Collision-induced dissociation pathways of yeast sphingolipids and their molecular profiling in total lipid extracts: a study by quadrupole TOF and linear ion trap-orbitrap mass spectrometry

The yeast Saccharomyces cerevisiae synthesizes three classes of sphingolipids: inositolphosphoceramides (IPCs), mannosyl-inositolphosphoceramides (MIPCs), and mannosyl-diinositolphosphoceramides (M(IP)2C). Tandem mass spectrometry of their molecular anions on a hybrid quadrupole time-of-flight (QqTOF) instrument produced fragments of inositol-containing head groups, which were specific for each lipid class. MS(n) analysis performed on a hybrid linear ion trap-orbitrap (LTQ Orbitrap) mass spectrometer with better than 3 ppm mass accuracy identified fragment ions specific for the amide-linked fatty acid and the long chain base moieties in individual molecular species. By selecting m/z of class-specific fragment ions for multiple precursor ion scanning, we profiled yeast sphingolipids in total lipid extracts on a QqTOF mass spectrometer. Thus, a combination of QqTOF and LTQ Orbitrap mass spectrometry lends itself to rapid, comprehensive and structure-specific profiling of the molecular composition of sphingolipids and glycerophospholipids in important model organisms, such as fungi and plants.     

Comprehensive Profiling for Histone H4of Human Liver Cells Using High Resolution LTQ-Orbitrap Mass Spectrometry

The post translational modifications of histone variants are playing an important role in the structure of chro‐ matin, the regulation of gene activities and the diagnosis of diseases, and conducting in‐depth researches and discovering new sites depend on new and rational analytical methods to some extent. In this work, the combinatorial method of high resolution LTQ‐Orbitrap mass spectrometry and multiple enzymes was employed to identify the post translational modifications (PTMs) of histone H4 of human liver cells. The novel methylation site, argnine 67 (R 67), was observed besides some sites reported previously such as lysine 31 (K 31), lysine 44 (K 44), argnine 55 (R 55) and lysine 59 (K 59) in the global domain. Meanwhile, various combinations of acetylation of lysine 5 (K 5), lysine 8 (K 8), lysine 12 (K 12), lysine 16 (K 16) and methylation of lysine 20 (K 20) in the NH₂‐terminal tails were also identified after the LC‐MS/MS analysis of trypsin, Arg‐C, Glu‐C and chymotrypsin digests.     

Evaluation of the LTQ-Orbitrap mass spectrometer for the analysis of polymerase chain reaction products

We have investigated the potential and robustness of the off‐line coupling of polymerase chain reaction (PCR) with electrospray ionization mass spectrometry (ESI‐MS), for further applications in the screening of single‐nucleotide polymorphisms (SNPs). This was based on recently reported data demonstrating that anion‐exchange solid‐phase extraction was the most efficient technique for efficiently desalting PCR products, with a recovery of ～70%. Results showed that this purification approach efficiently removes almost all the chemicals commonly added to PCR buffers. ESI‐MS analysis of a model 114‐bp PCR product performed on the LTQ‐Orbitrap instrument demonstrated that detection limits in the nM range along with an average mass measurement uncertainty of 9.15 ± 7.11 ppm can be routinely obtained using an external calibration. The PCR/ESI‐MS platform was able to detect just a few copies of a targeted oligonucleotide. However, it was shown that if two PCR products are present in a mixture in a ratio higher than 10 to 1, the lower abundance one might not be reproducibly detected. Applications to SNPs demonstrated that an LTQ‐Orbitrap with a resolution of 30 000 (at m/z 400) easily identified a single (A ? G) switch, i.e. a 16 Da difference, in binary mixtures of ～ 35 kDa PCR products. Complementary experiments also showed that the combination of endonucleases and ESI‐MS could be used to confirm base composition and sequence, and thus to screen for unknown polymorphisms in specific sequences. For example, a single (T ? A) switch (9 Da mass difference) was successfully identified in a 114‐bp PCR product. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

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.     

Complementary middle‐down and intact monoclonal antibody proteoform characterization by capillary zone electrophoresis – mass spectrometry

High‐resolution capillary zone electrophoresis – mass spectrometry (CZE‐MS) has been of increasing interest for the analysis of biopharmaceuticals. In this work, a combination of middle‐down and intact CZE‐MS analyses has been implemented for the characterization of a biotherapeutic monoclonal antibody (mAb) with a variety of post‐translational modifications (PTMs) and glycosylation structures. Middle‐down and intact CZE separations were performed in an acidified methanol‐water background electrolyte on a capillary with a positively charged coating (M7C4I) coupled to an Orbitrap mass spectrometer using a commercial sheathless interface (CESI). Middle‐down analysis of the IdeS‐digested mAb provided characterization of PTMs of digestion fragments. High resolution CZE enabled separation of charge variants corresponding to 2X‐deamidated, 1X‐deamidated, and non‐deamidated forms at baseline resolution. In the course of the middle‐down CZE‐MS analysis, separation of glycoforms of the F_C/2 fragment was accomplished due to hydrodynamic volume differences. Several identified PTMs were confirmed by CZE‐MS². Incorporation of TCEP‐HCl reducing agent in the sample solvent resulted in successful analysis of reduced forms without the need for alkylation. CZE‐MS studies on the intact mAb under denaturing conditions enabled baseline separation of the 2X‐glycosylated, 1X‐glycosylated, and aglycosylated populations as a result of hydrodynamic volume differences. The presence of a trace quantity of dissociated light chain was also detected in the intact protein analysis. Characterization of the mAb under native conditions verified identifications achieved via intact analysis and allowed for quantitative confirmation of proteoforms. Analysis of mAbs using CZE‐MS represents a complementary approach to the more conventional liquid‐chromatography – mass spectrometry‐based approaches.     

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.     

Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability

Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion‐mobility mass spectrometry (IM‐MS) and hydrogen/deuterium exchange mass spectrometry (HDX‐MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins.     

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.     

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 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.     

Automatic recalibration and processing of tandem mass spectra using formula annotation

High accuracy, high resolution tandem mass spectrometry (MS/MS) is becoming more common in analytical applications, yet databases of these spectra remain limited. Databases require good quality spectra with sufficient compound information, but processing, calibration, noise reduction and retrieval of compound information are time‐consuming tasks that prevent many contributions. We present a comprehensive workflow for the automatic processing of MS/MS using formula annotation for recalibration and cleanup to generate high quality spectra of standard compounds for upload to MassBank ( www.massbank.jp ). Compound information is retrieved via Internet services. Reference standards of 70 pesticides were measured at various collision energies on an LTQ‐Orbitrap XL to develop and evaluate the workflow. A total of 944 resulting spectra are now available on MassBank. Evidence of nitrogen adduct formation during MS/MS fragmentation processes was found, highlighting the benefits high accuracy MS/MS offers for spectral interpretation. A database of recalibrated, cleaned‐up spectra resulted in the most correct spectra ranked in first place, regardless of whether the search spectra were recalibrated or not, whereas the average rank of the correct molecular formula was improved from 2.55 (uncalibrated) to 1.53 when using recalibrated MS/MS data. The workflow is available as an R package RMassBank capable of generating MassBank records from raw MS and MS/MS data and can be adjusted to process data acquired with different settings and instruments. This workflow is a vital step towards addressing the need for more high quality, high accuracy MS/MS spectra in spectral databases and provides important information for spectral interpretation. Copyright © 2012 John Wiley & Sons, Ltd.     

Precise and global identification of phospholipid molecular species by an Orbitrap mass spectrometer and automated search engine Lipid Search

In the present research, we have established a new lipidomics approach for the comprehensive and precise identification of molecular species in a crude lipid mixture using a LTQ Orbitrap mass spectrometer (MS) and reverse-phase liquid chromatography (RPLC) combination with our newly developed lipid search engine “Lipid Search”. LTQ Orbitrap provides high mass accuracy MS spectra by Fourier-transform (FT) mass spectrometer mode and can perform rapid MSⁿ by ion trap (IT) mass spectrometer mode. In this study, the negative ion mode was selected to detect fragment ions from phospholipids, such as fatty acid anions, by MS2 or MS3. We selected the specific detection approach by neutral loss survey-dependent MS3, for the identification of molecular species of phosphatidylcholine, sphingomyelin and phosphatidylserine. Identification of molecular species was performed by using both the high mass accuracy of the mass spectrometric data obtained from FT mode and structural data obtained from fragments in IT mode. Some alkylacyl and alkenylacyl species have the same m/z value as molecular-related ions and fragment ions, thus, direct acid hydrolysis analysis was performed to identify alkylacyl and alkenylacyl species, and then the RPLC–LTQ Orbitrap method was applied. As a result, 290 species from mouse liver and 248 species from mouse brain were identified within six different classes of phospholipid, only those in manually detected and confirmed. Most of all manually detected mass peaks were also automatically detected by “Lipid Search”. Adding to differences in molecular species in different classes of phospholipids, many characteristic differences in molecular species were detected in mouse liver and brain. More variable number of saturated and monounsaturated fatty acid-containing molecular species were detected in mouse brain than liver.     

Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer

Mass and top-down analyses of 150-kDa monoclonal immunoglobulin gamma (IgG) antibodies were performed on an Orbitrap analyzer. Three different sample delivery methods were tested including (1) infusion of an off-line desalted IgG sample using nano-electrospray; (2) on-line desalting followed by a step elution with a high percentage of organic solvent; and (3) reversed-phase HPLC separation and on-line mass and top-down analyses of disulfide isoforms of an IgG2 antibody. The accuracy of mass measurements of intact antibody was within ±2 Da (15 ppm). The glycoforms of intact IgG antibodies separated by 162 Da were baseline resolved. In-source fragmentation of the intact antibodies produced mainly 115 residue fragments including N-terminal variable domains of heavy and light chains. The sequence coverage (the number of cleavages) was greatly increased after reduction of disulfide bonds and HPLC/MS/MS analysis of light and heavy chains using collision-induced dissociation in the ion trap of the LTQ-Orbitrap. This is an attractive alternative to peptide mapping for characterization and monitoring of post-translational modifications attributed to minimal sample preparation, high speed of the mass/top-down analysis, and relatively minor method-induced sample modifications.     

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.     

Comparison of LC and LC/MS methods for quantifying <Emphasis Type="Italic">N</Emphasis>-glycosylation in recombinant IgGs

High-performance liquid chromatography (LC) and liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-MS) methods with various sample preparation schemes were compared for their ability to identify and quantify glycoforms in two different production lots of a recombinant monoclonal IgG1 antibody. IgG1s contain a conserved N-glycosylation site in the fragment crystallizable (Fc) subunit. Six methods were compared: (1) LC/ESI-MS analysis of intact IgG, (2) LC/ESI-MS analysis of the Fc fragment produced by limited proteolysis with Lys-C, (3) LC/ESI-MS analysis of the IgG heavy chain produced by reduction, (4) LC/ESI-MS analysis of Fc/2 fragment produced by limited proteolysis and reduction, (5) LC/MS analysis of the glycosylated tryptic fragment (293EEQYNSTYR301) using extracted ion chromatograms, and (6) normal phase HPLC analysis of N-glycans cleaved from the IgG using PNGase F. The results suggest that MS quantitation based on the analysis of Fc/2 (4) is accurate and gives results that are comparable to normal phase HPLC analysis of N-glycans (6).     

Food contaminant analysis at ultra‐high mass resolution: application of hybrid linear ion trap – orbitrap mass spectrometry for the determination of the polyether toxins,azaspiracids, in shellfish

The biotoxins, azaspiracids (AZAs), from marine phytoplankton accumulate in shellfish and affect human health by causing severe gastrointestinal disturbance, diarrhea, nausea and vomiting. Specific and sensitive methods have been developed and validated for the determination of the most commonly occurring azaspiracid analogs. An LTQ Orbitrap mass spectrometer is a hybrid instrument that combines linear ion trap (LIT) mass spectrometry (MS) with high‐resolution Fourier transform (FT) MS and this was exploited to perform simultaneous ultra‐high‐resolution full‐scan MS analysis and collision‐induced dissociation (CID) tandem mass spectrometry (MS/MS). Using the highest mass resolution setting (100 000 FWHM) in full‐scan mode, the methodology was validated for the determination of six AZAs in mussel (Mytilus galloprovincialis) tissue extracts. Ultra‐high mass resolution, together with a narrow mass tolerance window of ±2 mDa, dramatically improved detection sensitivity. In addition to employing chromatographic resolution to distinguish between the isomeric azaspiracid analogs, AZA1/AZA6 and AZA4/AZA5, higher energy collisionally induced dissociation (HCD) fragmentation on selected precursor ions were performed in parallel with full‐scan FTMS. Using HCD MS/MS, most precursor and product ion masses were determined within 1 ppm of the theoretical m/z values throughout the mass spectral range and this enhanced the reliability of analyte identity. For the analysis of mussels (M. galloprovincialis), the method limit of quantitation (LOQ) was 0.010 µg/g using full‐scan FTMS and this was comparable with the LOQ (0.007 µg/g) using CID MS/MS. The repeatability data were; intra‐day RSD% (1.8–4.4%; n = 6) and inter‐day RSD% (4.7–8.6%; n = 3). Application of these methods to the analysis of mussels (M. edulis) that were naturally contaminated with azaspiracids, using high‐resolution full‐scan Orbitrap MS and low‐resolution CID MS/MS, produced equivalent quantitative data. Copyright © 2010 John Wiley & Sons, Ltd.     

The development and assessment of high‐throughput mass spectrometry‐based methods for the quantification of a nanoparticle drug delivery agent in cellular lysate

The safe use of lipid‐based drug delivery agents requires fast and sensitive qualitative and quantitative assessment of their cellular interactions. Many mass spectrometry (MS) based analytical platforms can achieve such task with varying capabilities. Therefore, four novel high‐throughput MS‐based quantitative methods were evaluated for the analysis of a small organic gene delivery agent: N,N‐bis(dimethylhexadecyl)‐1,3‐propane‐diammonium dibromide (G16‐3). Analysis utilized MS instruments that detect analytes using low‐resolution tandem MS (MS/MS) analysis (i.e. QTRAP or linear ion trap in this work) or high‐resolution MS analysis (i.e. time of flight (ToF) or Orbitrap). Our results indicate that the validated fast chromatography (FC)‐QTRAP‐MS/MS, FC‐ LTQ‐Orbitrap‐MS, desorption electrospray ionization‐collision‐induced dissociation (CID)‐MS/MS and matrix assisted laser desorption ionization‐ToF/ToF‐MS MS methods were superior in the area of method development and sample analysis time to a previously developed liquid chromatography (LC)‐CID‐MS/MS. To our knowledge, this is the first evaluation of the abilities of five MS‐based quantitative methods that target a single pharmaceutical analyte. Our findings indicate that, in comparison to conventional LC‐CID‐MS/MS, the new MS‐based methods resulted in a (1) substantial reduction in the analysis time, (2) reduction in the time required for method development and (3) production of either superior or comparable quantitative data. The four new high‐throughput MS methods, therefore, were faster, more efficient and less expensive than a conventional LC‐CID‐MS/MS for the quantification of the G16‐3 analyte within tissue culture. When applied to cellular lysate, no significant change in the concentration of G16‐3 gemini surfactant within PAM212 cells was observed between 5 and 53 h, suggesting the absence of any metabolism/excretion from PAM212 cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous quantification and semi‐quantification of amentoflavone and its metabolites in human intestinal bacteria by liquid chromatography Orbitrap high‐resolution mass spectrometry

A quick, easy, effective method followed by ultra‐high‐pressure liquid chromatography coupled with linear ion trap–Orbitrap tandem mass spectrometry (UHPLC‐LTQ‐Orbitrap MS) was developed for the simultaneous identification and quantification of the metabolites produced by amentoflavone (AMF) in human intestinal bacteria from human feces. The method validated for quantification of AMF concerning precision, accuracy, recovery, matrix effect, stability and limits showed acceptable results. Compared with blank human intestinal bacteria chromatography, three metabolites were identified based on high‐accuracy protonated precursors and multi‐stage mass spectrometry (MSⁿ ) using the proposed strategy. At the same time, a new method was developed for semi‐quantification of three metabolites. We describe the trend over 24 h of concentration–time curves for AMF and its metabolites. Moreover, the main metabolic pathway of AMF was clarified in human intestinal bacteria. The method was validated and successfully applied to the detection and quantification of AMF and its metabolites.