Compatibility of electron ionization and soft ionization methods in gas chromatography/orthogonal time‐of‐flight mass spectrometry

Orthogonal acceleration time‐of‐flight (oa‐TOF) mass spectrometry (MS) was coupled to gas chromatography (GC) to measure ion yields (ratio of ion counts to number of neutrals entering the ion source) and signal‐to‐noise (S/N) in the electron ionization (EI) mode (hard ionization) as well as in the soft ionization modes of chemical ionization (CI), electron capture negative ion chemical ionization (NICI) and field ionization (FI). Mass accuracies of the EI and FI modes were also investigated. Sixteen structurally diverse volatile organic compounds were chosen for this study. The oa‐TOF mass analyzer is highly suited for FI MS and provided an opportunity to compare the sensitivity of this ionization method to the more conventional ionization methods. Compared to the widely used quadrupole mass filter, the oa‐TOF platform offers significantly greater mass accuracy and therefore the possibility of determining the empirical formula of analytes. The findings of this study showed that, for the instrument used, EI generated the most ions with the exception of compounds able to form negative ions readily. Lower ion yields in the FI mode were generally observed but the chromatograms displayed greater S/N and in many cases gave spectra dominated by a molecular ion. Ion counts in CI are limited by the very small apertures required to maintain sufficiently high pressures in the ionization chamber. Mass accuracy for molecular and fragment ions was attainable at close to manufacturer's specifications, thus providing useful information on molecular ions and neutral losses. The data presented also suggests a potentially useful instrumental combination would result if EI and FI spectra could be collected simultaneously or in alternate scans during GC/MS. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of a new electron ionization/field ionization ion source for gas chromatography/time‐of‐flight mass spectrometry

We have developed a combined EI/FI source for gas chromatography/orthogonal acceleration time‐of‐flight mass spectrometry (GC/oaTOFMS). In general, EI (electron ionization) and FI (field ionization) mass spectra are complementary: the EI mass spectrum contains information about fragment ions, while the FI mass spectrum contains information about molecular ions. Thus, the comparative study of EI and FI mass spectra is useful for GC/MS analyses. Unlike the conventional ion sources for FI and EI measurements, the newly developed source can be used for both measurements without breaking the ion source vacuum or changing the ion source. Therefore, the combined EI/FI source is more preferable than the conventional EI or FI ion source from the viewpoint of the reliability of measurements and facility of operation. Using the combined EI/FI source, the complementarity between EI and FI mass spectra is demonstrated experimentally with n‐hexadecane (100 pg): characteristic fragment ions for the n‐alkane such as m/z 43, 57, 71, and 85 are obtained in the EI mass spectrum, while only the parent peak of m/z 226 (M⁺) without any fragment ions is observed in the FI mass spectrum. Moreover, the field desorption (FD) measurement is also demonstrated with poly(ethylene glycol)s M600 (10 ng) and M1000 (15 ng). Signals of [M+H]⁺, [M+Na]⁺ and [M+K]⁺ are clearly detected in the FD mass spectra. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of multiple pesticide residues in teas by gas chromatography with accurate time‐of‐flight mass spectrometry

In this work, gas chromatography tandem with electron ionization and full‐scan high‐resolution mass spectrometry with a time‐of‐flight mass analyzer was evaluated for analyzing pesticide residues in teas. The relevant aspects for mass spectrometry analysis, including the resolution and mass accuracy, acquisition rate, temperature of ion source, were investigated. Under acquisition condition in 2‐GHz extended dynamic range mode, accurate mass spectral library including 184 gas chromatography detectable pesticides was established and retrieval parameters were optimized. The mass spectra were consistent over a wide concentration range (three orders) with good match values to those of NIST (EI‐quadrupole). The methodology was verified by the validation of 184 pesticides in four tea matrices. A wide linear range (1–1000 μg/kg) was obtained for most compounds in four matrices. Limit of detection, limit of quantification, and limit of identification values acquired in this study could satisfy the requirements for maximum residue levels prescribed by the European Community. Recovery studies were performed at three concentrations (10, 50, and 100 μg/kg). Most of the analytes were recovered at an acceptable range of 70–120% with relative standard deviations ≤ 20% in four matrices. The potential extension of qualitative screening scope makes gas chromatography tandem with electron ionization and mass spectrometry with a time‐of‐flight mass analyzer a more powerful tool compared with gas chromatography with tandem mass spectrometry.     

Gas chromatography/time‐of‐flight mass spectrometry of triacetone triperoxide based on femtosecond laser ionization

Triacetone triperoxide (TATP), which is used as an explosive in acts of terrorism, was measured by means of gas chromatography/multiphoton ionization/time‐of‐flight mass spectrometry using a deep‐ultraviolet (deep‐UV) femtosecond laser as an ionization source. The fragmentation process was investigated by changing the intensity of the laser at the center axis of a molecular beam. A molecular ion was observed using a femtosecond laser, and the ratio of the intensities of the molecular and fragment ions decreased as the intensity of the laser increased. These results suggest that TATP can be efficiently ionized using a deep‐UV, ultrashort optical pulse. Furthermore, fragmentation was accelerated by excess energy supplied through higher‐order multiphoton processes under a strong radiation field. The detection limits obtained using the molecular ion and two dominant fragment ions, C₂H₃O⁺ and CH, were determined to be 670, 83 and 150 pg, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Analyzing multiple pesticides in tobacco leaf using gas chromatography with quadrupole time‐of‐flight mass spectrometry

A method combining gas chromatography with quadrupole time‐of‐flight mass spectrometry has been developed for the simultaneous analysis of multiple pesticide residues in tobacco leaf. The retention index and high accurate masses of ions from the first‐stage and the second‐stage mass spectra of each pesticide were collected for qualitation and quantification. A total of 115 pesticides were evaluated. The extract from organic tobacco leaf was used as a model matrix. The limit of detection was <10 ng/mL, and the limit of quantification was in the range of 1–20 ng/mL for 95% of the tested pesticides. The correlation coefficients were >0.9900 for all tested pesticides. At three concentrations (10, 50, and 100 ng/mL), most compounds presented satisfactory recoveries ranging from 70 to 120% and good precision <20%. Finally, three tobacco leaf samples collected from a local market were analyzed. A total of three pesticides were found, including dimethachlon, triadimenol, and flumetralin. Each pesticide was confirmed by the presence of three ions at the expected retention index and mass. In conclusion, gas chromatography with quadrupole time‐of‐flight mass spectrometry appears to be one of the most efficient tools for the analysis of pesticide residues in tobacco leaf.     

Quantification of hepcidin using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Hepcidin is known to be a key systemic iron‐regulatory hormone which has been demonstrated to be associated with a number of iron disorders. Hepcidin concentrations are increased in inflammation and suppressed in hemochromatosis. In view of the role of hepcidin in disease, its potential as a diagnostic tool in a clinical setting is evident. This study describes the development of a matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) assay for the quantitative determination of hepcidin concentrations in clinical samples. A stable isotope labeled hepcidin was prepared as an internal standard and a standard quantity was added to urine samples. Extraction was performed with weak cation‐exchange magnetic nanoparticles. The basic peptides were eluted from the magnetic nanoparticles using a matrix solution directly onto a target plate and analyzed by MALDI‐TOF MS to determine the concentration of hepcidin. The assay was validated in charcoal stripped urine, and good recovery (70–80%) was obtained, as were limit of quantitation data (5 nmol/L), accuracy (RE <10%), precision (CV <21%), within ‐day repeatability (CV <13%) and between‐day repeatability (CV <21%). Urine hepcidin levels were 10 nmol/mmol creatinine in healthy controls, with reduced levels in hereditary hemochromatosis (P < 0.000005) and elevated levels in inflammation (P < 0.0007). In summary a validated method has been developed for the determination of hepcidin concentrations in clinical samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Modern MALDI time‐of‐flight mass spectrometry

This paper focuses on development of time‐of‐flight (TOF) mass spectrometry in response to the invention of matrix‐assisted laser desorption/ionization (MALDI). Before this breakthrough ionization technique for nonvolatile molecules, TOF was generally considered as a useful tool for exotic studies of ion properties but was not widely applied to analytical problems. Improved TOF instruments and software that allow the full potential power of MALDI to be applied to difficult biological applications are described. A theoretical approach to the design and optimization of MALDI‐TOF instruments for particular applications is presented. Experimental data are provided that are in excellent agreement with theoretical predictions of resolving power and mass accuracy. Data on sensitivity and dynamic range using kilohertz laser rates are also summarized. These results indicate that combinations of high‐performance MALDI‐TOF and TOF‐TOF with off‐line high‐capacity separations may ultimately provide throughput and dynamic range several orders of magnitude greater than those currently available with electrospray LC‐MS and MS‐MS. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of the chemical constituents in Hongjingtian injection by liquid chromatography quadrupole time‐of‐flight mass spectrometry

Hongjingtian injection is made from Rhodiola wallichiana and used in the treatment of stable angina pectoris associated with coronary heart disease. In this study, the chemical constituents in Hongjingtian injection were comprehensively studied using liquid chromatography quadrupole time‐of‐flight mass spectrometry. A total of 49 compounds were identified or assumed, including 10 organic acids, nine phenylethanoids, 10 phenylpropanoids, two flavonoid glycosides, seven monoterpene glycosides, seven octylglycosides and four other types of compounds. The structures of seven compounds were confirmed by comparing their retention times, MS and UV spectra with the corresponding authentic standards. Amongst the 49 compounds, 35 were firstly found in R. wallichiana, while they have been reported in other species of the genus Rhodiola, including Rhodiola crenulata, Rhodiola sacra, Rhodiola rosea and Rhodiola kirilowii. The possible fragmentation pathways in the mass spectrometry of the major types of compounds are proposed and summarized. Our study demonstrates a rapid method for characterizing the chemical constituents present in the Hongjingtian injection, which could also be applied to the identification of chemical constituents in other TCM formulae containing R. wallichiana.     

The application of gas chromatography/atmospheric pressure chemical ionisation time‐of‐flight mass spectrometry to impurity identification in Pharmaceutical Development

Accurate mass measurement (used to determine elemental formulae) is an essential tool for impurity identification in pharmaceutical development for process understanding. Accurate mass liquid chromatography/mass spectrometry (LC/MS) is used widely for these types of analyses; however, there are still many occasions when gas chromatography (GC)/MS is the appropriate technique. Therefore, the provision of robust technology to provide accurate mass GC/MS (and GC/MS/MS) for this type of activity is essential. In this report we describe the optimisation and application of a newly available atmospheric pressure chemical ionisation (APCI) interface to couple GC to time‐of‐flight (TOF) MS. To fully test the potential of the new interface the APCI source conditions were optimised, using a number of standard compounds, with a variety of structures, as used in synthesis at AstraZeneca. These compounds were subsequently analysed by GC/APCI‐TOF MS. This study was carried out to evaluate the range of compounds that are amenable to analysis using this technique. The range of compounds that can be detected and characterised using the technique was found to be extremely broad and include apolar hydrocarbons such as toluene. Both protonated molecules ([M + H]⁺) and radical cations (M^+.) were observed in the mass spectra produced by APCI, along with additional ion signals such as [M + H + O]⁺. The technique has been successfully applied to the identification of impurities in reaction mixtures from organic synthesis in process development. A typical mass accuracy of 1–2 mm/zunits (m/z 80–500) was achieved allowing the reaction impurities to be identified based on their elemental formulae. These results clearly demonstrate the potential of the technique as a tool for problem solving and process understanding in pharmaceutical development. The reaction mixtures were also analysed by GC/electron ionisation (EI)‐MS and GC/chemical ionisation (CI)‐MS to understand the capability of GC/APCI‐MS relative to these two firmly established techniques. Copyright © 2010 John Wiley & Sons, Ltd.     

Potential of monitoring isotopologues by quantitative gas chromatography with time‐of‐flight mass spectrometry for metabolomic assay

Because of the extreme complexity of metabolomic samples, the effectiveness of quantitative gas chromatography with time‐of‐flight mass spectrometry depends substantially on the expansion of the linear dynamic range. Facing the existence of numerous saturated detector signals, a data processing method based on monitoring isotopologues has been developed. The monoisotopic ion kept the high mass spectrometry sensitivity, and the less abundant isotopologue ions extended the linear dynamic range. This alternative method was proved to extend the linear dynamic range to five orders of magnitude successfully and overcome the quantitative problems induced by the ion detector saturation. Finally, to validate the applicability, the method was applied to a metabolomic assay of Alzheimer's disease. Comparing with the traditional monoisotopic method, the use of monitoring isotopologues helped us to discover an additional eight metabolites with significant difference and to conduct a more reliable principal component analysis as well. The results demonstrated that monitoring isotopologues in quantitative gas chromatography with time‐of‐flight mass spectrometry could improve the authenticity of metabolomic analysis.     

The detection of various opiates and benzodiazepines by comprehensive two‐dimensional gas chromatography/time‐of‐flight mass spectrometry

A technique using comprehensive two‐dimensional gas chromatography/time‐of‐flight mass spectrometry (GC × GC/TOFMS) is applied to qualitative and quantitative drug testing. Human serum was ‘spiked’ with known quantities of benzodiazepines and a ‘street heroin’ mixture including some of the major metabolites and impurities. The sample components were extracted from the matrix by solid‐phase extraction (SPE). Constituents containing polar hydroxyl and/or secondary amine groups were derivatised with N‐methyl‐N‐(tert‐butyldimethyl)trifluoroacetamide (MTBSTFA) to improve the chromatographic performance. An orthogonal separation of the matrix constituents was achieved by coupling a DB‐5ms (5% phenyl) to a BPX50 (50% phenyl) GC column. The eluant was focused onto the second column by a twin‐stage cryo‐modulator. Rapid 6 s modulation times were achieved by transfer from a 30 m × 0.25 mm (length × internal diameter) to a 2 m × 0.1 mm column. TOFMS with rapid spectral acquisition (≤500 spectra/s) was employed in the mass range m/z 40–650. A clean mass spectrum was obtained for each analyte using mass spectral deconvolution software. The sensitivity and repeatability of the method were evaluated by the preparation of calibration standards for two benzodiazepines, flunitrazepam and its major metabolite 7‐aminoflunitrazepam (7‐amino‐FN), in the concentration range 5–1000 ng/mL. The limits of detection (LODs) and limits of quantitation (LOQs), calculated by repeat injections (×10) of the lowest standard, were 1.6 and 5.4 ng/mL (flunitrazepam); 2.5 and 8.5 ng/mL (7‐amino‐FN), respectively. There is scope to extend this protocol to screen a large number of drugs and metabolites stored in a library database. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of triacetone triperoxide by gas chromatography/mass spectrometry and gas chromatography/tandem mass spectrometry by electron and chemical ionization

The explosive triacetone triperoxide (TATP) has been analyzed by gas chromatography/mass spectrometry (GC/MS) and sub-nanogram detection limits are reported by ammonia positive ion chemical ionization (PICI), electron ionization (EI) and methane negative ion chemical ionization (NICI). Analysis by methane PICI and ammonia NICI gave detection limits in the low nanogram range. Analyses were carried out on (linear) quadrupole and ion trap instruments. Analysis of TATP by PICI using ammonia reagent gas is the preferred analytical method, producing low limits of detection as well as an abundant (greater than 60% of base peak) diagnostic adduct ion at m/z 240 corresponding to [TATP + NH4]+. Isolation of the [TATP + NH4]+ ion with subsequent collision-induced dissociation (CID) produces extremely low abundance product ions at m/z values greater than 60, and the m/z 223 ion corresponding to [TATP + H]+ was not observed. Density functional theory (DFT) calculations at the B88LYP/DVZP level indicate that dissociation of the complex to form NH4+ and TATP occurs at energies lower than peroxide bond dissociation, while protonation of TATP leads to cleavage of the ring structure. These results provide a method for pico-gram detection levels of TATP using commercial instrumentation commonly available in forensic laboratories. As a point of comparison, a detection limit of 15 ng was obtained by flame ionization detection.     

Pharmaceutical metabolite profiling using quadrupole/ion mobility spectrometry/time‐of‐flight mass spectrometry

The use of hybrid quadrupole ion mobility spectrometry time‐of‐flight mass spectrometry (Q/IMS/TOFMS) in the metabolite profiling of leflunomide (LEF) and acetaminophen (APAP) is presented. The IMS drift times (T_d) of the drugs and their metabolites were determined in the IMS/TOFMS experiments and correlated with their exact monoisotopic masses and other in silico generated structural properties, such as connolly molecular area (CMA), connolly solvent‐excluded volume (CSEV), principal moments of inertia along the X, Y and Z Cartesian coordinates (MI‐X, MI‐Y and MI‐Z), inverse mobility and collision cross‐section (CCS). The correlation of T_d with these parameters is presented and discussed. IMS/TOF tandem mass spectrometry experiments (MS² and MS³) were successfully performed on the N‐acetyl‐p‐benzoquinoneimine glutathione (NAPQI‐GSH) adduct derived from the in vitro microsomal metabolism of APAP. As comparison, similar experiments were also performed using hybrid triple quadrupole linear ion trap mass spectrometry (QTRAPMS) and quadrupole time‐of‐flight mass spectrometry (QTOFMS). The abilities to resolve the product ions of the metabolite within the drift tube and fragment the ion mobility resolved product ions in the transfer travelling wave‐enabled stacked ring ion guide (TWIG) demonstrated the potential applicability of the Q/IMS/TOFMS technique in pharmaceutical metabolite profiling. Copyright © 2009 John Wiley & Sons, Ltd.