Determination of the metabolites of gestrinone in human urine by high performance liquid chromatography, liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

Gestrinone was studied by high performance liquid chromatography (HPLC) for screening and by gas chromatography/mass spectrometry (GC/MS) for confirmation. When the chromatograms of blank, spiked urine and dosed urine were compared by HPLC, two unknown metabolites were found and these were excreted as the conjugated forms. Metabolites 1 and 2 were tested by LC/MS and LC/MS/MS and both had parent ions at m/z 325. The fragment ion of metabolite 1 was at m/z 263 and ions for metabolite 2 were m/z 307 [MH - H(2)O](+), 289, 279 and 241. LC/MS/MS of m/z 263 as the parent ion of metabolite 1 gave fragment ions at m/z 245 and 217, which were assumed to be [263 - H(2)O](+) and [235 - H(2)O](+), respectively. The trimethylsilyl (TMS)-enol-TMS ether derivative of gestrinone displayed three peaks in its GC/MS chromatogram, formed by tautomerism.     

Application of a linear ion trap/orbitrap mass spectrometer in metabolite characterization studies: Examination of the human liver microsomal metabolism of the non-tricyclic anti-depressant nefazodone using data-dependent accurate mass measurements

We report herein, facile metabolite identification workflow on the anti-depressant nefazodone, which is derived from accurate mass measurements based on a single run/experimental analysis. A hybrid LTQ/orbitrap mass spectrometer was used to obtain accurate mass full scan MS and MS/MS in a data-dependent fashion to eliminate the reliance on a parent mass list. Initial screening utilized a high mass tolerance ( approximately 10 ppm) to filter the full scan MS data for previously reported nefazodone metabolites. The tight mass tolerance reduces or eliminates background chemical noise, dramatically increasing sensitivity for confirming or eliminating the presence of metabolites as well as isobaric forms. The full scan accurate mass analysis of suspected metabolites can be confirmed or refuted using three primary tools: (1) predictive chemical formula and corresponding mass error analysis, (2) rings-plus-double bonds, and (3) accurate mass product ion spectra of parent and suspected metabolites. Accurate mass characterization of the parent ion structure provided the basis for assessing structural assignment for metabolites. Metabolites were also characterized using parent product ion m/z values to filter all tandem mass spectra for identification of precursor ions yielding similar product ions. Identified metabolite parent masses were subjected to chemical formula calculator based on accurate mass as well as bond saturation. Further analysis of potential nefazodone metabolites was executed using accurate mass product ion spectra. Reported mass measurement errors for all full scan MS and MS/MS spectra was <3 ppm, regardless of relative ion abundance, which enabled the use of predictive software in determining product ion structure. The ability to conduct biotransformation profiling via tandem mass spectrometry coupled with accurate mass measurements, all in a single experimental run, is clearly one of the most attractive features of this methodology.     

Mass spectrometric characterization of toremifene metabolites in human urine by liquid chromatography-tandem mass spectrometry with different scan modes

The metabolism and excretion of toremifene were investigated in one healthy male volunteer after a single oral administration of 120 mg toremifene citrate. Different liquid chromatographic/tandem mass spectrometric (LC/MS/MS) scanning techniques were carried out for the characterization of the metabolites in human urine for doping control purposes. The potential characteristic fragmentation pathways of toremifene and its major metabolites were presented. An approach for the metabolism study of toremifene and its analogs by liquid chromatography-tandem mass spectrometry was established. Five different LC/MS/MS scanning methods based on precursor ion scan (precursor ion scan of m/z 72.2, 58.2, 44.2, 45.2, 88.2 relative to five metabolic pathways) in positive ion mode were assessed to recognize the metabolites. Based on product ion scan and precursor ion scan techniques, the metabolites were proposed to be identified as 4-hydroxy-toremifene (m/z 422.4), 4'-hydroxy-toremifene (m/z 422.4), α-hydroxy-toremifene (m/z 422.4), 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2), 3-hydroxy-4-methoxy-toremifene (m/z 456.2), dihydroxy-dehydro-toremifene (m/z 440.2), 3,4-dihydroxy-toremifene (m/z 438.2), N-demethyl-4-hydroxy-toremifene (m/z 408.3), N-demethyl-3-hydroxy-4-methoxy-toremifene (m/z 438.3). In addition, a new metabolite with a protonated molecule at m/z 390.3 was detected in all urine samples. The compound was identified by LC/MS/MS as N-demethyl-4,4'-dihydroxy-tamoxifene. The results indicated that 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2) and N-demethyl-4,4'-dihydroxy-tamoxifene (m/z 390.3) were major metabolites in human urine.     

Targeted quantitative bioanalysis in plasma using liquid chromatography/high-resolution accurate mass spectrometry: an evaluation of global selectivity as a function of mass resolving power and extraction window, with comparison of centroid and profile modes

There is a growing interest in exploring the use of liquid chromatography coupled with full-scan high resolution accurate mass spectrometry (LC/HRMS) in bioanalytical laboratories as an alternative to the current practice of using LC coupled with tandem mass spectrometry (LC/MS/MS). Therefore, we have investigated the theoretical and practical aspects of LC/HRMS as it relates to the quantitation of drugs in plasma, which is the most commonly used matrix in pharmacokinetics studies. In order to assess the overall selectivity of HRMS, we evaluated the potential interferences from endogenous plasma components by analyzing acetonitrile-precipitated blank human plasma extract using an LC/HRMS system under chromatographic conditions typically used for LC/MS/MS bioanalysis with the acquisition of total ion chromatograms (TICs) using 10 k and 20 k resolving power in both profile and centroid modes. From each TIC, we generated extracted ion chromatograms (EICs) of the exact masses of the [M + H](+) ions of 153 model drugs using different mass extraction windows (MEWs) and determined the number of plasma endogenous peaks detected in each EIC. Fewer endogenous peaks are detected using higher resolving power, narrower MEW, and centroid mode. A 20 k resolving power can be considered adequate for the selective determination of drugs in plasma. To achieve desired analyte EIC selectivity and simultaneously avoid missing data points in the analyte EIC peak, the MEW used should not be too wide or too narrow and should be a small fraction of the full width at half maximum (FWHM) of the profile mass peak. It is recommended that the optimum MEW be established during method development under the specified chromatographic and sample preparation conditions. In general, the optimum MEW, typically ≤ ±20 ppm for 20 k resolving power, is smaller for the profile mode when compared with the centroid mode.     

Discovering metabolites of post-harvest fungicides in citrus with liquid chromatography/time-of-flight mass spectrometry and ion trap tandem mass spectrometry

In this study, we benefit from the combination of liquid chromatography (LC)/time-of-flight (TOF) MS accurate mass measurements to generate elemental compositions of ions and LC/ion trap multiple MS (MSn) providing complementary structural information, which is useful for the elucidation of unknown organic compounds at trace levels in complex food extracts. We have applied this approach to investigate different citrus fruits extracts, and we have identified two post-harvest fungicides (imazalil and prochloraz), the main degradation product of imazalil ([M + H]+, m/z 257) and a non-previously reported prochloraz degradation product ([M + H]+, m/z 282). The database-mediated identification of the parent compounds was based on the generated elemental composition obtained from accurate mass measurements and additional qualitative information from the high resolution chlorine isotopic clusters of both the protonated molecules (imazalil, [M + H]+ 297.0556, <0.1 ppm error, 2-Cl; prochloraz, [M + H]+ 376.0381, 1.9 ppm error, 3-Cl) and their characteristic fragments ions (imazalil: m/z 255 and 159; prochloraz: m/z 308 and 266). The correlation between the structural information provided by ion trap MS/MS fragmentation pathways of the parent species and the TOF accurate mass elemental composition data of the degradation products were the key to elucidate the structures of the degradation products of both post-harvest fungicides. Finally, where standards were not available (prochloraz), further confirmation was obtained by synthesizing the proposed degradation product by acid hydrolysis of the parent standard and confirmation by LC/TOF-MS.     

Integrated rapid resolution liquid chromatography–tandem mass spectrometric approach for screening and identification of metabolites of the potential anticancer agent 3,6,7-trimethoxyphenanthroindolizidine in rat urine

An integrated approach combining data acquisition using MS^E and multi-period product ion scan (mpMS/MS), with high-resolution characteristic extracted ion chromatograms (hcXIC) as a data mining method, was developed for in vivo drug metabolites screening and identification. This approach is illustrated by analyzing metabolites of a potential anticancer agent, 3,6,7-trimethoxyphenanthroindolizidine (CAT) in rat urine based on rapid resolution liquid chromatography combined with tandem mass spectrometry (RRLC–MS/MS). Untargeted full-scan MS^E enabled the high-throughput acquisition of potential metabolites, and targeted mpMS/MS contributed to the sensitivity and specificity of the acquisition of molecules of interest. The data processing method hcXIC, based on the structure of CAT, was shown to be highly effective for the metabolite discovery. Through the double-filtering effect of the characteristic ion and accurate mass, conventional extracted ion chromatograms that contained a substantial number of false-positive peaks were simplified into chromatograms essentially free of endogenous interferences. As a result, 21 metabolites were detected in rat urine after oral administration of CAT. Based on the characteristic fragmentation patterns of the phenanthroindolizidine alkaloid, the structures of 9 metabolites were identified. Furthermore, the interpretation of the MS/MS spectra of these metabolites enabled the determination of demethylation position as well as the differentiation between N-oxidized and hydroxylated metabolites.     

Determination and excretion study of gestrinone in human urine by high performance liquid chromatography and gas chromatography/mass spectrometry

Gestrinone was studied by HPLC for screening and by GC/MS for confirmation. Three unknown peaks were found by HPLC which are probably the metabolites of gestrinone, and conjugated gestrinone in dosed human urine. The metabolites and gestrinone were excreted as the conjugated forms. The total amounts of metabolite 1 and conjugated gestrinone, recovered after 48 h, were 0.20 and 0.32 mg, respectively. When metabolite 1 was tested by LC/MS and LC/MS/MS, the parent ion was m/z 327, [MH](+), and fragment ions were seen at m/z 309 [MH - H(2)O](+), 291 [MH - 2H(2)O](+), 283, 263 and 239. The TMS-enol-TMS ether derivative of gestrinone has three peaks in the GC/MS chromatogram formed by tautomerism. The reproducibility of the derivatization method was stable and recoveries were over 87% when spiked into blank urine.     

Feasibility of different mass spectrometric techniques and programs for automated metabolite profiling of tramadol in human urine

The purpose of the study was to determine the advantages of different mass spectrometric instruments and commercially available metabolite identification programs for metabolite profiling. Metabolism of tramadol hydrochloride and the excretion of it and its metabolites into human urine were used as a test case because the metabolism of tramadol is extensive and well known. Accurate mass measurements were carried out with a quadrupole time-of-flight mass spectrometer (Q-TOF) equipped with a LockSpray dual-electrospray ionization source. A triple quadrupole mass spectrometer (QqQ) was applied for full scan, product ion scan, precursor ion scan and neutral loss scan measurements and an ion trap instrument for full scan and product ion measurements. The performance of two metabolite identification programs was tested. The results showed that metabolite programs are time-saving tools but not yet capable of fully automated metabolite profiling. Detection of non-expected metabolites, especially at low concentrations in a complex matrix, is still almost impossible. With low-resolution instruments urine samples proved to be challenging even in a search for expected metabolites. Many false-positive hits were obtained with the automated searching and manual evaluation of the resulting data was required. False positives were avoided by using the higher mass accuracy Q-TOF. Automated programs were useful for constructing product ion methods, but the time-consuming interpretation of mass spectra was done manually. High-quality MS/MS spectra acquired on the QqQ instrument were used for confirmation of the tramadol metabolites. Although the ion trap instrument is of undisputable benefit in MS(n), the low mass cutoff of the ion trap made the identification of tramadol metabolites difficult. Some previously unreported metabolites of tramadol were found in the tramadol urine sample, and their identification was based solely on LC/MS and LC/MS/MS measurements.     

Targeted tandem mass spectrometry for high-throughput comparative proteomics employing NanoLC-FTICR MS with external ion dissociation

Targeted tandem mass spectrometry (MS/MS) is an attractive proteomic approach that allows selective identification of peptides exhibiting abundance differences, e.g., between culture conditions and/or diseased states. Herein, we report on a targeted LC-MS/MS capability realized with a hybrid quadrupole-7 tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer that provides data-dependent ion selection, accumulation, and dissociation external to the ICR trap, and a control software that directs intelligent MS/MS target selection based on LC elution time and m/z ratio. We show that the continuous on-the-fly alignment of the LC elution time during the targeted LC-MS/MS experiment, combined with the high mass resolution of FTICR MS, is crucial for accurate selection of targets, whereas high mass measurement accuracy MS/MS data facilitate unambiguous peptide identifications. Identification of a subset of differentially abundant proteins from Shewanella oneidensis grown under suboxic versus aerobic conditions demonstrates the feasibility of such approach.     

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.     

Characterization of metabolites of Celecoxib in rabbits by liquid chromatography/tandem mass spectrometry

The metabolism of the anti-inflammatory drug Celecoxib in rabbits was characterized using liquid chromatography (LC)/tandem mass spectrometry (MS/MS) with precursor ion and constant neutral loss scans followed by product ion scans. After separation by on-line liquid chromatography, the crude urine samples and plasma and fecal extracts were analyzed with turbo-ionspray ionization in negative ion mode using a precursor ion scan of m/z 69 (CF(3)) and a neutral loss scan of 176 (dehydroglucuronic acid). The subsequent product ion scans of the [M - H] ions of these metabolites yielded the identification of three phase I and four phase II metabolites. The phase I metabolites had hydroxylations at the methyl group or on the phenyl ring of Celecoxib, and the subsequent oxidation product of the hydroxymethyl metabolite formed the carboxylic acid metabolite. The phase II metabolites included four positional isomers of acyl glucuronide conjugates of the carboxylic acid metabolite. These positional isomers were caused by the alkaline pH of the rabbit urine and were not found in rabbit plasma. The chemical structures of the metabolites were characterized by interpretation of their product ion spectra and comparison of their LC retention times and the product ion spectra with those of the authentic synthesized standards.     

An introduction to hybrid ion trap/time‐of‐flight mass spectrometry coupled with liquid chromatography applied to drug metabolism studies

Metabolism studies play an important role at various stages of drug discovery and development. Liquid chromatography combined with mass spectrometry (LC/MS) has become a most powerful and widely used analytical tool for identifying drug metabolites. The suitability of different types of mass spectrometers for metabolite profiling differs widely, and therefore, the data quality and reliability of the results also depend on which instrumentation is used. As one of the latest LC/MS instrumentation designs, hybrid ion trap/time‐of‐flight MS coupled with LC (LC‐IT‐TOF‐MS) has successfully integrated ease of operation, compatibility with LC flow rates and data‐dependent MSⁿ with high mass accuracy and mass resolving power. The MSⁿ and accurate mass capabilities are routinely utilized to rapidly confirm the identification of expected metabolites or to elucidate the structures of uncommon or unexpected metabolites. These features make the LC‐IT‐TOF‐MS a very powerful analytical tool for metabolite identification. This paper begins with a brief introduction to some basic principles and main properties of a hybrid IT‐TOF instrument. Then, a general workflow for metabolite profiling using LC‐IT‐TOF‐MS, starting from sample collection and preparation to final identification of the metabolite structures, is discussed in detail. The data extraction and mining techniques to find and confirm metabolites are discussed and illustrated with some examples. This paper is directed to readers with no prior experience with LC‐IT‐TOF‐MS and will provide a broad understanding of the development and utility of this instrument for drug metabolism studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of 1-hydroxypyrene glucuronide in tissue of marine polychaete Nereis diversicolor by liquid chromatography/ion trap multiple mass spectrometry

1-Hydroxypyrene glucuronide is identified as the single major aqueous metabolite of the tetracyclic aromatic hydrocarbon pyrene, in tissue from a deposit-feeding marine polychaete, Nereis diversicolor. Identification was performed using an ion trap mass spectrometer fitted with an atmospheric pressure chemical ionization (APCI) probe and connected to a high-performance liquid chromatography/diode array detector (HPLC/DAD) system. Besides 1-hydroxypyrene, the 339-nm UV trace of tissue samples from pyrene-exposed worms showed only one dominant peak that could be related to pyrene metabolism. Negative APCI-MS of this supposed 1- hydroxypyrene conjugate gave a characteristic signal at m/z 429 corresponding to the molecular ion of 1-hydroxypyrene glucuronide plus eluent adducts ([M - H + 2H(2)O](-)). Fragmentation pathways were studied by isolating the abundant ion at m/z 429 in the ion trap and performing multiple mass spectrometric experiments (MS(n)). The fragmentations observed were consistent with the proposed identification. Two low intensity LC peaks that could be related to pyrene metabolism by their DAD absorption spectra were also present in the 339-nm UV chromatogram of tissue samples. However, these peaks could not be identified by their mass spectra in negative ion mode due to ion suppression by very abundant co-eluting impurities. The present method shows that LC/MS(n) is a fast and useful analytical tool for identification of aqueous polycyclic aromatic hydrocarbon biotransformation products in samples from relatively small marine invertebrates with limited sample preparation.     

Strategies to avoid false negative findings in residue analysis using liquid chromatography coupled to time-of-flight mass spectrometry

Liquid chromatography coupled to orthogonal acceleration time-of-flight mass spectrometry (LC/TOF) provides an attractive alternative to liquid chromatography coupled to triple quadrupole mass spectrometry (LC/MS/MS) in the field of multiresidue analysis. The sensitivity and selectivity of LC/TOF approach those of LC/MS/MS. TOF provides accurate mass information and a significantly higher mass resolution than quadrupole analyzers. The available mass resolution of commercial TOF instruments ranging from 10 000 to 18 000 full width at half maximum (FWHM) is not, however, sufficient to completely exclude the problem of isobaric interferences (co-elution of analyte ions with matrix compounds of very similar mass). Due to the required data storage capacity, TOF raw data is commonly centroided before being electronically stored. However, centroiding can lead to a loss of data quality. The co-elution of a low intensity analyte peak with an isobaric, high intensity matrix compound can cause problems. Some centroiding algorithms might not be capable of deconvoluting such partially merged signals, leading to incorrect centroids.Co-elution of isobaric compounds has been deliberately simulated by injecting diluted binary mixtures of isobaric model substances at various relative intensities. Depending on the mass differences between the two isobaric compounds and the resolution provided by the TOF instrument, significant deviations in exact mass measurements and signal intensities were observed. The extraction of a reconstructed ion chromatogram based on very narrow mass windows can even result in the complete loss of the analyte signal. Guidelines have been proposed to avoid such problems. The use of sub-2 microm HPLC packing materials is recommended to improve chromatographic resolution and to reduce the risk of co-elution. The width of the extraction mass windows for reconstructed ion chromatograms should be defined according to the resolution of the TOF instrument. Alternative approaches include the spiking of the sample with appropriate analyte concentrations. Furthermore, enhanced software, capable of deconvoluting partially merged mass peaks, may become available.     

Use of high‐resolution mass spectrometry to investigate a metabolite interference during liquid chromatography/tandem mass spectrometric quantification of a small molecule in toxicokinetic study samples

During routine liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalysis of a small molecule analyte in rat serum samples from a toxicokinetic study, an unexpected interfering peak was observed in the extracted ion chromatogram of the internal standard. No interfering peaks were observed in the extracted ion chromatogram of the analyte. The dose‐dependent peak area response and peak area response versus time profiles of the interfering peak suggested that it might have been related to a metabolite of the dosed compound. Further investigation using high‐resolution mass spectrometry led to unequivocal identification of the interfering peak as an N‐desmethyl metabolite of the parent analyte. High‐resolution mass spectrometry (HRMS) was also used to demonstrate that the interfering response of the metabolite in the multiple reaction monitoring (MRM) channel of the internal standard was due to an isobaric relationship between the ¹³C‐isotope of the metabolite and the internal standard (i.e., common precursor ion mass), coupled with a metabolite product ion with identical mass to the product ion used in the MRM transition of the internal standard. These results emphasize (1) the need to carefully evaluate internal standard candidates with regard to potential interferences from metabolites during LC/MS/MS method development, validation and bioanalysis of small molecule analytes in biological matrices; (2) the value of HRMS as a tool to investigate unexpected interferences encountered during LC/MS/MS analysis of small molecules in biological matrices; and (3) the potential for interference regardless of choice of IS and therefore the importance of conducting assay robustness on incurred in vitro or in vivo study samples. Copyright © 2010 John Wiley & Sons, Ltd.     

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).     

Mass defect filter technique and its applications to drug metabolite identification by high‐resolution mass spectrometry

Identification of drug metabolites by liquid chromatography/mass spectrometry (LC/MS) involves metabolite detection in biological matrixes and structural characterization based on product ion spectra. Traditionally, metabolite detection is accomplished primarily on the basis of predicted molecular masses or fragmentation patterns of metabolites using triple‐quadrupole and ion trap mass spectrometers. Recently, a novel mass defect filter (MDF) technique has been developed, which enables high‐resolution mass spectrometers to be utilized for detecting both predicted and unexpected drug metabolites based on narrow, well‐defined mass defect ranges for these metabolites. This is a new approach that is completely different from, but complementary to, traditional molecular mass‐ or MS/MS fragmentation‐based LC/MS approaches. This article reviews the mass defect patterns of various classes of drug metabolites and the basic principles of the MDF approach. Examples are given on the applications of the MDF technique to the detection of stable and chemically reactive metabolites in vitro and in vivo. Advantages, limitations, and future applications are also discussed on MDF and its combinations with other data mining techniques for the detection and identification of drug metabolites. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid metabolite identification with sub parts-per-million mass accuracy from biological matrices by direct infusion nanoelectrospray ionization after clean-up on a ZipTip and LTQ/Orbitrap mass spectrometry

Metabolite identification studies remain an integral part of pre-clinical and clinical drug development programs. Analysis of biological matrices, such as plasma, urine, feces and bile, pose challenges due to the large amounts of endogenous components that can mask a drug and its metabolites. Although direct infusion nanoelectrospray using capillaries has been used routinely for proteomic studies, metabolite identification has traditionally employed liquid chromatographic (LC) separation prior to analysis. A method is described here for rapid metabolite profiling in biological fluids that involves initial sample clean-up using pipette tips packed with reversed-phase material (i.e. ZipTips) to remove matrix components followed by direct infusion nanoelectrospray on an LTQ/Orbitrap mass spectrometer using a protonated polydimethylcyclosiloxane cluster ion for internal calibration. We re-examined samples collected from a prazosin metabolism study in the rat. Results are presented that demonstrate that sub parts-per-million accuracies can be achieved on molecular ions, facilitating identification of metabolites, and on product ions, facilitating structural assignments. The data also show that the high-resolution measurements (R = 100 000 at m/z 400) enable metabolites of interest to be resolved from endogenous components. The extended analysis times available with nanospray enables signal averaging for 1 min or more that is valuable when metabolites are present in low concentrations as encountered here in plasma and brain. Using this approach, the metabolic fate of a drug can be quickly obtained. A limitation of this approach is that metabolites that are structural isomers cannot be distinguished, although such information can be collected by LC/MS during follow-on experiments. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Determination of ricin by nano liquid chromatography/mass spectrometry after extraction using lactose-immobilized monolithic silica spin column

Ricin is a glycosylated proteinous toxin that is registered as toxic substance by Chemical Weapons convention. Current detection methods can result in false negatives and/or positives, and their criteria are not based on the identification of the protein amino acid sequences. In this study, lactose-immobilized monolithic silica extraction followed by tryptic digestion and liquid chromatography/mass spectrometry (LC/MS) was developed as a method for rapid and accurate determination of ricin. Lactose, which was immobilized on monolithic silica, was used as a capture ligand for ricin extraction from the sample solution, and the silica was supported in a disk-packed spin column. Recovery of ricin was more than 40%. After extraction, the extract was digested with trypsin and analyzed by LC/MS. The accurate masses of molecular ions and MS/MS spectra of the separated peptide peaks were measured by Fourier transform-MS and linear iontrap-MS, respectively. Six peptides, which were derived from the ricin A-(m/z 537.8, 448.8 and 586.8) and B-chains (m/z 701.3, 647.8 and 616.8), were chosen as marker peptides for the identification of ricin. Among these marker peptides, two peptides were ricin-specific. This method was applied to the determination of ricin from crude samples. The monolithic silica extraction removed most contaminant peaks from the total ion chromatogram of the sample, and the six marker peptides were clearly detected by LC/MS. It takes about 5 h for detection and identification of more than 8 ng/ml of ricin through the whole handling, and this procedure will be able to deal with the terrorism using chemical weapon.     

Investigation of an enhanced resolution triple quadrupole mass spectrometer for high-throughput liquid chromatography/tandem mass spectrometry assays

Triple quadrupole mass spectrometers, when operated in multiple reaction monitoring (MRM) mode, offer a unique combination of sensitivity, specificity, and dynamic range. Consequently, the triple quadrupole is the workhorse for high-throughput quantitation within the pharmaceutical industry. However, in the past, the unit mass resolution of quadrupole instruments has been a limitation when interference from matrix or metabolites cannot be eliminated. With recent advances in instrument design, triple quadrupole instruments now afford mass resolution of less than 0.1 Dalton (Da) full width at half maximum (FWHM). This paper describes the evaluation of an enhanced resolution triple quadrupole mass spectrometer for high-throughput bioanalysis with emphasis on comparison of selectivity, sensitivity, dynamic range, precision, accuracy, and stability under both unit mass (1 Da FWHM) and enhanced (相似文献