Isotopic pattern and accurate mass determination in urine drug screening by liquid chromatography/time-of-flight mass spectrometry

An efficient method was developed for toxicological drug screening in urine by liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry. The method relies on a large target database of exact monoisotopic masses representing the elemental formulae of reference drugs and their metabolites. Mass spectral identification is based on matching measured accurate mass and isotopic pattern (SigmaFit) of a sample component with those in the database. Data post-processing software was developed for automated reporting of findings in an easily interpretable form. The mean and median of SigmaFit for true-positive findings were 0.0066 and 0.0051, respectively. The mean and median of mass error absolute values for true-positive findings were 2.51 and 2.17 ppm, respectively, corresponding to 0.65 and 0.60 mTh. For routine screening practice, a SigmaFit tolerance of 0.03 and a mass tolerance of 10 ppm were chosen. Ion abundance differences from urine extracts did not affect the accuracy of the automatically acquired SigmaFit or mass values. The results show that isotopic pattern matching by SigmaFit is a powerful means of identification in addition to accurate mass measurement.     

Intercomparison study on accurate mass measurement of small molecules in mass spectrometry

The results from an intercomparison of accurate mass measurement of a small molecule (molecular weight 475 Da) across a broad range of mass spectrometers are reported. The intercomparison was designed to evaluate the relative capabilities and the optimum methodology of the diverse range of mass spectrometers currently used to record accurate mass measurements. The data will be used as a basis for developing guidance on accurate mass measurement. The need for guidance has resulted from the continued growth in the use of accurate mass measurements for assignment of elemental formula in the chemical and biochemical industries. This has been fuelled by a number of factors and includes the rapid pace of instrument development, which has enabled accurate mass measurements to be made in a less costly, yet robust fashion. The data from the intercomparison will allow us to compare those protocols that produced excellent accuracy and precision with those that produced poorer accuracy and/or precision for each type of mass spectrometer. The key points for best practice will then be established from this comparison for each type of mass spectrometer and accurate mass measurement technique. A compound was sent to the participating laboratories (in the UK, Europe, and USA), the identity of which was not revealed. Each laboratory was asked to record a minimum of five repeat mass measurements of the molecular species using their local protocols and their preferred ionization technique or techniques. To the best of our knowledge there were no interfering (unresolved) ions that originated from the sample. A questionnaire was also completed with the experimental work. The information from the questionnaires was used to evaluate the protocols used to record the measurements. Forty-five laboratories have reported their results. To summarize the performance of mass spectrometers in the intercomparison, magnetic sector field mass spectrometers used in peak matching mode and FTMS reported the highest mean mass measurement accuracy (88 and 83%, respectively, achieved < or =1 ppm). Magnetic sector field mass spectrometers used in voltage scanning produced 60% of the mean mass measurements with accuracy < or =1 ppm. Magnetic sector field mass spectrometers used in magnet scanning modes, quadrupole-TOF and TOF instruments generally achieved mean mass measurement accuracy between 5 and 10 ppm. The two low resolution triple quadrupoles used in the inter-comparison produced mean mass measurement accuracy of <2 ppm. The precision of the data from each instrument and experiment type is an important consideration when evaluating their relative capabilities. Using both the precision and accuracy, it will be possible to define the uncertainty associated with the elemental formulae derived from accurate mass measurements. Therefore, a thorough statistical evaluation of the data is underway and will be presented in a subsequent publication.     

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).     

Accurate mass measurements and ultrahigh-resolution: evaluation of different mass spectrometers for daily routine analysis of small molecules in negative electrospray ionization mode

Six mass spectrometers based on different mass analyzer technologies, such as time-of-flight (TOF), hybrid quadrupole-TOF (Q-TOF), orbitrap, Fourier transform ion cyclotron resonance (FT-ICR), and triple quadrupole (QqQ), installed at independent laboratories have been tested during a single day of work for the analysis of small molecules in negative electrospray ionization (ESI) mode. The uncertainty in the mass measurements obtained from each mass spectrometer has been determined by taking the precision and accuracy of replicate measurements into account. The present study is focused on calibration processes (before, after, and during the mass measurement), the resolving power of the mass spectrometers, and the data processing for obtaining elemental formulae. The mass range between m/z 100 and 600 has been evaluated with a mix of four standards. This mass range includes small molecules usually detected in food and environmental samples. Negative ESI has been tested as there is almost no data on accurate mass (AM) measurements in this mode. Moreover, it has been used because it is the ESI mode for analysis of many compounds, such as pharmaceutical, herbicides, and fluorinated compounds. Natural organic matter has been used to demonstrate the significance of ultrahigh-resolution in complex mixtures. Sub-millidalton accuracy and precision have been obtained with Q-TOF, FT-ICR, and orbitrap achieving equivalent results. Poorer accuracy and precision have been obtained with the QqQ used: 11 mDa root-mean-square error and 6–11 mDa standard deviation. Some advice and requirements for daily AM routine analysis are also discussed here.     

The validation of exact mass measurements for small molecules using FT-ICRMS for improved confidence in the selection of elemental formulas

The confidence in an individual measurement is the most important factor when selecting the elemental formula candidates from the list of possible elemental compositions following an exact mass measurement. It is the single mass measurement capability rather than the averaged mass measurement potential of the mass spectrometer that is the critical factor when validating the exact mass measurements of small molecules. Here, an experimental protocol has been established to determine the frequency of exact mass measurement by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICRMS) at known relative ion abundance ratios (RA). This in turn allows for statements about the confidence limit for any single exact mass measurement to be made. This is particularly crucial for a high throughput, automated environment where operator intervention is required to be minimal and repeat analyses are to be avoided. The relative ion abundance calculations are essential to determine the working ranges for specific sample ion abundances. Further, it has been shown that if the sample ion abundance is low, then the ion abundance range for the calibration file does not need to be exactly or closely matched, again benefiting the high throughput application.     

Fourier transform ion cyclotron resonance mass spectrometry for the characterisation of kavalactones in the kava plant: elemental formulae confirmation by dual spray accurate mass measurement and structural confirmation by infrared multiphoton dissociation and sustained off-resonance irradiation collision induced dissociation

Roots and extracts of the kava plant have been used in herbal medicine to treat sleep disturbances, stress and anxiety, although reported cases of liver toxicity led to many countries restricting its sale. The detection of the presence of kava in many medicinal products requires the use of methods capable of identifying the kavalactones with high certainty. Here, we describe the use of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) for the characterisation of six kavalactones (kavain, dihydrokavain, methysticin, dihydromethysticin, yangonin and desmethoxyyangonin) utilising accurate mass measurement for the determination of their elemental formulae and product ion MS (both sustained off-resonance irradiation collision-induced dissociation and infrared multiphoton dissociation (SORI-CID and IRMPD) for structural confirmation. High performance liquid chromatography/FT-ICR-MS with a dual spray system for internal calibration of mass spectra was employed for accurate mass measurement and the determination of elemental formulae of the kavalactones in both standards and a root extract to confirm the presence of the kavalactones in the root powder. Mass accuracy of < 1 ppm was achieved. For structural confirmation, the IRMPD and SORI-CID spectra of the kavalactones in standards and a kava root powder extract were compared. Accurate mass measurement of the product ions was also conducted by external calibration and the elemental formula determined to aid with structural confirmation. The presence of the same fragment ions detected in the standards as in the extract further confirmed the presence of the kavalactones in the kava root powder with high certainty.     

Mass spectral characterization of phloroglucinol derivatives hyperforin and adhyperforin

Active phloroglucinol constituents of Hypericum perforatum (St. John's wort) extracts, hyperforin and adhyperforin, have been studied following ion activation using tandem mass spectrometry (MS/MS) and complemented by accurate mass measurements. These two compounds were readily analyzed as protonated and deprotonated molecules with electrospray ionization. MS/MS and MS3 data from a quadrupole-linear ion trap tandem mass spectrometer were employed to elucidate fragmentation pathways. Fourier transform ion cyclotron resonance measurements afforded excellent mass accuracies for the confirmation of elemental formulae of product ions formed via infrared multiphoton dissociation and sustained off-resonance irradiation collision-induced dissociation. Fragmentation schemes have been devised for the dissociation of hyperforin and adhyperforin in negative and positive ion modes. This information is expected to be especially valuable for the characterization of related compounds, such as degradation products, metabolites and novel synthetic analogs of hyperforin.     

Analysis of carbon and nitrogen co-metabolism in yeast by ultrahigh-resolution mass spectrometry applying 13C- and 15N-labeled substrates simultaneously

Alternative metabolic pathways inside a cell can be deduced using stable isotopically labeled substrates. One prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of single-element isotopes, mainly (13)C. Here, we demonstrate the application of direct infusion nanospray, ultrahigh-resolution Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) for metabolic studies using differently labeled elemental isotopes simultaneously--i.e., (13)C and (15)N--in amino acids of a total protein hydrolysate. The optimized strategy for the analysis of metabolism by a hybrid linear ion trap-FTICR-MS comprises the collection of multiple adjacent selected ion monitoring scans. By limiting both the width of the mass range and the number of ions entering the ICR cell with automated gain control, sensitive measurements of isotopologue distribution were possible without compromising mass accuracy and isotope intensity mapping. The required mass-resolving power of more than 60,000 is only achievable on a routine basis by FTICR and Orbitrap mass spectrometers. Evaluation of the method was carried out by comparison of the experimental data to the natural isotope abundances of selected amino acids and by comparison to GC/MS results obtained from a labeling experiment with (13)C-labeled glucose. The developed method was used to shed light on the complexity of the yeast Saccharomyces cerevisiae carbon-nitrogen co-metabolism by administering both (13)C-labeled glucose and (15)N-labeled alanine. The results indicate that not only glutamate but also alanine acts as an amino donor during alanine and valine synthesis. Metabolic studies using FTICR-MS can exploit new possibilities by the use of multiple-labeled elemental isotopes.     

Isotope pattern evaluation for the reduction of elemental compositions assigned to high-resolution mass spectral data from electrospray ionization fourier transform ion cyclotron resonance mass spectrometry

The number of possible chemical formulae assigned to an accurate determined mass was significantly reduced by comparing spectral and theoretical isotope patterns based on mass measurement obtained with an ultrahigh-resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer (ESI-FTICR-MS) at high field intensity (7 T). Reduction is performed by rating congruency between experimental and theoretical pattern intensity and mass, and filtering out compositions with insufficient user-definable results. The methods used for isotope pattern simulation, peak searching, and comparison will be briefly described and evaluated on molecule ion signals of 25 compounds (300-1000 Da) applying a mass accuracy of +/-5 ppm, a set of eight elements with constant constraints (C0-200H0-1000N0-15O0-15S0-2Cl0-2Br0-2Ru0-1), natural isotope abundances and experimental resolution (full width at half maximum).     

Accurate mass measurement using Fourier transform ion cyclotron resonance mass spectrometry for structure elucidation of designer drug analogs of tadalafil, vardenafil and sildenafil in herbal and pharmaceutical matrices

Phosphodiesterase type 5 (PDE-5) inhibitors are a class of drugs used primarily in the treatment of erectile dysfunction. The Food and Drug Administration (FDA) approved PDE-5 inhibitors include sildenafil citrate, vardenafil hydrochloride and tadalafil. In this study, accurate mass measurements were made by electrospray ionization (ESI) using Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) to elucidate the structures of sildenafil, tadalafil and vardenafil analogs that were found in products marketed as dietary supplements. Initial detection of these analogs was accomplished through routine screening of suspect samples by liquid chromatography/electrospray ionization multi-stage mass spectrometry (LC/ESI-MS(n)) on a low-resolution ion trap instrument. The chromatographic behavior and mass spectrometric fragmentation patterns observed were often similar to those observed for FDA approved PDE-5 inhibitors. The mass accuracy and resolving power associated with FTICRMS allows for the determination of elemental compositions. Elucidation of the product ion structures for the analogs was accomplished through the use of accurate mass measurements with the aid of Mass Frontier software (version 4.0). Using FTICRMS, accurate masses with measurement errors averaging <0.4 ppm were achieved, allowing assignment of one possible elemental formula to each fragment ion. The mass measurement errors associated with [M + H](+) for the analogs aminotadalafil, piperidino vardenafil, hydroxyacetildenafil and piperidino acetildenafil were 0.1, 0.0, 0.1 and 0.5 ppm, respectively. Based on the accuracy of the measurements, structural assignments could be made with a high degree of confidence.     

Second hydrogen atom abstraction by molecular ions

We report the observation of a new physical phenomenon of the addition of 2 hydrogen atoms to molecular ions thus forming [M + 2H]⁺ ions. We demonstrate such second hydrogen atom abstraction onto the molecular ions of pentaerythritol and trinitrotoluene (TNT). We used both gas chromatography mass spectrometry (GC‐MS) with supersonic molecular beam (SMB) with methanol added into its make‐up gas and electron ionization (EI) liquid chromatography mass spectrometry (LC‐MS) with SMB with methanol as the LC solvent. We found that the formation of methanol clusters resulted upon EI in the formation of dominant protonated pentaerythritol ion at m/z = 137 plus about 70% relative abundance of pentaerythritol molecular ion with 2 additional hydrogen atoms at m/z = 138 which is well above the 5.7% natural C¹³ isotope abundance of protonated pentaerythritol. Similarly, we found an abundant protonated TNT ion at m/z = 228 and a similar abundance of TNT molecular ion with 2 additional hydrogen atoms at m/z = 229. Upon the use of deuterated methanol (CD₃OD) as the solvent, we observed an abundant m/z = 231 (M + 2D)⁺ of TNT with 2 deuterium atoms. We found such abundant second hydrogen atom abstraction with butylglycolate and at low abundances in dioctylphthalate, Vitamin K3, phenazine, and RDX. At this time, we are unable to report the magnitude and frequency of occurrence of this phenomenon in standard electrospray LC‐MS. This observation could have important implications on the provision of elemental formula from mass spectra that are involved with protonated molecules. Accordingly, while accurate mass measurements can serve for the generation of elemental formula, their further support and improvement via isotope abundance analysis are questionable. Consequently, if a given compound can be analyzed by both GC‐MS and LC‐MS, its GC‐MS analysis can be superior for the provision of accurate elemental formulae if its EI mass spectrum exhibits abundant molecular ions such as with GC‐MS with SMB (also known as cold EI).     

Isotope abundance analysis methods and software for improved sample identification with supersonic gas chromatography/mass spectrometry

We present newly developed isotope abundance analysis (IAA) methods and software which are used to derive elemental formula information from experimental mass spectral data of molecular ion isotopomeric abundances. The software, using a novel method, can also be used to automatically confirm or reject NIST library search results, thereby significantly improving the confidence level in sample identifications. In the case of IAA confirmation of the NIST library results, sample identification is unambiguous, since the confirmation is achieved by two independent sets of data and analytical methods. In the case of a rejection, such as when the molecule is not included in the library's databases, the IAA software independently provides a list of elemental formulae with declining order of matching to the isotopomeric experimental data, in a similar way to accurate mass measurements with costly instruments. IAA is ideally applicable to gas chromatography/mass spectrometry (GC/MS) (and liquid chromatography/electron ionization mass spectrometry (LC/EI-MS)) with a supersonic molecular beam (SMB) since it requires a trustworthy and highly abundant true molecular ion that is unique to the SMB-MS systems, plus the absence of self chemical ionization and vacuum background noise, again unique features of GC/SMB-MS. The various features of the IAA methods and software are described, their performance is demonstrated with the analysis of experimental GC-SMB-MS data and the IAA concept is compared with accurate mass alternatives. The combination of IAA and GC/SMB-MS is believed to be superior to accurate mass GC/MS in view of the general availability of trustworthy molecular ions for an extended range of compounds.     

Paradigms in isotope dilution mass spectrometry for elemental speciation analysis

Isotope dilution mass spectrometry currently stands out as the method providing results with unchallenged precision and accuracy in elemental speciation. However, recent history of isotope dilution mass spectrometry has shown that the extent to which this primary ratio measurement method can deliver accurate results is still subject of active research. In this review, we will summarize the fundamental prerequisites behind isotope dilution mass spectrometry and discuss their practical limits of validity and effects on the accuracy of the obtained results. This review is not to be viewed as a critique of isotope dilution; rather its purpose is to highlight the lesser studied aspects that will ensure and elevate current supremacy of the results obtained from this method.     

Modern Russian mass spectrometers for the atomic industry

Product quality control within the nuclear fuel cycle is a subject of special concern in Russia nowadays. Earlier, mass spectrometers of foreign production were commonly used for elemental and isotope analysis of samples. Currently, a series of domestic mass spectrometers MTI-350 has been developed and their production has been organized in Russia. The series comprises an automated MTI-350G mass spectrometer for the isotope analysis of uranium hexafluoride; thermal ionization MTI-350T mass spectrometer for the isotope analysis of uranium, plutonium, and mixed oxide (MOX) fuels; an MTI-350GS mass spectrometer for controlling the production of uranium hexafluoride; and an MTI-350GP mass spectrometer for the determination of the impurity concentration in uranium hexafluoride. The article considers operation principles, analytical characteristics, and the advantages of the above mass spectrometers.     

Regioisomeric analysis of triacylglycerols using silver-ion liquid chromatography-atmospheric pressure chemical ionization mass spectrometry: comparison of five different mass analyzers

Silver-ion high-performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionization mass spectrometry (APCI-MS) is used for the regioisomeric analysis of triacylglycerols (TGs). Standard mixtures of TG regioisomers are prepared by the randomization reaction from 8 mono-acid TG standards (tripalmitin, tristearin, triarachidin, triolein, trielaidin, trilinolein, trilinolenin and tri-gamma-linolenin). In total, 32 different regioisomeric doublets and 11 triplets are synthesized, separated by silver-ion HPLC using three serial coupled chromatographic columns giving a total length of 75cm. The retention of TGs increases strongly with the double bond (DB) number and slightly for regioisomers having more DBs in sn-1/3 positions. DB positional isomers (linolenic vs. γ-linolenic acids) are also separated and their reverse retention order in two different mobile phases is demonstrated. APCI mass spectra of all separated regioisomers are measured on five different mass spectrometers: single quadrupole LC/MSD (Agilent Technologies), triple quadrupole API 3000 (AB SCIEX), ion trap Esquire 3000 (Bruker Daltonics), quadrupole time-of-flight micrOTOF-Q (Bruker Daltonics) and LTQ Orbitrap XL (Thermo Fisher Scientific). The effect of different types of mass analyzer on the ratio of [M+H-R(i)COOH](+) fragment ions in APCI mass spectra is lower compared to the effect of the number of DBs, their position on the acyl chain and the regiospecific distribution of acyl chains on the glycerol skeleton. Presented data on [M+H-R(i)COOH](+) ratios measured on five different mass analyzers can be used for the direct regioisomeric determination in natural and biological samples.     

Automated neutral loss and data dependent energy resolved "pseudo MS3" for the targeted identification, characterization and quantitative analysis of methionine- containing peptides

A strategy involving the fixed-charge sulfonium ion derivatization, stable isotope labeling, capillary high- performance liquid chromatography and automated data dependent neutral loss scan mode tandem mass spectrometry (MS/MS) and "pseudo multiple mass spectrometry (MS(3))" product ion scans in a triple quadrupole mass spectrometer has been developed for the "targeted" gas-phase identification, characterization and quantitative analysis of low abundance methionine-containing peptides present within complex protein digests. Selective gas-phase "enrichment" and identification is performed via neutral loss scan mode MS/MS, by low energy collision-induced dissociation of the derivatized methionine side chain, resulting in the formation of a single characteristic product ion. Structural characterization of identified peptides is then achieved by automatically subjecting the characteristic neutral loss product ion to further dissociation by data dependent product ion scan mode pseudo MS(3) under higher collision energy conditions. Quantitative analysis is achieved by measurement of the abundances of characteristic product ions formed by sequential neutral loss scan mode MS/MS experiments from "light" ((12)C) and "heavy" ((13)C) stable isotope encoded fixed-charge derivatized peptides. In contrast to MS-based quantitative analysis strategies, the neutral loss scan mode MS/MS method employed here was able to achieve accurate quantification for individual peptides at levels as low as 100 fmol and at abundance ratios ranging from 0.1 to 10, present within a complex protein digest.     

Effect of post-excitation radius on ion abundance, mass measurement accuracy, and isotopic distributions in Fourier transform ion cyclotron resonance mass spectrometry

We report an evaluation of a modern Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) instrument to determine the general trend of post-excitation radius on total ion abundance, mass measurement accuracy, and isotopic distributions for internally calibrated mass spectra. The optimum post-excitation radius was determined using total ion abundance, mass measurement accuracy (MMA), and isotope ratios. However, despite the utility of internal calibration for achieving ultimate MMA, the internal calibrant ions were insufficient for compensating for sub-optimum ICR cell conditions. The findings presented herein underscore the importance of determining the optimal post-excitation radius in FT-ICR-MS to achieve high ion abundance (low limits of detection), high MMA, and valid isotopic distributions.     

Identification of glucuronide conjugates of ketobemidone and its phase I metabolites in human urine utilizing accurate mass and tandem time-of-flight mass spectrometry

The glucuronide conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone were identified in human urine post-intravenous administration of Ketogan Novum. The human urine was extracted on a mixed-mode solid-phase micro-column before analysis with liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) and tandem MS (MS/MS). Accurate mass and collision-induced dissociation product ion spectra were used for identification of the glucuronide conjugates. Two different TOF mass spectrometers were used and the accurate mass measurements were performed on three separate days with each instrument. The accuracy of the mass measurements was better than 2.1 ppm for two out of three conjugates and the inter-day relative standard deviation was within +/-0.00049%. The MS/MS fragmentation patterns of the conjugates were in accordance with those of the synthetic aglycones and included peaks originating from the [M + H](+) ion of the respective aglycone.     

Rapid 18O analysis of small water and CO2 samples using a continuous-flow isotope ratio mass spectrometer

High-frequency throughput is often needed in isotopic studies in biological and medical fields. Here we report that high-precision oxygen isotope ratio measurements of water (+/-0.13 per thousand) were rapidly and routinely made on small samples (40-100 microL) using an isotope ratio mass spectrometer operated in continuous-flow mode. Simple modifications to existing instrumentation allow for rapid manual analyses of dilute CO2 (10% CO2/90% N2), including the addition of a septum port and water trap prior to the gas chromatography (GC) column (elemental analyzer column in this study) and the extension of fused-silica capillary tubing between the mass spectrometer source and the effluent tubing from the GC column (located within the CONFLO unit on Finnigan mass spectrometers). We routinely analyzed 20 small-volume samples per hour using this technique, without sacrificing precision of the oxygen isotope ratio measurement.     

Mass calibration of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer including the rise time of the delayed extraction pulse

To utilize fully modern MALDI-TOF and TOF/TOF mass spectrometers with mass resolution exceeding 10,000 and 2 ppm precision of flight time measurements for high mass accuracy, the model of ion motion used in the mass calibration equation must be expanded. The standard three-term equation providing up to 5-10 ppm (rms) mass accuracy with internal standards was modified with an additional term accounting for the finite rise time of the high-voltage extraction pulse. This new four-term calibration equation minimizes the effect of systematic error resulting from the fact that ion velocities are mass dependent due to the rise time of the extraction pulse. Applying this new calibration equation to a mass spectrum obtained in an axial MALDI-TOF MS containing 70 peaks (sodiated PEG), each with a signal-to-noise ratio greater than 100, a mass accuracy of 1.6 ppm (rms) was obtained over the mass range 1.0-4.0 kDa compared with 3.6 ppm (rms) with the standard three-term equation. The physical basis of the effects of the finite extraction pulse rise time on mass calibration is examined for axial MALDI-TOF mass spectrometers, as well as for orthogonal acceleration TOF mass spectrometers.