triclosan was performed by using Sprague‐Dawley (SD) rat liver S9 and microsome, while the in vivo metabolism was investigated on SD rats. Twelve metabolites were identified by using in‐source fragmentation from high‐performance liquid chromatography/negative atmospheric pressure chemical ionization ion trap mass spectrometry (HPLC/APCI‐ITMS) analysis. Compared to electrospray ionization mass spectrometry (ESI‐MS) and tandem mass spectrometry (MS/MS) that gave little fragmentation for triclosan and its metabolites, the in‐source fragmentation under APCI provided intensive fragmentations for the structural identifications. The in vitro metabolic rate of triclosan was quantitatively determined by using HPLC/ESI‐ITMS with the monitoring of the selected triclosan molecular ion. The metabolism results indicated that glucuronidation and sulfonation were the major pathways of phase II metabolism and the hydroxylated products were the major phase I metabolites. Moreover, glucose, mercapturic acid and cysteine conjugates of triclosan were also observed in the urine samples of rats orally administrated with triclosan. Copyright © 2010 John Wiley & Sons, Ltd.     

In vivo metabolism study of rhubarb decoction in rat using high-performance liquid chromatography with UV photodiode-array and mass-spectrometric detection: a strategy for systematic analysis of metabolites from traditional Chinese medicines in biological samples

High-performance liquid chromatography with diode-array detection (HPLC-DAD) and tandem mass spectrometry (HPLC-MS/MS) was used for separation and identification of metabolites in rat urine, bile and plasma after oral administration of rhubarb decoction. Based on the proposed strategy, 91 of the 113 potential metabolites were tentatively identified or characterized. Besides anthraquinones metabolites, gallic acid, (-)-epicatechin and (+)-catechin metabolites were also detected and characterized in these biological samples. Our results indicated that glucuronidation and sulfation were the main metabolic pathways of anthraquinones, while methylation, glucuronidation and sulfation were the main metabolic pathways of gallic acid, (-)-epicatechin and (+)-catechin. Phase I reactions (e.g., hydroxylation and reduction) played a relatively minor role compared to phase II reactions in metabolism of phenolic compounds of rhubarb decoction. The identification and structure elucidation of these metabolites provided essential data for further pharmacological and clinical studies of rhubarb and related preparations. Moreover, the results of the present investigations clearly indicated the relevance and usefulness of the combination of chromatographic, spectrophotometric, and mass-spectrometric analysis to detect and identify metabolites.     

Metabolite profiling of l‐isocorypalmine in rat urine,plasma, and feces after oral administration using high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry

l ‐Isocorypalmine, an active alkaloid compound isolated from Rhizoma Corydalis yanhusuo, has been reported to possess biological activity for treating cocaine use disorder. A high‐performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry method was established for identification of the metabolites of l ‐isocorypalmine in urine, plasma and feces samples of rats after a single intragastric gavage of l ‐isocorypalmine at a dose of 15 mg/kg. As a result, a total of 21 metabolites (six phase ? metabolites and fifteen phase II metabolites) were detected and tentatively identified by mass spectrometry and fragment ions from tandem mass spectrometry spectra. All metabolites were present in the urine samples, nine metabolites were found in the plasma samples and three metabolites were found in the feces samples. Results indicated that metabolic pathways of l ‐isocorypalmine included oxidation, dehydrogenation, demethylation, sulfate conjugation, and glucuronide conjugation. In addition, glucuronidation was the major metabolic reaction. Results of this investigation could provide significant experimental basis for efficacy, safety and action mechanism of l ‐isocorypalmine, which will be advantageous to new drug development for treating cocaine addiction.     

Application of on-line nano-liquid chromatography/mass spectrometry in metabolite identification studies

Metabolite identification is an important part of the drug discovery and development process. High sensitivity is necessary to identify metabolic products in vitro and in vivo. The most common method utilizes standard high-performance liquid chromatography (4.6 mm i.d. column and 1 mL/min flow rate) coupled to tandem mass spectrometry (HPLC/MS/MS). We have developed a method that utilizes a nano-LC system coupled to a high-resolution tandem mass spectrometer to identify metabolites from in vitro and in vivo samples. Using this approach, we were able to increase the sensitivity of analysis by approximately 1000-fold over HPLC/MS. In vitro samples were analyzed after simple acetonitrile precipitation, centrifugation, and dilution. The significant improvement in sensitivity enabled us to conduct experiments at very low substrate concentrations (0.01 μM), and very low incubation volumes (20 μL). In vivo samples were injected after simple dilution without any pre-purification. All the metabolites identified by conventional HPLC/MS/MS were also identified using the nano-LC method. This study demonstrates a very sensitive approach to identifying phase I and II metabolites with throughput and separation equivalent to the standard HPLC/MS/MS method.     

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

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

Metabolites identification of Huo Luo Xiao Ling Dan in rat urine by UPLC coupled with electrospray ionization time‐of‐flight mass spectrometry

Huo Luo Xiao Ling Dan (HLXLD), a Chinese herbal formula, is used in folk medicine for the treatment of arthritis and other chronic inflammatory diseases. However, the in vivo integrated metabolism of its multiple components remains unknown. In this paper, an ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐Q‐TOF‐MS) method was developed for detection and identification of HLXLD metabolites in rat urine at high and normal clinical dosages. The prototype constituents and their metabolites in urine were analyzed. The mass measurements were accurate within 8 ppm, and subsequent fragment ions offered higher quality structural information for interpretation of the fragmentation pathways of various compounds. A total of 85 compounds were detected in high dosages urine samples by a highly sensitive extracted ion chromatograms method, including 31 parent compounds and 54 metabolites. Our results indicated that phase 2 reactions (e.g. glucuronidation, glutathionidation and sulfation) were the main metabolic pathways of lactones, alkaloids and flavones, while phase I reactions (e.g. hydrogenation and hydroxylation) were the major metabolic reaction for coumarins, paeoniflorin and iridoids. This investigation provided important structural information on the metabolism of HLXLD and provided scientific evidence to obtain a more comprehensive metabolic profile. Copyright © 2015 John Wiley & Sons, Ltd.     

Automated software-guided identification of new buspirone metabolites using capillary LC coupled to ion trap and TOF mass spectrometry

The identification and structure elucidation of drug metabolites is one of the main objectives in in vitro ADME studies. Typical modern methodologies involve incubation of the drug with subcellular fractions to simulate metabolism followed by LC-MS/MS or LC-MS(n) analysis and chemometric approaches for the extraction of the metabolites. The objective of this work was the software-guided identification and structure elucidation of major and minor buspirone metabolites using capillary LC as a separation technique and ion trap MS(n) as well as electrospray ionization orthogonal acceleration time-of-flight (ESI oaTOF) mass spectrometry as detection techniques.Buspirone mainly underwent hydroxylation, dihydroxylation and N-oxidation in S9 fractions in the presence of phase I co-factors and the corresponding glucuronides were detected in the presence of phase II co-factors. The use of automated ion trap MS/MS data-dependent acquisition combined with a chemometric tool allowed the detection of five small chromatographic peaks of unexpected metabolites that co-eluted with the larger chromatographic peaks of expected metabolites. Using automatic assignment of ion trap MS/MS fragments as well as accurate mass measurements from an ESI oaTOF mass spectrometer, possible structures were postulated for these metabolites that were previously not reported in the literature.     

The identification of three human metabolites of a peptide-doxorubicin conjugate using HPLC-MS-MS in positive and negative ionization modes

A peptide-doxorubicin conjugate (I) is a drug candidate that has been evaluated for the treatment of prostate cancer. During the high-performance liquid chromatographic (HPLC)-fluorescence analysis of clinical samples for compound I and its two known metabolites [doxorubicin (II) and leucine-doxorubicin (III)], additional metabolites are observed in postdose human plasma extracts. Using neutral loss, precursor, and product ion tandem mass spectrometric (MS-MS) experiments, two of these metabolites are identified as doxorubicinol (IV) and leucine-doxorubicinol (V), the active 13-hydroxy metabolites of doxorubicin and leucine-doxorubicin, respectively. A third metabolite, 7-deoxydoxorubicinol aglycone (VI), is detected using single-ion monitoring at m/z 399 in the negative ionization mode. The product ion mass spectrum of this metabolite contains a major fragment at m/z 351, resulting from the loss of water and formaldehyde from the pseudomolecular ion. An HPLC-MS-MS method for simultaneous analysis of II, III, IV, V, and VI is developed utilizing gradient HPLC with a combination of positive/negative ionization MS in the multiple reaction monitoring mode and monitoring the appropriate MS-MS transitions. Using this methodology, rat, dog, and human plasma metabolite profiles are compared and found to be qualitatively similar. Simultaneous fluorescence and MS detection experiments confirm that the peaks observed in the HPLC-fluorescence chromatograms of plasma extracts correspond to each of the five metabolites (II-VI).     

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

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

Rapid identification of phase I and II metabolites of artemisinin antimalarials using LTQ‐Orbitrap hybrid mass spectrometer in combination with online hydrogen/deuterium exchange technique

Artemisinin drugs have become the first‐line antimalarials in areas of multi‐drug resistance. However, monotherapy with artemisinin drugs results in comparatively high recrudescence rates. Autoinduction of CYP‐mediated metabolism, resulting in reduced exposure, has been supposed to be the underlying mechanism. To better understand the autoinduction of artemisinin drugs, we evaluated the biotransformation of artemisinin, also known as Qing‐hao‐su (QHS), and its active derivative dihydroartemisinin (DHA) in vitro and in vivo, using LTQ‐Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange high‐resolution (HR)‐LC/MS (mass spectrometry) for rapid structural characterization. The LC separation was improved allowing the separation of QHS parent drugs and their metabolites from their diastereomers. Thirteen phase I metabolites of QHS have been identified in liver microsomal incubates, rat urine, bile and plasma, including six deoxyhydroxylated metabolites, five hydroxylated metabolites, one dihydroxylated metabolite and deoxyartemisinin. Twelve phase II metabolites of QHS were detected in rat bile, urine and plasma. DHA underwent similar metabolic pathways, and 13 phase I metabolites and 3 phase II metabolites were detected. Accurate mass data were obtained in both full‐scan and MS/MS mode to support assignments of metabolite structures. Online H/D exchange LC‐HR/MS experiments provided additional evidence in differentiating deoxydihydroxylated metabolites from mono‐hydroxylated metabolites. The results showed that the main phase I metabolites of artemisinin drugs are hydroxylated and deoxyl products, and they will undergo subsequent phase II glucuronidation processes. This study also demonstrated the effectiveness of online H/D exchange LC‐HR/MSⁿ technique in rapid identification of drug metabolites. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

Identification of metabolites of isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate in rats by high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate (IDHP) is an investigational new drug having the capacity for treating ailments in the cardiovascular and cerebrovascular system. In this work, a rapid and sensitive method using high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐ESI‐Q‐TOF‐MS) was developed to reveal the metabolic profile of IDHP in rats after oral administration. The method involved pretreatment of the samples by formic acid–methanol solution (v/v, 5:95), chromatographic separation by an Agilent Eclipse XDB‐C₁₈ column (150 × 4.6 mm i.dx., 5 μm) and online identification of the metabolites by Q‐TOF‐MS equipped with electrospray ionizer. A total of 16 metabolites from IDHP, including four phase I metabolites and 12 phase II metabolites, were detected and tentatively identified from rat plasma, urine and feces. Among these metabolites, Danshensu (DSS), a hydrolysis product of IDHP, could be further transformed to 11 metabolites. These results indicated that DSS was the main metabolite of IDHP in rats and the major metabolic pathways of IDHP in vivo were hydrolysis, O‐methylation, sulfation, glucuronidation and reduction. The results also demonstrated that renal route was the main pathway of IDHP clearance in rat. The present study provided valuable information for better understanding the efficacy and safety of IDHP. Copyright © 2015 John Wiley & Sons, Ltd.     

Ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry for robust, high-throughput quantitative analysis of an automated metabolic stability assay, with simultaneous determination of metabolic data

The application of ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC/TOFMS) for high-throughput analysis of a 96-well plate based metabolic stability assay has been investigated. Full-scan data were acquired, with run times of 2.5-3.5 min, from which narrow window extracted ion chromatograms were generated, producing quantitative data for the test compound equivalent to that obtained by high-performance liquid chromatography with tandem mass spectrometric detection on a triple quadrupole instrument (HPLC/MS/MS). Sensitivity and mass accuracy were maintained over the analysis of >300 samples. Additionally, the UPLC/TOFMS datasets obtained gave access to metabolic route information, at no cost in terms of sensitivity for the test compound.     

Determination of methotrexate in environmental samples by solid phase extraction and high performance liquid chromatography: ultraviolet or tandem mass spectrometry detection?

In order to assess exposure levels of hospital personnel involved in the preparation and administration of antineoplastic drugs, environmental monitoring should be carried out. Wipe samples, pads, gloves and air samples should be collected at the end of each work shift, properly treated and then analysed using instrumental techniques which are sufficiently sensitive and specific to detect even trace amounts of drug. In this study, a method using high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS), incorporating solid phase extraction (SPE), was validated for determination of methotrexate (MTX) in wipe and air samples. Each step of the method was first developed and optimised using ultraviolet detection (UV), and afterwards tandem mass spectrometry was used to obtain a lower limit of quantitation when the expected drug level was less than the analytical UV detection limit. SPE enabled a 20-fold preconcentration of the analyte when using HPLC/UV and a further 30-fold preconcentration was obtained when analysing samples by HPLC/MS/MS. For example, the limit of quantitation (LLQ) was lowered from 3000 ng on wipe (direct injection onto an HPLC/UV system) to 5 ng on wipe (SPE plus HPLC/MS/MS). 7-hydroxymethotrexate was used as internal standard to assess precision and accuracy.     

Metabolites of an orally active antimicrobial prodrug, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime, identified by liquid chromatography/tandem mass spectrometry

DB75 (2,5-bis(4-amidinophenyl)furan) is a promising antimicrobial agent against African trypanosomiasis and Pneumocystis carinii pneumonia. However, it suffers from poor oral activity in rodent models for both infections. In contrast, a novel prodrug of DB75, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), has excellent oral activity. DB289 is currently undergoing clinical investigation as a candidate drug to treat primary stage African trypanosomiasis and Pneumocystis carinii pneumonia. In this study, metabolites of DB289 formed after incubation with freshly isolated rat hepatocytes were characterized using liquid chromatography/ion trap mass spectrometry. Administration of DB289 and octadeuterated DB289 in a 1 : 1 mixture greatly facilitated metabolite identification by providing isotope patterns with twin ions separated by 8 m/z units in the ratio 1 : 1, in the extracted ion chromatograms of molecular ions and in the product ion mass spectra of metabolites. Ten metabolites were identified. Series of O-demethylations and N-dehydroxylations led to the metabolic activation of DB289 to DB75 with the production of four intermediate phase I metabolites. Phase II glucuronidation and sulfation led to the formation of four glucuronide and one sulfate metabolites.     

Mass spectrometrically detected directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry for the identification of xenobiotic metabolites in maize plants

Reconstructed ion chromatograms have been used to identify relevant high performance liquid chromatography (HPLC) peaks in a directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry (HPLC/NMR/MS) experiment. This has been applied to a study of the metabolism of a model compound, 5-nitropyridone (2-hydroxy-5-nitropyridine), in maize plants grown hydroponically. By monitoring the on-flow reconstructed ion chromatogram corresponding to the 5-nitropyridone fragment at m/z 143, and additional molecular ions corresponding to metabolites identified as products from similar compounds, relevant peaks were identified rapidly for subsequent stopped-flow 1H NMR spectroscopic analysis. The combination of coupled HPLC/NMR/MS enabled the direct identification of three metabolites, namely the N-glucoside, N-malonylglucoside, and O-malonylglucoside. This work demonstrates the power of HPLC/NMR/MS for the structural elucidation of xenobiotic metabolites in complex biological matrices (such as plant material) with minimal sample preparation. In particular, using mass spectrometry for the initial identification of relevant HPLC peaks allows the analysis of complex samples without the necessity for other spectroscopic markers, such as 19F NMR signal for fluorinated compounds or UV spectroscopy for molecules with strong UV chromophores.