Genotyping single nucleotide polymorphisms using intact polymerase chain reaction products by electrospray quadrupole mass spectrometry

Both single nucleotide polymorphisms (SNPs) and mutations are commonly observed in the gene encoding the tumor suppressor protein, p53. SNPs occur at specific locations within genes whereas mutations may be distributed across large regions of genes. When determining nucleotide differences, mass spectrometry is the only method other than Sanger sequencing which offers direct structural information. Electrospray ionization (ESI) quadrupole mass spectrometry (MS) analysis of intact polymerase chain reaction (PCR) products was performed following a simple purification and on-line heating to limit ion adduction. The PCR products were amplified directly from genomic DNA rather than plasmids, as in our previous work. Two known polymorphisms of the p53 gene were genotyped. A cytosine (C) or guanine (G) transversion, designated C <--> G (G <--> C on the opposite strand), were each detected by a 40.0 Da change upon ESI quadrupole MS analysis. Using known PCR products as standards, the genotypes determined for 10 human samples corresponded with restriction fragment length polymorphism (RFLP) analysis. Cytosine/thymine (T) transitions, designated C <--> T (G <--> A on the opposite strand), were also genotyped by ESI-MS. This SNP is discriminated by a 15.0 Da change on one strand (C <--> T) and a 16.0 Da change on the other (G <--> A). Appropriate sample preparation and instrumental configuration (including heated sample inlet syringe and MS source), to limit adducts, are both vital for successful ESI quadrupole MS analysis of intact PCR products.     

Improved characterization of tomato polyphenols using liquid chromatography/electrospray ionization linear ion trap quadrupole Orbitrap mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry

Tomato (Lycopersicon esculentum Mill.) is the second most important fruit crop worldwide. Tomatoes are a key component in the Mediterranean diet, which is strongly associated with a reduced risk of chronic degenerative diseases. In this work, we use a combination of mass spectrometry (MS) techniques with negative ion detection, liquid chromatography/electrospray ionization linear ion trap quadrupole‐Orbitrap‐mass spectrometry (LC/ESI‐LTQ‐Orbitrap‐MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) on a triple quadrupole, for the identification of the constituents of tomato samples. First, we tested for the presence of polyphenolic compounds through generic MS/MS experiments such as neutral loss and precursor ion scans on the triple quadrupole system. Confirmation of the compounds previously identified was accomplished by injection into the high‐resolution system (LTQ‐Orbitrap) using accurate mass measurements in MS, MS² and MS³ modes. In this way, 38 compounds were identified in tomato samples with very good mass accuracy (<2 mDa), three of them, as far as we know, not previously reported in tomato samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Electrospray ionization quadrupole time-of-flight mass spectrometry and quadrupole mass spectrometry for genotyping single nucleotide substitutions in intact polymerase chain reaction products in K-ras and p53

Single nucleotide polymorphisms (SNPs) and mutations were genotyped for both homozygous and heterozygous PCR products of p53, a tumor suppressor gene, and K-ras, an oncogene, using electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) and ESI-quadrupole MS analysis. Mass accuracy was adequate for both instruments to detect genetic changes in homozygous PCR products, including the most difficult to distinguish (adenine [A] --> thymine [T] transversion). However, for the detection of A --> T shifts (9.0 Da difference) in heterozygous PCR products, the increased resolution of ESI-Q-TOFMS proved essential. Although, greater mass differences in heterozygotes (e.g. cytosine [C] <--> T or guanine [G] <--> A) can be discriminated using ESI-quadrupole MS analysis.     

Structural characterization of intact antibodies by high‐resolution LTQ Orbitrap mass spectrometry

Recombinant monoclonal antibodies (MAbs) can be heterogeneous due to modifications that can occur during expression, purification or during storage. These large multichain proteins (～150 kDa) are structurally challenging for detailed characterization to identify the sites of modifications. We report the use of LTQ Orbitrap mass spectrometry to accurately measure the average masses of individual glycoforms by direct infusion of an intact antibody. To identify the site‐specific modification of methionines in the antibody caused by forced oxidation, we used a ‘middle‐down’ approach. The antibody was subjected to limited digestion using the endoproteinase Lys‐C and reduced to generate Fab heavy chain, single chain Fc and light chain fragments (～25 kDa each). These species were subjected to on‐line liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis using an LTQ Orbitrap, where these large precursors were dissociated by higher‐energy collisions in the C‐trap. High resolution and accuracy achieved for resulting fragments allowed us to show in a site‐specific manner that only the methionines in the Fc heavy chain were oxidized under the studied conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

Heterobifunctional isotope‐labeled amine‐reactive photo‐cross‐linker for structural investigation of proteins by matrix‐assisted laser desorption/ionization tandem time‐of‐flight and electrospray ionization LTQ‐Orbitrap mass spectrometry

Chemical cross‐linking combined with a subsequent enzymatic digestion and mass spectrometric analysis of the created cross‐linked products presents an alternative approach to assess low‐resolution protein structures. By covalently connecting pairs of functional groups within a protein or a protein complex a set of structurally defined interactions is built up. We synthesized the heterobifunctional amine‐reactive photo‐cross‐linker N‐succinimidyl p‐benzoyldihydrocinnamate as a non‐deuterated (SBC) and doubly deuterated derivative (SBDC). Applying a 1:1 mixture of SBC and SBDC for cross‐linking experiments aided the identification of cross‐linked amino acids in the mass spectra based on the characteristic isotope patterns of fragment ions. The cross‐linker was applied to the calcium‐binding protein calmodulin with a subsequent analysis of cross‐linked products by nano‐high‐performance liquid chromatography matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry (nano‐HPLC/MALDI‐TOF/TOF‐MS) and nano‐HPLC/nano‐electrospray ionization (ESI)‐LTQ‐Orbitrap‐MS. Copyright © 2009 John Wiley & Sons, Ltd.     

Two‐injection workflow for a liquid chromatography/LTQ‐Orbitrap system to complete in vivo biotransformation characterization: demonstration with buspirone metabolite identification

The relatively high background matrix in in vivo samples typically poses difficulties in drug metabolite identification, and causes repeated analytical runs on unit resolution liquid chromatography/mass spectrometry (LC/MS) systems before the completion of biotransformation characterization. Ballpark parameter settings for the LTQ‐Orbitrap are reported herein that enable complete in vivo metabolite identification within two HPLC/MS injections on the hybrid LTQ‐Orbitrap data collection system. By setting the FT survey full scan at 60K resolution to trigger five dependent LTQ MS² scans, and proper parameters of Repeat Duration, Exclusion Duration and Repeat Count for the first run (exploratory), the Orbitrap achieved the optimal parallel data acquisition capability and collected maximum number of product ion scans. Biotransformation knowledge based prediction played the key role in exact mass ion extraction and multiple mass defect filtration when the initial data was processed. Meanwhile, product ion extraction and neutral loss extraction of the initial dependent data provided additional bonus in identifying metabolites. With updated parent mass list and the data‐dependent setting to let only the ions on the parent mass list trigger dependent scans, the second run (confirmatory) ensures that all precursor ions of identified metabolites trigger not only dependent product ion scans, but also at or close to the highest concentration of the eluted metabolite peaks. This workflow has been developed for metabolite identification of in vivo or ADME studies, of which the samples typically contain a high level of complex matrix. However, due to the proprietary nature of the in vivo studies, this workflow is presented herein with in vitro buspirone sample incubated with human liver microsomes (HLM). The major HLM‐mediated biotransformation on buspirone was identified as oxidation or hydroxylation since five mono‐ (+16 Da), seven di‐ (+32 Da) and at least three tri‐oxygenated (+48 Da) metabolites were identified. Besides the metabolites 1‐pyrimidinylpiperazine (1‐PP) and hydroxylated 1‐PP that formed by N‐dealkylation, a new metabolite M308 was identified as the result of a second N‐dealkylation of the pyrimidine unit. Two new metabolites containing the 8‐butyl‐8‐azaspiro[4,5]decane‐7,9‐dione partial structure, M240 and M254, were also identified that were formed apparently due to the first N‐dealkylation of the 1‐PP moiety. Copyright © 2009 John Wiley & Sons, Ltd.     

Multi‐mycotoxin analysis in complex biological matrices using LC‐ESI/MS: Experimental study using triple stage quadrupole and LTQ‐Orbitrap

In the present study, we report the application of LC‐MS based on two different LC‐MS systems to mycotoxin analysis. The mycotoxins were extracted with an ACN/water/acetic acid mixture and directly injected into a LC‐MS/MS system without any dilution procedure. First, a sensitive and reliable HPLC‐ESI‐MS/MS method using selected reaction monitoring on a triple quadrupole mass spectrometer (TSQ Quantum Ultra AM) has been developed for determining 32 mycotoxins in crude extracts of wheat and maize. This method was operated both in positive and in negative ionization modes in two separate chromatographic runs. The method was validated by studies of spiked recoveries, linearity, matrix effect, intra‐assay precision and sensitivity. Further, we have developed and evaluated a method based on accurate mass measurements of extracted target ions in full scan mode using micro‐LC‐LTQ‐Orbitrap as a tool for fast quantitative analysis. Both instruments exhibited very high sensitivity and repeatability in positive ionization mode. Coupling of micro‐LC to Orbitrap technology was not applicable to the negatively ionizable compounds. The LC triple quadrupole MS method has proved to be stable in quantitation, as it is with respect to the matrix effects of grain samples.     

Metabolite analysis in Curcuma domestica using various GC‐MS and LC‐MS separation and detection techniques

The metabolic profile of polar (methanol) and non‐polar (hexane) extracts of Curcuma domestica, a widely used medicinal plant, was established using various different analytical techniques, including GC‐FID, GC‐MS, HR‐GC‐MS and analytical HPLC‐ESI‐MS/MS by means of LTQ‐Orbitrap technology. The major non‐volatile curcuminoids curcumin, demethoxycurcumin and bisdemethoxycurcumin were identified when their chromatographic and precursor ion masses were compared with those of authentic standard compounds. In this paper we describe for the first time a GC/MS‐based method for metabolic profiling of the hydrophilic extract. We also identified 61 polar metabolites as TMS derivatives. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparison of different signal thresholds on data dependent sampling in orbitrap and LTQ mass spectrometry for the identification of peptides and proteins in complex mixtures

We evaluate the effect of ion-abundance threshold settings for data-dependent acquisition on a hybrid LTQ-Orbitrap mass spectrometer, analyzing features such as the total number of spectra collected, the signal to noise ratio of the full MS scans, the spectral quality of the tandem mass spectra acquired, and the number of peptides and proteins identified from a complex mixture. We find that increasing the threshold for data-dependent acquisition generally decreases the quantity but increases the quality of the spectra acquired. This is especially true when the threshold setting is set above the noise level of the full MS scan. We compare two distinct experimental configurations: one where full MS scans are acquired in the Orbitrap analyzer while tandem MS scans are acquired in the LTQ analyzer, and one where both full MS and tandem MS scans are acquired in the LTQ analyzer. We examine the number of spectra, peptides, and proteins identified under various threshold conditions, and we find that the optimal threshold setting is at or below the respective noise level of the instrument regardless of whether the full MS scan is performed in the Orbitrap or in the LTQ analyzer. When comparing the high-throughput identification performance of the two analyzers, we conclude that, used at optimal threshold levels, the LTQ and the Orbitrap identify similar numbers of peptides and proteins. The higher scan speed of the LTQ, which results in more spectra being collected, is roughly compensated by the higher mass accuracy of the Orbitrap, which results in improved database searching and peptide validation software performance.     

Application of high‐resolution ESI and MALDI mass spectrometry to metabolite profiling of small interfering RNA duplex

We investigated the application of a high‐resolution Orbitrap mass spectrometer equipped with an electrospray ionization (ESI) source and a matrix‐assisted laser desorption/ionization‐time‐of‐flight (MALDI‐TOF) mass spectrometer to the metabolite profiling of a model small interfering RNA (siRNA) duplex TSR#34 and compared their functions and capabilities. TSR#34 duplex was incubated in human serum in vitro, and the duplex and its metabolites were then purified by ion exchange chromatography in order to remove the biological matrices. The fraction containing the siRNA duplex and its metabolites was collected and desalted and then subjected to high‐performance liquid chromatography (HPLC) equipped with a reversed phase column. The siRNA and its metabolites were separated into single strands by elevated chromatographic temperature and analyzed using the ESI‐Orbitrap or the MALDI‐TOF mass spectrometer. Using this method, the 5' and/or 3' truncated metabolites of each strand were detected in the human serum samples. The ESI‐Orbitrap mass spectrometer enabled differentiation between two possible RNA‐based sequences, a monoisotopic molecular mass difference which was less than 2 Da, with an intrinsic mass resolving power. In‐source decay (ISD) analysis using a MALDI‐TOF mass spectrometer allowed the sequencing of the RNA metabolite with characteristic fragment ions, using 2,4‐dihydroxyacetophenone (2,4‐DHAP) as a matrix. The ESI‐Orbitrap mass spectrometer provided the highest mass accuracy and the benefit of on‐line coupling with HPLC for metabolite profiling. Meanwhile, the MALDI‐TOF mass spectrometer, in combination with 2,4‐DHAP, has the potential for the sequencing of RNA by ISD analysis. The combined use of these methods will be beneficial to characterize the metabolites of therapeutic siRNA compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitation of small molecules using high‐resolution accurate mass spectrometers – a different approach for analysis of biological samples

The quantitative capabilities of a linear ion trap high‐resolution mass spectrometer (LTQ‐Orbitrap™) were investigated using full scan mode bracketing the m/z range of the ions of interest and utilizing a mass resolution (mass/FWHM) of 15000. Extracted ion chromatograms using a mass window of ±5–10 mmu centering on the theoretical m/z of each analyte were generated and used for quantitation. The quantitative performance of the LTQ‐Orbitrap™ was compared with that of a triple quadrupole (API 4000) operating using selected reaction monitoring (SRM) detection. Comparable assay precision, accuracy, linearity and sensitivity were observed for both approaches. The concentrations of actual study samples from 15 Merck drug candidates reported by the two methods were statistically equivalent. Unlike SRM being a tandem mass spectrometric (MS/MS)‐based detection method, a high resolution mass spectrometer operated in full scan does not need MS/MS optimization. This approach not only provides quantitative results for compounds of interest, but also will afford data on other analytes present in the sample. An example of the identification of a major circulating metabolite for a preclinical development study is demonstrated. Copyright © 2009 John Wiley & Sons, Ltd.     

Investigation of uremic analytes in hemodialysate and their structural elucidation from accurate mass maps generated by a multi‐dimensional liquid chromatography/mass spectrometry approach

Historically, structural elucidation of unknown analytes by mass spectrometry alone has involved tandem mass spectrometry experiments using electron ionization. Most target molecules for bioanalysis in the metabolome are unsuitable for detection by this previous methodology. Recent publications have used high‐resolution accurate mass analysis using an LTQ‐Orbitrap with the more modern approach of electrospray ionization to identify new metabolites of known metabolic pathways. We have investigated the use of this methodology to build accurate mass fragmentation maps for the structural elucidation of unknown compounds. This has included the development and validation of a novel multi‐dimensional LC/MS/MS methodology to identify known uremic analytes in a clinical hemodialysate sample. Good inter‐ and intra‐day reproducibility of both chromatographic stages with a high degree of mass accuracy and precision was achieved with the multi‐dimensional liquid chromatography/tandem mass spectrometry (LC/MS/MS) system. Fragmentation maps were generated most successfully using collision‐induced dissociation (CID) as, unlike high‐energy CID (HCD), ions formed by this technique could be fragmented further. Structural elucidation is more challenging for large analytes >270 Da and distinguishing between isomers where their initial fragmentation pattern is insufficiently different. For small molecules (<200 Da), where fragmentation data may be obtained without loss of signal intensity, complete structures can be proposed from just the accurate mass fragmentation data. This methodology has led to the discovery of a selection of known uremic analytes and two completely novel moieties with chemical structural assignments made. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification and fragmentation pathways of caffeine metabolites in urine samples via liquid chromatography with positive electrospray ionization coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry

Liquid chromatography (LC) with positive ion electrospray ionization (ESI+) coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) was employed for the simultaneous determination of caffeine and its metabolites in human urine within a single chromatographic run. LC/ESI‐FTICRMS led to the unambiguous determination of the molecular masses of the studied compounds without interference from other biomolecules. A systematic and comprehensive study of the mass spectral behaviour of caffeine and its fourteen metabolites by tandem mass spectrometry (MS/MS) was performed, through in‐source ion trap collision‐induced dissociation (CID) of the protonated molecules, [M+H]⁺. A retro‐Diels‐Alder (RDA) process along with ring‐contraction reactions were the major fragmentation pathways observed during CID. The base peak of xanthine precursors originates from the loss of methyl isocyanate (CH₃NCO, 57 Da) or isocyanic acid (HNCO, 43 Da), which in turn lose a CO unit. Also uric acid derivatives shared a RDA rearrangement as a common fragmentation process and a successive loss of CO₂ or CO. The uracil derivatives showed a loss of a ketene unit (CH₂CO, 42 Da) from the protonated molecule along with the loss of H₂O or CO. To assess the potential of the present method three established metabolite ratios to measure P450 CYP1A2, N‐acetyltransferase and xanthine oxidase activities were evaluated by a number of identified metabolites from healthy human urine samples after caffeine intake. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolism of TM‐2, a potential antitumor drug,in rats by using LC–MS

TM‐2 (13‐(N‐Boc‐3‐i‐butylisoserinoyl‐4,10‐β‐diacetoxy‐2‐α‐benzoyloxy‐5‐β‐20‐epoxy‐1,13‐α‐dihydroxy‐9‐oxo‐19‐norcyclopropa[g]tax‐11‐ene) is a novel semisynthetic taxane derivative. Our previous study suggested that TM‐2 is a promising antitumor analogue. In this paper, the metabolism of TM‐2 was investigated in rats following intravenous administration. Two different types of mass spectrometry—hybrid linear trap quadrupole orbitrap (LTQ‐Orbitrap) mass spectrometry and triple‐quadrupole tandem (QQQ) mass spectrometry—were employed to acquire structural information of TM‐2 metabolites. A total of 17 components were identified as the metabolites of TM‐2 in bile, feces, and urine samples. Accurate mass measurement using LC–LTQ‐Orbitrap‐MS was used to determine the accurate mass data and elemental composition of metabolites thereby confirming the proposed structures of the metabolites. The metabolites proposed were mainly oxidates of TM‐2, including methoxy, hydroxyl, dihydroxy, and trihydroxyl analogues. The major metabolic pathway of TM‐2 was the hydroxylation of the taxane ring or the lateral chain. These important metabolic data serve as a useful resource to support further research of TM‐2.     

Identification of NTBC metabolites in urine from patients with hereditary tyrosinemia type 1 using two different mass spectrometric platforms: triple stage quadrupole and LTQ‐Orbitrap

The objective of our work was to identify known and unknown metabolites of the drug NTBC (2‐(2‐nitro‐4‐trifluoromethylbenzoyl)‐1,3‐cyclohexanedione) in urine from patients during the treatment of hereditary tyrosinemia type 1 (HT‐1) disease, a severe inborn error of tyrosine metabolism. Two different mass spectrometric techniques, a triple stage quadrupole and an LTQ‐Orbitrap (Fourier transform mass spectrometry (FTMS)), were used for the identification and the structural elucidation of the detected metabolites. Initially, the mass spectrometric (MS) approach consisted of the precursor ion scan detection of the selected product ions, followed by the corresponding collision‐induced dissociation (CID) fragmentation analysis (MS²) for the targeted selected reaction monitoring (SRM) mode. Subsequently, accurate and high‐resolution full scan and MS/MS measurements were performed on the possible metabolites using the LTQ‐Orbitrap. Final confirmation of the identified metabolites was achieved by measuring commercially supplied or laboratory‐synthesized standards. Altogether six metabolites, including NTBC itself, were extracted, detected and identified. In addition, two new NTBC metabolites were unambiguously identified as amino acid conjugates, namely glycine‐NTBC and β‐alanine‐NTBC. These identifications were based on their characteristics of chromatographic retention times, protonated molecular ions, elemental compositions, product ions (using CID and higher‐energy C‐trap dissociation (HCD) techniques) and synthesized references. The applied MS strategy, based on two different MS platforms (LC/MS/MS and FTMS), allowed the rapid identification analysis of the drug metabolites from human extracts and could be used for pharmaceutical research and drug development. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid screening and determination of 4‐chloroamphetamine in saliva by paper spray‐mass spectrometry and capillary electrophoresis‐mass spectrometry

A novel drug‐screening system, consisting of paper spray‐MS (PS‐MS) and a CE‐ESI‐MS method was developed. This system can be easily switched either to PS‐MS for rapidly screening samples or to the traditional CE‐ESI‐MS method for separation and to obtain detailed mass spectral information, while sharing the same mass spectrometer. In the former case, when a sharp (15°‐tip) chromatography paper was used, the optimized distance from the paper tip to the mass inlet was 7.7 mm, whereas the optimized distance for the CE‐ESI tip was ～13.5 mm. Using 4‐chloroamphetamine as a model compound, the LODs for PS‐MS and CE‐ESI‐MS were determined to ～0.1 and 0.25 ppm, respectively. Comparisons of results obtained using PS‐MS and CE‐ESI‐MS and the experimental conditions are described.     

ESIprot: a universal tool for charge state determination and molecular weight calculation of proteins from electrospray ionization mass spectrometry data

Electrospray ionization (ESI) ion trap mass spectrometers with relatively low resolution are frequently used for the analysis of natural products and peptides. Although ESI spectra of multiply charged protein molecules also can be measured on this type of devices, only average spectra are produced for the majority of naturally occurring proteins. Evaluating such ESI protein spectra would provide valuable information about the native state of investigated proteins. However, no suitable and freely available software could be found which allows the charge state determination and molecular weight calculation of single proteins from average ESI‐MS data. Therefore, an algorithm based on standard deviation optimization (scatter minimization) was implemented for the analysis of protein ESI‐MS data. The resulting software ESIprot was tested with ESI‐MS data of six intact reference proteins between 12.4 and 66.7 kDa. In all cases, the correct charge states could be determined. The obtained absolute mass errors were in a range between ?0.2 and 1.2 Da, the relative errors below 30 ppm. The possible mass accuracy allows for valid conclusions about the actual condition of proteins. Moreover, the ESIprot algorithm demonstrates an extraordinary robustness and allows spectral interpretation from as little as two peaks, given sufficient quality of the provided m/z data, without the necessity for peak intensity data. ESIprot is independent from the raw data format and the computer platform, making it a versatile tool for mass spectrometrists. The program code was released under the open‐source GPLv3 license to support future developments of mass spectrometry software. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of metabolites of a novel histamine H(2)-receptor antagonist, lafutidine, in human liver microsomes by liquid chromatography coupled with ion trap mass spectrometry

The metabolism of lafutidine in human liver microsomes was studied using liquid chromatography/ion trap mass spectrometry with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources. A total of 14 metabolites were identified including hydroxylated lafutidine and sulfonyl lafutidine as the major metabolites. The chemical properties and the MS(n) behaviors of lafutidine and all of its identified metabolites were studied in detail. Lafutidine had a fragmentation pattern as a result of homolytic bond cleavage in the MS/MS spectrum. This cleavage can form an odd-electron ion with the loss of furan-2-ylmethyl radical (-81 Da with a proton shift), which then sequentially loses neutral groups in the MS(3) spectrum. This fragmentation sequence was also observed from the metabolites with the unchanged sulfinyl moiety. When the sulfinyl moiety was oxidized to the sulfonyl moiety, this fragmentation sequence did not exist, which could be used to identify S-oxidation metabolites of lafutidine. In general, N-oxides could produce distinct [M+H-O](+) ions under LC/APCI-MS due to the thermal activation in the desolvation region of the API source, which could be used to identify N-oxidation metabolites of lafutidine. In order to avoid the possibility of false positives, the MS/MS spectrum of the [M+H-O](+) ion was compared with that of the non-N-oxidation metabolites or parent drug in the APCI source. If they were consistent, the structure could be finally confirmed. The exact masses for lafutidine and lafutidine N-oxide fragment ions were determined using an LTQ/Orbitrap mass spectrometer.     

Metabolite identification in rat brain microdialysates by direct infusion nanoelectrospray ionization after desalting on a ZipTip and LTQ/Orbitrap mass spectrometry

Analyzing brain microdialysate samples by mass spectrometry is challenging due to the high salt content of the artificial cerebral spinal fluid (aCSF), low analyte concentrations and small sample volumes collected. A drug and its major metabolites can be examined in brain microdialysates by targeted approaches such as selected reaction monitoring (SRM) which provides selectivity and high sensitivity. However, this approach is not well suited for metabolite profiling in the brain which aims to determine biotransformation pathways. Identifying minor metabolites, or metabolites that arise from brain metabolism, remains a challenge and, for a drug in early discovery, identification of metabolites present in the brain can provide useful information for understanding the pharmacological activity and potential toxicological liabilities of the drug. A method is described here for rapid metabolite profiling in brain microdialysates that involves sample clean‐up using C18 ZipTips to remove salts followed by direct infusion nanoelectrospray with an LTQ/Orbitrap mass spectrometer using real‐time internal recalibration. Full scan mass spectra acquired at high resolving power (100 K at m/z 400) were examined manually and with mass defect filtering. Metabolite identification was aided by sub‐parts‐per‐million mass accuracy and structural characterization was accomplished by tandem mass spectrometry (MS/MS) experiments in the Orbitrap or LTQ depending on the abundance of the metabolite. Using this approach, brain microdialysate samples from rats dosed with one of four CNS drugs (imipramine, reboxetine, citalopram or trazodone) were examined for metabolites. For each drug investigated, metabolites, some of which not previously reported in rat brain, were identified and characterized. Copyright © 2009 John Wiley & Sons, Ltd.