Isotope abundance analysis for improved sample identification with tandem mass spectrometry

Tandem mass spectrometry (MS/MS) is widely used for trace level sample analysis in complex mixtures. However, sample identification in MS/MS is challenging and not as trustworthy as with electron ionization (EI) mass spectral libraries. This paper presents a novel method for the combination of isotope abundance analysis (IAA) and EI‐MS/MS for improved sample identification even at trace level in complex matrices. Accordingly, the first quadrupole is scanned in a narrow range around the molecular ion group of isotopomers such as M⁺, [M+1]⁺ and [M+2]⁺, Q2 serves for collision‐induced dissociation to produce product ions while Q3 transfers the major sample product ions with low resolution, thus encompassing and uniformly transmitting all the product ion isotopomers. IAA can then be used to derive elemental formula information from the cleansed experimental data. IAA‐MS/MS was experimentally tested with perfluorotributylamine and a very good matching factor of 995 (out of 1000) was obtained for IAA on m/z 502, 503 and 504 (fragment ion isotopomers) while Q3 transmitted the m/z 264 product ion with a mass window of 6 m/z units. The IAA‐MS/MS method was further tested with the pesticide diazinon on its molecular ions m/z 304, 305 and 306 while Q3 was locked on its m/z 179 product ion with a mass window of 6 m/z units. Again, very good matching factors were obtained, even for 40 pg diazinon on‐column during its GC/MS analysis (match = 981). IAA‐MS/MS combines the traditional benefits of MS/MS in the removal of matrix interferences with the IAA power of elemental analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Liquid chromatography tandem mass spectrometry method for the quantitation of mycophenolate mofetil in human plasma: Application to a bioequivalence study and metabolite identification

We established a sensitive, selective, and rapid analytical method for the quantitation and pharmacokinetic investigation of mycophenolate mofetil in human plasma. To our knowledge, this is the first method that characterizes presence of mycophenolate mofetil glucuronide in clinical samples through tandem mass spectrometry detection and resolves mycophenolate mofetil from its glucuronide metabolite. Liquid chromatography coupled to tandem mass spectrometry detection in positive ion mode was selected to provide optimal selectivity and sensitivity. Due to the ionizable characteristics of the mycophenolate mofetil, a mixed‐mode cation‐exchange disposable extraction cartridge was prudently chosen. The chromatographic separation was achieved on Luna^® C18(2) (100×4.60 mm) column using mobile phase consisting of a mixture of 1±0.05 mM ammonium formate in water, titrated to pH 3.1±0.1 with formic acid, and methanol (20:80, v/v), at a flow rate of 0.7 mL/min. The detection was led at m/z ratios of 434.4→ 114.2 and 438.4→ 118.3, for mycophenolate mofetil and mycophenolate mofetil‐D4, respectively. The developed method was linear between 40.2–4986.0 pg/mL. All validation parameters were within the defined limits. The validated method was then successfully applied for the evaluation of bioequivalence parameters of mycophenolate mofetil after an oral administration of 500 mg mycophenolate mofetil tablet to healthy male Indian volunteers.     

Liquid chromatography/tandem mass spectrometry for the identification and determination of trichothecenes in maize

A reliable, sensitive and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to determine four trichothecene mycotoxins (nivalenol, deoxynivalenol, fusarenon X and 3-acetyldeoxynivalenol) in maize. Sample preparation was performed by extracting the analytes with a mixture of acetonitrile and water, followed by a solid-phase extraction with Carbograph-4 cartridges as the purification step. For the LC/MS/MS analysis two interfacing systems, Turbo IonSpray (TISP) and atmospheric pressure chemical ionization (APCI), were compared in both negative and positive ion modes. LC and MS parameters were optimized to achieve better results and sensitivity. The effect of mobile phase modifiers such as ammonium acetate and formic acid on the ionization yield was also evaluated. The best results were obtained using the electrospray ionization (ESI) interface in negative ion mode and the multiple reaction monitoring mode (MRM) for the quantitation. The detection limits ranged between 10 ng/g for fusarenon X and 1.5 ng/g for deoxynivalenol. A linear working range was achieved with a standard deviation between 3 and 10% and recovery rates from the maize samples above 81%. The procedure was applied to the analysis of a set of maize samples collected from farms located in different areas of northern and central Italy. The investigated samples turned out to be contaminated primarily with deoxynivalenol and, to a minor extent, with its derivatives.     

In vitro metabolite identification using fast gradient high performance liquid chromatography combined with tandem mass spectrometry.

Metabolite identification can provide critical information for further synthesis during the lead optimization stage of drug discovery. Fast chromatographic gradient methodology was explored to reduce the analysis time for metabolite identification by liquid chromatography/tandem mass spectrometry while maintaining chromatographic separation of all components of interest. Short HPLC columns (2 cm) containing 3 microm particles were used and the chromatographic conditions involved elevated flow rates (1.0-2.5 mL/min) and fast, full range linear gradients (<2 min). Detection was accomplished by fast scanning triple quadrupole mass spectrometers or a quadrupole time-of-flight mass spectrometer. Analysis of liver microsomal incubations of several compounds indicates that separation of isomeric metabolites as well as the parent compound is feasible with analysis times not exceeding 2 minutes.     

Liquid chromatography-thermospray mass spectrometry of DNA adducts formed with mitomycin C, porfiromycin and thiotepa

High-performance liquid chromatography (HPLC) and thermospray mass spectrometry were combined for the analysis of DNA adducts formed from the interaction of the anticancer drugs mitomycin C, porfiromycin and thiotepa with calf thymus DNA. The adducts formed from reaction of mitomycin C and porfiromycin with DNA were separated from unmodified nucleosides by HPLC on a C18 column and identified by thermospray mass spectrometry. Thiotepa DNA adducts readily depurinated from DNA and were chromatographed and identified by thermospray liquid chromatography-mass spectrometry as the modified bases without the ribose moiety attached. The utility of thermospray mass spectrometry for the identification of microgram quantities of nucleoside adducts and depurinated base adducts of these anticancer drugs was demonstrated.     

High-throughput, accurate mass liquid chromatography/tandem mass spectrometry on a quadrupole time-of-flight system as a 'first-line' approach for metabolite identification studies

Throughput for drug metabolite identification studies has been increased significantly by the combined use of accurate mass liquid chromatography/tandem mass spectrometry (LC/MS/MS) data on a quadrupole time-of-flight (QTOF) system and targeted data analysis procedures. Employed in concert, these tools have led to the implementation of a semi-automated high-throughput metabolite identification strategy that has been incorporated successfully into lead optimization efforts in drug discovery. The availability of elemental composition data on precursor and all fragment ions in each spectrum has greatly enhanced confidence in ion structure assignments, while computer-based algorithms for defining sites of biotransformation based upon mass shifts of diagnostic fragment ions have facilitated identification of positions of metabolic transformation in drug candidates. Adoption of this technology as the 'first-line' approach for in vitro metabolite profiling has resulted in the analysis of as many as 21 new chemical entities on one day from diverse structural classes and therapeutic programs.     

High-performance liquid chromatography-thermospray mass spectrometry of prostaglandin and thromboxane acetyl derivatives.

A quantitative analysis of prostaglandin-related substances has been developed. Hydroxyl groups of prostaglandin and thromboxane were acetylated by acetic anhydride, the mixture was partially purified on a Sep-Pak C18 cartridge and analysed by high-performance liquid chromatography combined with thermospray mass spectrometry. Using this method, twenty kinds of prostaglandin derivative could be detected simultaneously within 11 min on a selected-ion monitoring detection chromatogram without a gradient system. Generally, the base ion, [M + H - n(60)]+, is produced through elimination of acetic acid (n = number of the hydroxyl group of prostaglandin or thromboxane). The detection limit for these derivatives was ca. 0.2 pmol at the levels of prostaglandin-related substances prior to derivatization. They could be analysed in the range 0.5-10 pmol. The assay was successfully applied to prostaglandin-related substances in human seminal fluid and rat brain.     

Liquid chromatography/tandem mass spectrometry method for simultaneous determination of risperidone and its active metabolite 9-hydroxyrisperidone in human plasma

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for simultaneous quantification of risperidone (RSP) and its active metabolite 9-hydroxyrisperidone (9-OH-RSP) in human plasma. The analytes were extracted from human plasma by using the protein precipitation extraction technique. Methyl risperidone was used as internal standard for RSP and 9-OH-RSP. A Betasil C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 411.28 --> 191.15 for RSP and m/z 427.30 --> 207.10 for 9-OH-RSP. The method involves a simple extraction, isocratic chromatography conditions and mass spectrometric detection that enable detection at sub-nanogram levels. The proposed method has been validated with a linear range of 0.10-15.0 ng/mL for RSP and 9-OH-RSP. The intrarun and interrun precision and accuracy values were within 15%. The overall recoveries for RSP and 9-OH-RSP were 82.1% and 83.2%, respectively. The total analysis time was as low as 3.0 min only. The developed method was applied for the determination of the pharmacokinetic parameters of RSP and 9-OH-RSP following a single oral administration of a 1 mg RSP tablet in 24 healthy male volunteers.     

High-performance liquid chromatography-thermospray mass spectrometry of hydroperoxy polyunsaturated fatty acid acetyl derivatives.

A method for the analysis of hydroperoxy polyunsaturated fatty acids was developed. The hydroperoxy groups were acetylated by acetic anhydride, and the mixture was partially purified on a Sep-Pak C18 cartridge and analysed by high-performance liquid chromatography with thermospray mass spectrometry. Generally, the base ion, [M+H - n(60)]+ or [M+H - n(60) - n(H2O)]+, is produced through elimination of acetic acid or water (n = number of hydroperoxy groups). The detection limit for these derivatives was ca. 1 pmol at concentrations of hydroperoxy polyenoic acids prior to derivatization. Using this method, many hydroxy and hydroperoxy polyunsaturated fatty acid derivatives could be detected simultaneously within 30 min on a selected-ion monitoring detection chromatogram without a gradient system. The assay was successfully applied to hydroxy and hydroperoxy polyunsaturated fatty acids from an incubation mixture of rat brain homogenate to which polyunsaturated fatty acids had been added.     

Data requirements for protein identification using chemically-assisted fragmentation and tandem mass spectrometry

Many laboratories identify proteins by searching tandem mass spectrometry data against genomic or protein sequence databases. These database searches typically use the measured peptide masses or the derived peptide sequence and, in this paper, we focus on the latter. We study the minimum peptide sequence data requirements for definitive protein identification from protein sequence databases. Accurate mass measurements are not needed for definitive protein identification, even when a limited amount of sequence data is available for searching. This information has implications for the mass spectrometry performance (and cost), data base search strategies and proteomics research.     

Improved mass accuracy for tandem mass spectrometry

With the emergence of top-down proteomics, the ability to achieve high mass measurement accuracy on tandem MS/MS data will be beneficial for protein identification and characterization. (FT-ICR) Fourier transform ion cyclotron resonance mass spectrometers are the ideal instruments to perform these experiments with their ability to provide high resolution and mass accuracy. A major limitation to mass measurement accuracy in FT-ICR instruments arises from the occurrence of space charge effects. These space charge effects shift the cyclotron frequency of the ions, which compromises the mass measurement accuracy. While several methods have been developed that correct these space charge effects, they have limitations when applied to MS/MS experiments. It has already been shown that additional information inherent in electrospray spectra can be used for improved mass measurement accuracy with the use of a computer algorithm called DeCAL (deconvolution of Coulombic affected linearity). This paper highlights a new application of the strategy for improved mass accuracy in tandem mass analysis. The results show a significant improvement in mass measurement accuracy on complex electron capture dissociation spectra of proteins. We also demonstrate how the improvement in mass accuracy can increase the confidence in protein identification from the fragment masses of proteins acquired in MS/MS experiments.     

Hybrid mass spectrometers for tandem mass spectrometry

Mass spectrometers that use different types of analyzers for the first and second stages of mass analysis in tandem mass spectrometry (MS/MS) experiments are often referred to as "hybrid" mass spectrometers. The general goal in the design of a hybrid instrument is to combine different performance characteristics offered by various types of analyzers into one mass spectrometer. These performance characteristics may include mass resolving power, the ion kinetic energy for collision-induced dissociation, and speed of analysis. This paper provides a review of the development of hybrid instruments over the last 30 years for analytical applications.     

pFind 2.0: a software package for peptide and protein identification via tandem mass spectrometry

This paper describes the pFind 2.0 software package for peptide and protein identification via tandem mass spectrometry. Firstly, the most important feature of pFind 2.0 is that it offers a modularized and customized platform for third parties to test and compare their algorithms. The developers can create their own modules following the open application programming interface (API) standards and then add it into workflows in place of the default modules. In addition, to accommodate different requirements, the package provides four automated workflows adopting different algorithm modules, executing processes and result reports. Based on this design, pFind 2.0 provides an automated target-decoy database search strategy: The user can just specify a certain false positive rate (FPR) and start searching. Then the system will return the protein identification results automatically filtered by such an estimated FPR. Secondly, pFind 2.0 is also of high accuracy and high speed. Many pragmatic preprocessing, peptide-scoring, validation, and protein inference algorithms have been incorporated. To speed up the searching process, a toolbox for indexing protein databases is developed for high-throughput applications and all modules are implemented under a new architecture designed for large-scale parallel and distributed searching. An experiment on a public dataset shows that pFind 2.0 can identify more peptides than SEQUEST and Mascot at the 1% FPR. It is also demonstrated that this version of pFind 2.0 has better usability and higher speed than its previous versions. The software and more detailed supplementary information can both be accessed at http://pfind.ict.ac.cn/.     

Rapid identification of saponins in plant extracts by electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/tandem mass spectrometry

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) and liquid chromatography coupled with on-line mass spectrometry (LC/MS/MS) were applied to characterize saponins in crude extracts from Panax ginseng. The MS(n) data of the [M - H](-) ions of saponins can provide structural information on the sugar sequences of the saccharide chains and on the sapogins of saponins. By ESI-MS(n), non-isomeric saponins and isomeric saponins with different aglycones can be determined rapidly in plant extracts. LC/MS/MS is a good complementary analytical tool for determination of isomeric saponins. These approaches constitute powerful analytical tools for rapid screening and structural assignment of saponins in plant extracts.     

Rapid screening and identification of cytochalasins by electrospray tandem mass spectrometry

The cytochalasin class of fungal metabolites was analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) with the aim of developing a methodology for their rapid identification in microbial extracts. ESI-MS analyses of reference cytochalasins were performed and several product ions were produced in MS/MS experiments on parent ions that are structurally characteristic. A precursor ion search was performed to detect cytochalasins in an ethyl acetate extract of fungal strain RK97-F21. Three cytochalasins were detected and one of the components was identified as epoxycytochalasin H by comparing the tandem mass spectra of the product ions with those of reference compounds. This finding was further validated by LC/MS and LC/MS/MS experiments.     

Detection and identification of carcinogen-peptide adducts by nanoelectrospray tandem mass spectrometry

Nanoelectrospray (nanoES) tandem mass spectrometry was used to examine covalently modified peptides in crude enzymatic digests of human serum albumin (HSA) that had been exposed to either benzo[a]pyrene diol epoxide (B[a]PDE, 1), chrysene diol epoxide (CDE, 2), 5-methylchrysene diol epoxide (5MeCDE, 3), or benzo[g]chrysene diol epoxide (B[g]CDE, 4). The low flow rates of nanoES (～20 nL/min) allowed several MS/MS experiments to be optimized and performed on a single sample with very little sample consumption (～30 min analysis time/µL sample). Initially, nanoES was compared with conventional LC/MS/MS analysis of carcinogen-peptide adducts. For example, nanoES analysis of an unseparated digest of B[a]PDE-treated serum albumin revealed the same peptides (RRHPY and RRHPY-FYAPE) that were previously shown, by LC/MS/MS, to be adducted with B[a]PDE. In addition, nanoES could detect unstable peptide adducts that might not otherwise have been directly observable. Finally, nanoES was shown to be an effective way to screen mixtures of modified and unmodified peptides for which no chromatographic information is available.