Parallel high-throughput accurate mass measurement using a nine-channel multiplexed electrospray liquid chromatography ultraviolet time-of-flight mass spectrometry system

A nine-channel multiplexed electrospray (MUX) liquid chromatography ultraviolet time-of-flight mass spectrometry (LC/UV/TOFMS) system has been used to simultaneously measure accurate masses of eluting components from eight parallel gradient LC columns. Accuracies better than 5 and 10 ppm were achieved for 50 and 80% of samples, respectively, from a single batch analysis of ten plates (960 samples) of a Fmoc-Asp(OtBu)-OH and reserpine mixture. Combinatorial library compounds were analyzed using this parallel high-throughput system in both positive and negative modes to rigorously verify expected products and identify side products. A mass accuracy of 10 ppm root mean square (RMS) is routinely obtained for combinatorial library samples from this high-throughput accurate mass LC/MS system followed by automated data processing. This mass accuracy is critical in revealing combinatorial synthesis problems that would be missed by unit mass measurement.     

Accurate mass measurement of DNA oligonucleotide ions using high-resolution time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) time-of-flight mass spectrometry (TOFMS) play an essential role in the analysis of biological molecules, not only peptides and proteins, but also DNA and RNA. Tandem mass spectrometry used for sequence analysis has been a major focus of technological developments in mass spectrometry, but accurate mass measurements by high-resolution TOFMS are equally important. This paper describes the role that high mass measurement accuracy can play in DNA composition assignment and discusses the influence of several parameters on mass measurement accuracy in both MALDI and ESI mass spectra. Five oligonucleotides (5-13mers) were used to test the resolving power and mass measurement accuracy obtained with MALDI and ESI instruments with reflectron TOF mass analyzers. The results from the experimental studies and additional theoretical calculations provide a basis to predict the practical utility of high-resolution TOFMS for the analysis of larger oligonucleotides.     

Multi-detection of corticosteroids in sports doping and veterinary control using high-resolution liquid chromatography/time-of-flight mass spectrometry

A liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) method was developed using the latest high-resolution LC column technology, the ultra performance liquid chromatography (UPLC), and electrospray ionization (ESI) in the positive ion mode. Gradient UPLC separation conditions were optimized for a group of 22 analytes comprising 17 glucocorticosteroids, specific designer steroids such as tetrahydrogestrinone (THG) and specific beta2-agonists such as formoterol. The UPLC/TOFMS separation obtained required 5.5 min only for all the substances tested. Even the critical pair of dexamethasone and betamethasone isomers was almost completely resolved. Thanks to the over 10,000 full-width at half maximum (FWHM) mass resolution and high mass accuracy features of TOFMS 50 mDa window accurate mass chromatograms could be reconstructed for the individual analytes. Sensitive screening in human and calf urine samples fortified at the glucocorticosteroids minimum required performance limit (MRPL) of 30 microg L(-1) (human urine, sports doping) and 2 microg L(-1) (calf urine, veterinary control) could be obtained. The potential of UPLC/TOFMS for confirmatory analysis was shown by determining the accurate mass of all compounds and fragment ions upon in-source collision-induced dissociation (CID) at different energies. The exact mass measurement errors for all glucocorticosteroids were found to be within 6 ppm. Considering veterinary control, limits of detection (LOD) and limits of quantification (LOQ) were determined for most of the analytes in calf urine and found to range from 0.1 to 3.3 and from 0.4 to 4.4 microg L(-1), respectively. The method can be easily extended with other banned substances of interest, as demonstrated by the addition of 21 beta2-agonists to the original analyte mixture in urine, without causing any interferences.     

Evaluation of a High Resolving Power Time-of-Flight Mass Spectrometer for Drug Analysis in Terms of Resolving Power and Acquisition Rate

Liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) is applied increasingly to various fields of small molecule analysis. The moderate resolving power (RP) of standard TOFMS instruments poses a risk of false negative results when complex biological matrices are to be analyzed. In this study, the performance of a high resolving power TOFMS instrument (maXis by Bruker Daltonik, Bremen, Germany) was evaluated for drug analysis. By flow injection analysis of critical drug mixtures, including a total of 17 compounds with nominal masses of 212–415 Da and with mass differences of 8.8–23.5 mDa, RP varied from 34,400 to 51,900 (FWHM). The effect of acquisition rate on RP, mass accuracy, and isotopic pattern fit was studied by applying 1, 2, 5, 10, and 20 Hz acquisition rates in a 16 min gradient elution LC separation. All three variables were independent of the acquisition rate, with an average mass accuracy and isotopic pattern fit factor (mSigma) of 0.33 ppm and 5.9, respectively. The average relative standard deviation of RP was 1.8%, showing high repeatability. The performance was tested further with authentic urine extracts containing a co-eluting compound pair with a nominal mass of 296 Da and an 11.2 mDa mass difference. The authentic sample components were readily resolved and correctly identified by the automated data analysis. The average RP, mass accuracy, and isotopic pattern fit were 36,600, 0.9 ppm, and 7.3 mSigma, respectively.     

Toxicological screening of urine for drugs by liquid chromatography/time-of-flight mass spectrometry with automated target library search based on elemental formulas

The present study describes a novel approach for utilizing liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) in qualitative screening analysis. An LC/TOFMS method was developed for screening toxicologically relevant substances in urine samples. After solid phase extraction and LC separation, the method included full spectrum acquisition followed by automatic internal calibration, searching against a target library, and reporting positive identifications. The target library, containing 433 toxicologically relevant substances in the mass range of 105-734 Da, was created simply by entering the elemental formulas of substances into the instrument software for the calculation of their respective monoisotopic masses. In addition to parent drugs, the library contained selected urinary drug metabolites, based on their structures available in the literature. Identification was based on the exact masses of the compounds. The LC/TOFMS method provided 5-10 ppm mass accuracy for a majority of identified compounds in authentic urine samples. Compared with established thin-layer and gas chromatographic methods, the LC/TOFMS method produced similar findings in urine with the additional advantage of metabolite identification without actual reference substances.     

Sample preparation for high throughput accurate mass analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

An automated sample preparation for high throughput accurate mass determinations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been developed. Sample preparation was performed with an automated workstation and automated mass analyses were performed with a commercial MALDI-TOF mass spectrometer. The method was tested with a 41-sample library. MALDI-TOFMS was found to give the needed sensitivity, accurate mass measurement, and soft ionization necessary for structure confirmation, even of mixtures. A mass accuracy of 5 ppm or less was obtained in over 80% of known compound measurements. A mass accuracy better than 10 ppm was obtained for all measurements of known compounds. Analyses of parallel synthesis products resulted in 77% of the measurements with a mass accuracy of 5 ppm or better.     

Identification of anabolic steroids and derivatives using bioassay-guided fractionation, UHPLC/TOFMS analysis and accurate mass database searching

Biological tests can be used to screen samples for large groups of compounds having a particular effect, but it is often difficult to identify a specific compound when a positive effect is observed. The identification of an unknown compound is a challenge for analytical chemistry in environmental analysis, food analysis, as well as in clinical and forensic toxicology. In this study bioassay-guided fractionation, ultra high performance liquid chromatography combined with time-of-flight mass spectrometry (UHPLC/TOFMS) and accurate mass database searching was tested to detect and identify unknown androgens. Herbal mixtures and sport supplements were tested using an androgen bioassay and modifications in sample preparations were carried out in order to activate inactive pro-androgens, androgen esters and conjugated androgens to enable their detection in the bioassay. Two of the four herbal mixtures tested positive and bioassay-guided fractionation followed by UHPLC/TOFMS of positive fractions resulted in the identification of nortestosterone phenylpropionate, testosterone cyclohexanecarboxylate and methyltestosterone. Three of the four sport supplements reacted toxic in the bioassay or gave inconclusive results and were further investigated using UHPLC/TOFMS in combination with data processing software and an accurate mass database having approximately 40,000 entries. This accurate mass database was derived from the PubChem database on the internet and coupled to the TOFMS software. This resulted in the tentative identification of several androgens, including methylboldenone, testosterone and the androgen esters methyltestosterone propionate or testosterone isobutyrate, testosterone buciclate and methylenetestosterone acetate. The study showed that bioassay-guided fractionation in combination with UHPLC/TOFMS analysis is a useful procedure to detect, isolate and identify unknown androgens in suspected samples. As an alternative, the use of data processing software in combination with an accurate mass database and coupled on-line with the TOFMS instrument software enabled the identification of androgens and androgen esters in the chromatogram even without bioassay-guided fractionation.     

Correction of accurate mass measurement for target compound verification by quadrupole time-of-flight mass spectrometry

The aim of this work is to evaluate quadrupole/time-of-flight (QTOF) mass spectrometry for simultaneous measurement of accurate mass and quantification of a target by using a stable isotopically labeled internal standard. Mixtures of caffeine and (13)C(3)-caffeine (internal standard) at different concentration ratios were analyzed by capillary HPLC/QTOF. A calibration plot for quantification is linear over a factor of 20. Without invoking any correction scheme, the mass accuracy seriously degraded when the ratio of the mass standard to the test compound was not unity. The accuracy could be restored to approximately 2 ppm by using a quadratic function to correct the measured mass as a function of the measured signal ratio of target and internal calibrant.     

Automated accurate mass data processing using a gas chromatograph/time-of-flight mass spectrometer in drug discovery

A gas chromatograph/time-of-flight (GCT) mass spectrometer, with high mass measurement accuracy to within 5 ppm, has been used for the automated accurate mass analysis of multicomponent mixtures and drug discovery compounds. A multicomponent mixture was analyzed several times over the course of a week to assess the reproducibility and ruggedness of the automated method while operating the GCT in electron ionization mode. For example, the data for 31 radical cations generated via electron ionization was processed using automated software (i.e. OpenLynx) to provide for mass accuracies less than 5 ppm for nearly 100% of the ions from multiple injection data. Mass accuracies of the radical anions of polyaromatic hydrocarbons generated via negative chemical ionization, and protonated pyridines and quinolines generated via methane chemical ionization, were mainly less than 5 ppm from multiple injection data. In addition, the automated method has been used for the accurate mass analysis of drug discovery compounds.     

Detection, characterization and identification of major constituents in Zhimu-Baihe herb-pair extract by fast high-performance liquid chromatography and time-of-flight mass spectrometry through dynamic adjustment of fragmentor voltage

This work describes a novel methodology for unequivocal identification of chemical constituents in Zhimu‐Baihe herb‐pair (ZMBHHP). Compounds were removed from ZMBHHP by ultrasonic extraction with 70% ethanol, and then analyzed by fast high‐performance liquid chromatography (HPLC) coupled with time‐of‐flight mass spectrometry (TOFMS). The accurate‐mass capability of the TOF analyzer allowed reliable confirmation of the identity of the detected compounds, normally with mass errors below 3 ppm in routine analysis. This mass accuracy was sufficient to verify the elemental compositions of the chemical constituents in ZMBHHP. With dynamic adjustment of fragmentor voltage in TOFMS, an efficient ion transmission was achieved to obtain the best sensitivity and abundant fragmentation. By accurate mass measurements for each molecular ion and subsequent fragment ions, a reliable identification and differentiation of 24 saponins, 3 xanthones, 1 anthraquinone and 2 alkaloids was described here, including four groups of isomers. It is concluded that this fast and sensitive HPLC/ESI‐TOFMS technique is powerful in qualitative analysis of complex herbal medicines in terms of sensitivity, selectivity, resolving power, time savings and lower solvent consumption. Furthermore, the data gathered in this study may be helpful for understanding the synergistic nature of this herb pair in traditional Chinese medicine (TCM) and further pharmacokinetic studies of ZMBHHP. Copyright © 2010 John Wiley & Sons, Ltd.     

若干气溶胶单粒子化学成分的实时测量

利用激光解吸附电离飞行时间质谱技术获得了若干已知化学成分的气溶胶粒子的飞行时间质谱,分析标识了各类气溶胶粒子的特征离子谱峰,并对一些特征峰的形成机理进行了探讨。在此基础上,对烟花火药以及纸张燃烧产生的烟气气溶胶粒子进行了实时在线测量,通过对质谱图的分析,获得了有关此两类燃烧过程产生的烟气气溶胶单粒子的化学组成信息。     

Development of a simple and reliable accurate mass liquid chromatography/electrospray ionization mass spectrometry method for high-resolution accurate mass determinations of new drug entities on a triple quadrupole mass spectrometer

Accurate mass measurements are used to determine the elemental composition and formulae of molecules to confirm their identity or to assist in their characterization. Currently, the most widely used techniques for measuring exact masses employ magnetic sector instruments, Fourier transform ion cyclotron resonance mass spectrometers and lower resolution instruments such as time-of-flight (TOF) and quadrupole-TOF. This paper reports the accurate mass measurement using a triple quadrupole mass spectrometer. Indeed, the recently introduced triple quadrupole mass spectrometer, with unique enhanced mass-resolution capability, has demonstrated simple data acquisition methods and requires few experiments to measure exact masses with accuracy and determines elemental compositions of both protonated and deprotonated molecules. All the accurate mass measurements were performed using both positive and negative electrospray ionization in enhanced mass-resolution mode (peak width of 0.1 Th FWMH). Several new drug entities were investigated as simulated unknowns and analyzed by means of an accurate mass liquid chromatography/electrospray ionization mass spectrometry (AM-LC/ESI-MS) method. The accurate mass measurements resulted in only one proposed elemental composition for all tested compounds, using reasonable elemental limits and mass tolerance for the calculation. Moreover, all the experimentally determined accurate mass measurements gave satisfactory results in terms of accuracy (lower than 5 ppm).     

High-throughput bioanalysis with simultaneous acquisition of metabolic route data using ultra performance liquid chromatography coupled with time-of-flight mass spectrometry

The capability of ultra performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC/TOFMS) in the high-throughput quantitative analysis of a drug candidate in plasma has been investigated. Data obtained were compared with results from conventional analysis by high-performance liquid chromatography with tandem mass spectrometric detection on a triple quadrupole instrument (HPLC/MS/MS). The accuracies and precisions of the two approaches were comparable. The UPLC/TOFMS system displayed excellent robustness over the course of 276 injections of protein-precipitated plasma samples. With the instrumentation used, the limits of detection and quantification were approximately five-fold higher with UPLC/TOFMS than for HPLC/MS/MS. Nevertheless, the UPLC/TOFMS system proved adequate to quantify plasma concentrations of a drug molecule administered orally to rats at a pharmacologically relevant dose of 4 mg/kg. As well as providing quantitative data on the test compound, it was also possible to extract data for eight different metabolites, including several isomeric species (three +O and three +2O) from the UPLC/TOFMS data sets, using an analytical method with a 2.5-minute run time. Selectivity for the test compound and its metabolites was derived from the accurate mass capabilities of the TOF instrument, and no MS method development was required.     

大气压离子化技术/飞行时间质谱联用进展

综述了大气压离子化技术/飞行时间质谱联用技术及其应用的进展     

Ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry for robust, high-throughput quantitative analysis of an automated metabolic stability assay, with simultaneous determination of metabolic data

The application of ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC/TOFMS) for high-throughput analysis of a 96-well plate based metabolic stability assay has been investigated. Full-scan data were acquired, with run times of 2.5-3.5 min, from which narrow window extracted ion chromatograms were generated, producing quantitative data for the test compound equivalent to that obtained by high-performance liquid chromatography with tandem mass spectrometric detection on a triple quadrupole instrument (HPLC/MS/MS). Sensitivity and mass accuracy were maintained over the analysis of >300 samples. Additionally, the UPLC/TOFMS datasets obtained gave access to metabolic route information, at no cost in terms of sensitivity for the test compound.     

Screening and confirmation criteria for hormone residue analysis using liquid chromatography accurate mass time-of-flight, Fourier transform ion cyclotron resonance and orbitrap mass spectrometry techniques

An emerging trend is recognised in hormone and veterinary drug residue analysis from liquid chromatography tandem mass spectrometry (LC/MS/MS) based screening and confirmation towards accurate mass alternatives such as LC coupled with time-of-flight (TOF), Fourier transform ion cyclotron resonance (FTICR) or Fourier transform orbitrap (FT Orbitrap) MS. In this study, mass resolution and accuracy are discussed for LC/MS screening and confirmation of targeted analytes and for the identification of unknowns using the anabolic steroid stanozolol and the designer beta-agonist "Clenbuterol-R" as model substances. It is shown theoretically and experimentally that mass accuracy criteria without proper mass resolution criteria yield false compliant (false negative) results, both in MS screening and MS/MS confirmation of stanozolol. On the other hand, previous medium resolution accurate mass TOFMS/MS data of the designer beta-agonist were fully confirmed by high resolution FT Orbitrap MS(n) experiments. A discussion is initiated through a proposal for additional criteria for the use of accurate mass LC/MS technologies, to be implemented in Commission Decision 2002/657/EC.     

Application of time-of-flight mass spectrometry to the analysis of phototransformation products of diclofenac in water under natural sunlight

Exact mass capabilities of time-of-flight (TOF) mass spectrometry along with other mass spectrometric techniques have been evaluated to elucidate a complete range of dichlofenac phototransformation products. Photolysis experiments with diclofenac in water under direct solar irradiation were performed to characterise the main phototransformation products generated and to determine their stability. Photolysis experiments were performed in both demineralised water and reconstructed standard freshwater. Samples were extracted before analysis by solid phase extraction (SPE) with Oasis HLB and MAX cartridges. Separation and identification of the transformation products were accomplished by the combined use of gas chromatography-mass spectrometry (GC/MS) and liquid chromatography coupled with time-of-flight mass spectrometry (LC/TOFMS). Both techniques provided complementary information that enabled the identification of 13 phototransformation products. Six of them were identified by GC/MS through the structural information provided by the full scan mass spectra obtained under electron impact (EI) ionisation and the confirmation of the molecular mass provided by positive chemical ionisation (PCI) analyses. Accurate mass measurements obtained by LC/TOFMS provided the elucidation of seven polar transformation products. The low mass error observed (<2 ppm) enabled the assignment of highly probable empirical formulas as well as identification of a process dimerisation route. The photoproducts identified demonstrated that photolysis of diclofenac occurs by two main routes. One is the consequence of the initial photocyclisation of diclofenac into carbazole derivatives. The other route goes through the initial decarboxilation of diclofenac and further oxidation of the alkyl-chain, which are typical photolytic process reactions. The main photoproduct identified was 8-chloro-9H-carbazole-1yl-acetic acid.     

Ultra-performance liquid chromatography/time-of-flight mass spectrometry based metabolomics of raw and steamed Panax notoginseng

At present, metabolite profiling is of growing importance in herbal medicine fields such as breeding, formulation, quality control and clinical trials. This preliminary study indicated that ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOFMS)-based metabolomics allows direct detection of down-stream derivatives of metabolites, arising from the herbal formulation process. This analytical approach allows the discrimination and tentative authentication of unique biomarkers related to different herbal extracts using unsupervised multivariate principal component analysis (PCA). The tentative identification of biomarkers is complemented significantly by the accurate mass measurement of TOFMS and the high resolution and high retention time reproducibility rendered by UPLC. The application of this approach in herbal extract discrimination and ginsenoside biomarker discovery of raw and steamed Panax notoginseng (Burk.) F.H. Chen is demonstrated and discussed.     

Comparison of comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry and gas chromatography-mass spectrometry for the analysis of tobacco essential oils

A sample of tobacco essential oil was analyzed using gas chromatography-mass spectrometry (GC/MS) and comprehensive two-dimensional gas chromatography coupled to a time-of-flight mass spectrometry (GC × GC/TOFMS), respectively. In the GC/MS analysis, serially coupled columns were used. By comparing the GC/MS results with GC × GC/TOFMS results, many more components in the essential oil could be found within the two-dimensional separation space of GC × GC. The quantitative determination of components in the essential oil was performed by GC × GC with flame ionization detection (FID), using a method of multiple internal standards calibration.