Improved fourier-transform ion-cyclotron-resonance mass spectrometry of large biomolecules

Initial results from a Fourier-transform mass spectrometer with a 6.2 Tesla magnet using electrospray ionization show substantial improvements in resolution, mass accuracy, mass range, signal/noise, and tandem mass spectromehy capabilities compared to our earlier 2.8 T instrument that demonstrated the first unit resolution mass spectra of molecules as large as myoglobin (17 kDa). The new instrument exhibits greater than 10⁶ and 10⁵ resolving power for 8.6 and 29 kDa, respectively, proteins. Using an internal standard, the mass measuring error for myoglobin is less than 1 ppm. Nozzle-skimmer dissociation during electrospray of carbonic anhydrase (29 kDa) has yielded 38 fragment ions for which both mass and charge are identifiable; of these, 21 have been assigned to expected oligopeptide fragments.     

Charge reduction lowers mass resolving power for isotopically resolved electrospray ionization Fourier transform ion cyclotron resonance mass spectra

Electrospray ionization of synthetic or biological macromolecules above ∼1–2 kDa in mass typically produces ions of multiple charge states. Several recent papers have illustrated charge reduction as a means to simplify low-resolution electrospray ionization mass spectra, at the cost of significant loss in signal-to-noise ratio. However, if mass resolving power is sufficiently high (as in Fourier transform ion cyclotron resonance mass spectrometry) to resolve the heavy-atom isotopic distribution, then charge reduction actually lowers mass resolving power by a factor proportional to the ion charge. For proteins or nucleic acids of 10–50 kDa in mass, reducing the charge state to unity thus lowers mass resolving power by a factor of 10–50. In other words, as long as it is possible to resolve the isotopic distributions, charge reduction has no advantages for electrospray ionization mass spectrometry and has the very serious disadvantage of greatly degraded mass resolving power. Copyright © 2001 John Wiley & Sons, Ltd.     

High mass accuracy and high mass resolving power FT-ICR secondary ion mass spectrometry for biological tissue imaging

Biological tissue imaging by secondary ion mass spectrometry has seen rapid development with the commercial availability of polyatomic primary ion sources. Endogenous lipids and other small bio-molecules can now be routinely mapped on the sub-micrometer scale. Such experiments are typically performed on time-of-flight mass spectrometers for high sensitivity and high repetition rate imaging. However, such mass analyzers lack the mass resolving power to ensure separation of isobaric ions and the mass accuracy for elemental formula assignment based on exact mass measurement. We have recently reported a secondary ion mass spectrometer with the combination of a C₆₀ primary ion gun with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for high mass resolving power, high mass measurement accuracy, and tandem mass spectrometry capabilities. In this work, high specificity and high sensitivity secondary ion FT-ICR MS was applied to chemical imaging of biological tissue. An entire rat brain tissue was measured with 150 μm spatial resolution (75 μm primary ion spot size) with mass resolving power (m/Δm _50%) of 67,500 (at m/z 750) and root-mean-square measurement accuracy less than two parts-per-million for intact phospholipids, small molecules and fragments. For the first time, ultra-high mass resolving power SIMS has been demonstrated, with m/Δm _50%?>?3,000,000. Higher spatial resolution capabilities of the platform were tested at a spatial resolution of 20 μm. The results represent order of magnitude improvements in mass resolving power and mass measurement accuracy for SIMS imaging and the promise of the platform for ultra-high mass resolving power and high spatial resolution imaging.

Top-down identification and characterization of biomolecules by mass spectrometry

The most widely used modern mass spectrometers face severe performance limitations with molecules larger than a few kDa. For far larger biomolecules, a common practice has been to break these up chemically or enzymatically into fragments that are sufficiently small for the instrumentation available. With its many sophisticated recent enhancements, this "bottom-up" approach has proved highly valuable, such as for the rapid, routine identification and quantitation of DNA-predicted proteins in complex mixtures. Characterization of smaller molecules, however, has always measured the mass of the molecule and then that of its fragments. This "top-down" approach has been made possible for direct analysis of large biomolecules by the uniquely high (>10(5)) mass resolving power and accuracy ( approximately 1 ppm) of the Fourier-transform mass spectrometer. For complex mixtures, isolation of a single component's molecular ions for MS/MS not only gives biomolecule identifications of far higher reliability, but directly characterizes sequence errors and post-translational modifications. Protein sizes amenable for current MS/MS instrumentation are increased by a "middle-down" approach in which limited proteolysis forms large (e.g., 10 kDa) polypeptides that are then subjected to the top-down approach, or by "prefolding dissociation." The latter, which extends characterization to proteins >200 kDa, was made possible by greater understanding of how molecular ion tertiary structure evolves in the gas phase.     

Electrospray ionization—Fourier transform ion cyclotron resonance mass spectrometry at 11.5 tesla: Instrument design and initial results

Initial results obtained using a new electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer operated at a magnetic field 11.5 tesla are presented. The new instrument utilized an electrostatic ion guide between the ESI source and FTICR trap that provided up to 5% overall transmission efficiency for light ions and up to 30% efficiency for heavier biomolecules. The higher magnetic field in combination with an enlarged FTICR ion trap made it possible to substantially improve resolving power and operate in a more robust fashion for large biopolymers compared to lower field instruments. Mass resolution up to 10⁶ has been achieved for intermediate size biopolymers such as bovine ubiquitin (8.6 kDa) and bovine cytochrome c (12.4 kDa) without the use of frequency drift correction methods. A mass resolution of 370,000 has been demonstrated for isotopically resolved molecular ions of bovine serum albumin (66.5 kDa). Comparative measurements were made with the same spectrometer using a lower field 3.5-tesla magnet allowing the performance gains to be more readily quantified. Further improvements in pumping capacity of the vacuum system and efficiency of ion transmission from the source are expected to lead to further substantial sensitivity gains.     

Unit resolution mass spectra of 112 kDa molecules with 3 Da accuracy

Previously, the highest molecular weight for a sample yielding a unit resolution mass spectrum was 67 kDa (marginal at 86 kDa), obtained with a 6. 2 T Fourier-transform mass spectrometer with electrospray ionization. Now with a 9. 4 T instrument, resolving power of 170,000 has been achieved for chondroitinase I (997 amino acids) and II (990 amino acids), making possible molecular weight assignments of 112,509 and 111,714, respectively, versus 112,508 and 111,713 calculated. Assisting these assignments was the noise reduction in the resolved isotopic peaks achieved by the time domain data sampling technique introduced by Senko, Marshall, and co-workers.     

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.     

An inductively coupled plasma-time-of-flight mass spectrometer for elemental analysis. Part I: Optimization and characteristics

An inductively coupled plasma-time-of-flight mass spectrometer (ICP-TOFMS) has been constructed and evaluated for elemental analysis. The instrument produces analog spectra similar to those from quadrupole inductively coupled plasma mass spectrometers. The large abundance of Ar ions is deflected away from the microchannel plate detector to reduce detector dead time and space-charge complications. The ICP-TOFMS, operated in a linear (nonreflecting) mode, currently has a resolving power of 500 (full width at half maximum). Present ion optics employed in the instrument require a trade-off between signal-to-noise ratio and resolving power. In addition, mass-dependent kinetic energies in the supersonic beam created in the ICP mass spectrometer interface cause a mass bias in the right-angle TOFMS because the ions must be steered to the detector to compensate for their velocity in the supersonic beam direction. In the current design the sampling duty cycle is only approximately 3%, thereby limiting sensitivity. However, positive potentials applied to the right-angle extraction region can increase sensitivity by a factor of 2–4 by slowing down the ions that enter the extraction zone. The transmission efficiency of the TOFMS is approximately 20% and is limited by divergence of the ion packet in the drift tube.     

Dynamics of ions of intact proteins in the Orbitrap mass analyzer

While allowing analysis of intact proteins without a theoretical upper mass limit, the Orbitrap mass analyzer demonstrates reduced resolving power as ion mass increases even at a constant mass-to-charge ratio. It is shown that this effect comes from the effects of ion scattering on background gas molecules. The main mechanisms causing decay of acquired transient appear to be fragmentation as well as accelerated dephasing of ion packets. Isotopic resolution of proteins including bovine serum albumin (MW 66.4 kDa) and transferrin (MW 78 kDa) has also been demonstrated. As a part of this study, detection of individual multiply-charged ions of myoglobin (MW 16.9 kDa) has been demonstrated. Quantized distribution of signal intensities for myoglobin ions well above the noise threshold was observed, with high mass accuracy and resolution of recorded individual ions used as an independent confirmation of correct assignment of signal to ions rather than to noise. The latter also allowed us to benchmark the sensitivity of image-current detection and explore in detail factors responsible for signal decay.     

Intact and top-down characterization of biomolecules and direct analysis using infrared matrix-assisted laser desorption electrospray ionization coupled to FT-ICR mass spectrometry

We report the implementation of an infrared laser onto our previously reported matrix-assisted laser desorption electrospray ionization (MALDESI) source with ESI post-ionization yielding multiply charged peptides and proteins. Infrared (IR)-MALDESI is demonstrated for atmospheric pressure desorption and ionization of biological molecules ranging in molecular weight from 1.2 to 17 kDa. High resolving power, high mass accuracy single-acquisition Fourier transform ion cyclotron resonance (FT-ICR) mass spectra were generated from liquid- and solid-state peptide and protein samples by desorption with an infrared laser (2.94 μm) followed by ESI post-ionization. Intact and top-down analysis of equine myoglobin (17 kDa) desorbed from the solid state with ESI post-ionization demonstrates the sequencing capabilities using IR-MALDESI coupled to FT-ICR mass spectrometry. Carbohydrates and lipids were detected through direct analysis of milk and egg yolk using both UV- and IR-MALDESI with minimal sample preparation. Three of the four classes of biological macromolecules (proteins, carbohydrates, and lipids) have been ionized and detected using MALDESI with minimal sample preparation. Sequencing of O-linked glycans, cleaved from mucin using reductive β-elimination chemistry, is also demonstrated.     

Performance evaluation of a high-field orbitrap mass analyzer

A new design of the Orbitrap^™ mass analyzer is presented. Higher frequencies of ion oscillations and hence higher resolving power over fixed acquisition time are achieved by decreasing the gap between the inner and outer Orbitrap electrodes, thus providing higher field strength for a given voltage. Experimental results confirm maximum FWHM resolving power in excess of 350,000 at m/z 524 and 600,000 at m/z 195, isotopic resolution of proteins above 40 kDa, and a single-shot dynamic range of 25,000. It was also found that mass shifts in the new design depend very little on space charge inside the analyzer. This performance was achieved using higher voltages and by careful balancing of construction tolerances and operation parameters, which appeared to vary in narrower ranges of tuning than for a standard Orbitrap analyzer.     

An improved calibration method for the matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resononance analysis of 15N-metabolically- labeled proteome digests using a mass difference approach

High mass measurement accuracy of peptides in enzymatic digests is critical for confident protein identification and characterization in proteomics research. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) can provide low or sub-ppm mass accuracy and ultrahigh resolving power. While for ESI-FT-ICR-MS, the mass accuracy is generally 1 ppm or better, with matrix-assisted laser desorption/ionization (MALDI)-FT-ICR-MS, the mass errors can vary from sub-ppm with internal calibration to over 100 ppm with conventional external calibration. A novel calibration method for (15)N-metabolically labeled peptides from a batch digest of a proteome is described which corrects for space charge induced frequency shifts in FT-ICR spectra without using an internal calibrant. This strategy utilizes the information from the mass difference between the (14)N/(15)N peptide peak pairs to correct for space charge induced mass shifts after data collection. A procedure for performing the mass correction has been written into a computer program and has been successfully applied to high-performance liquid chromatography-MALDI-FT- ICR-MS measurement of (15)N-metabolic labeled proteomes. We have achieved an average measured mass error of 1.0 ppm and a standard deviation of 3.5 ppm for 900 peptides from 68 MALDI-FT-ICR mass spectra of the proteolytic digest of a proteome from Methanococcus maripaludis.     

Fourier-transform electrospray instrumentation for tandem high-resolution mass spectrometry of large molecules

Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, New York, USA Mass spectrometry instrumentation providing unit resolution and lo-ppm mass accuracy for molecules larger than 10 kDa was first reported in 1991. This instrumentation has now been improved with a 6.2-T magnet replacing that of 2.8 T, a more efficient vacuum system, ion injection with controlled ion kinetic energies, accumulated ion trapping with an open-cylindrical ion cell, acquisition of 2M data points, and updated electrospray apparatus. The resulting capabilities include resolving power of 5 × 10⁵ for a 29-kDa protein, less than l-ppm mass measuring error, and dissociation of protein molecular ions to produce dozens of fragment ions whose exact masses can be identified from their mass-to-charge ratio values and isotopic peak spacing.     

Development of two‐dimensional gas chromatography (GC×GC) coupled with Orbitrap‐technology‐based mass spectrometry: Interest in the identification of biofuel composition

Comprehensive gas chromatography (GC) has emerged in recent years as the technique of choice for the analysis of volatile and semivolatile compounds in complex matrices. Coupling it with high‐resolution mass spectrometry (MS) makes a powerful tool for identification and quantification of organic compounds. The results obtained in this study showed a significant improvement by using GC×GC‐EI‐MS in comparison with GC‐EI‐MS; the separation of chromatogram peaks was highly improved, which facilitated detection and identification. However, the limitation of Orbitrap mass analyzer compared with time‐of‐flight analyzer is the data acquisition rate; the frequency average was about 25 Hz at a mass resolving power of 15.000, which is barely sufficient for the proper reconstruction of the narrowest chromatographic peaks. On the other hand, the different spectra obtained in this study showed an average mass accuracy of about 1 ppm. Within this average mass accuracy, some reasonable elemental compositions can be proposed and combined with characteristic fragment ions, and the molecules can be identified with precision. At a mass resolving power of 7.500, the scan rate reaches 43 Hz and the GC×GC‐MS peaks can be represented by more than 10 data points, which should be sufficient for quantification. The GC×GC‐MS was also applied to analyze a cellulose bio‐oil sample. Following this, a highly resolved chromatogram was obtained, allowing EI mass spectra containing molecular and fragment ions of many distinct molecules present in the sample to be identified.     

Generation and detection of multiply-charged peptides and proteins by matrix-assisted laser desorption electrospray ionization (MALDESI) fourier transform ion cyclotron resonance mass spectrometry

We report the coupling of a hybrid ionization source, matrix-assisted laser desorption electrospray ionization (MALDESI), to a Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS). The details of the source design and initial data are presented. Analysis of peptides and proteins ranging from 1 to 8.6 kDa resulted in high resolving power single-acquisition FT-ICR mass spectra with average charge-states highly correlated to those obtained by nanoESI, thus, providing strong evidence that the ESI process dictates the observed charge-state distribution. Importantly, unlike the recently introduced electrospray assisted laser desorption ionization (ELDI) source reported by Shiea and coworkers [1, 2], the data we have obtained to date rely on the use of an organic acid matrix. The results presented herein provide insight into the charging mechanism of this emerging ionization approach, while also expanding the utility of FT-ICR MS for top-down protein and complex mixture analysis.     

High-precision Ru isotopic measurements by multi-collector ICP-MS

Ruthenium isotopic data for a pure Aldrich ruthenium nitrate solution obtained using a Nu Plasma multi collector inductively coupled plasma-mass spectrometer (MC-ICP-MS) shows excellent agreement (better than 1 epsilon unit = 1 part in 10(4)) with data obtained by other techniques for the mass range between 96 and 101 amu. External precisions are at the 0.5-1.7 epsilon level (2sigma). Higher sensitivity for MC ICP-MS compared to negative thermal ionization mass spectrometry (N-TIMS) is offset by the uncertainties introduced by relatively large mass discrimination and instabilities in the plasma source-ion extraction region that affect the long-term reproducibility. Large mass bias correction in ICP mass spectrometry demands particular attention to be paid to the choice of normalizing isotopes. Because of its position in the mass spectrum and the large mass bias correction, obtaining precise and accurate abundance data for 104Ru by MC-ICP-MS remains difficult. Internal and external mass bias correction schemes in this mass range may show similar shortcomings if the isotope of interest does not lie within the mass range covered by the masses used for normalization. Analyses of meteorite samples show that if isobaric interferences from Mo are sufficiently large (Ru/Mo < 10(4)), uncertainties on the Mo interference correction propagate through the mass bias correction and yield inaccurate results for Ru isotopic compositions. Second-order linear corrections may be used to correct for these inaccuracies, but such results are generally less precise than N-TIMS data.     

Accuracy and long-term reproducibility of lead isotopic measurements by multiple-collector inductively coupled plasma mass spectrometry using an external method for correction of mass discrimination

The precision of isotopic measurements of Pb by thermal ionization mass spectrometry (TIMS) is limited by the fact that this element does not possess an invariant isotope ratio that can be used for the correction of mass fractionation by internal normalization. Multiple-collector inductively coupled plasma mass spectrometry (MC-ICPMS) can overcome this limitation, because with plasma ionization, elements with overlapping mass ranges are thought to display identical mass discrimination. With respect to Pb, this can be exploited by the addition of Tl to the sample solutions; the mass discrimination factor obtained for Tl can then be used for the correction of the measured Pb isotope ratios. In this article we present the results of a detailed study that investigates the accuracy and precision of such an external correction technique for mass discrimination based upon the results of multiple analyses of a mixed standard solution of NIST SRM-981 Pb and SRM-997 Tl. Our data indicate that normalization of the Pb isotope ratios to the certified isotopic composition of SRM-997 Tl produces Pb isotopic results that are significantly lower than recently published reference values by TIMS. This systematic offset can be eliminated by renormalization of the Pb data to a different Tl isotopic composition to obtain an empirically determined mass discrimination factor for Pb that generates accurate results. It is furthermore shown that a linear law is least suited for the correction of mass discrimination, whereas a power or exponential law function provide significantly more accurate and precise results. In detail, it appears that a power law may provide the most appropriate correction procedure, because the corrected Pb isotope ratios display less residual correlations with mass discrimination compared to the exponentially corrected data. Using an exponential or power law correction our results, obtained over a period of over seven months, display a precision (2σ) of better than 60 parts per million (ppm) for ²⁰⁸Pb/²⁰⁶Pb and ²⁰⁷Pb/²⁰⁶Pb and of better than 350 ppm for ²⁰⁶Pb/²⁰⁴Pb, ²⁰⁷Pb/²⁰⁴Pb/²⁰⁴Pb, and ²⁰⁸Pb/²⁰⁴Pb. This represents a significant improvement compared to conventional TIMS techniques and demonstrates the potential of MC-ICPMS for routine, high-precision measurements of Pb isotopic compositions.     

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.     

Metabolite identification by data-dependent accurate mass spectrometric analysis at resolving power of 60,000 in external calibration mode using an LTQ/Orbitrap

Performance evaluation of accurate mass measurement by the LTQ/Orbitrap, at a resolving power of 60,000 and in external calibration mode, indicated that the Orbitrap is capable of providing high mass accuracy of <2 ppm for over 24 h post-calibration. This, together with limited trade-off between sensitivity and resolving power plus a wide dynamic range for mass accuracy, suggested that the LTQ/Orbitrap is an ideal analytical tool for structural elucidation of metabolites. The application of the LTQ/Orbitrap to identification of human liver microsomal metabolites of carvedilol was evaluated, using parent mass list triggered data-dependent multiple-stage accurate mass analysis, at a resolving power of 60,000 in external calibration mode. A metabolite identification workflow was developed to utilize chemical formulas from high-resolution accurate mass measurements to confirm structures of product ions of a drug proposed by Mass Frontier, illustrated by identification of structures used to establish lineage of product ions of carvedilol, which later served as a template for identification of its metabolites. A total of 58 in vitro metabolites of carvedilol were detected using 5-ppm mass tolerance filters for theoretical m/z of protonated molecules of predicted metabolites in addition to product ions and neutral mass losses diagnostic of carvedilol. The chemical formulas with unsaturation numbers calculated from the accurate m/z of precursor and product ions can be used to assign, with a high degree of confidence, the structures of metabolites and the sites of metabolism. The mass accuracies obtained for all full scan MS and MSn spectra were <2 ppm. The majority of the metabolites identified agreed with those previously reported except for those that have not been reported before. For example, several glutathione conjugates of carvedilol were reported for the first time, which may explain the reported hepatotoxicity during clinical trials and recent clinical use.     

Full-scan accurate mass selectivity of ultra-performance liquid chromatography combined with time-of-flight and orbitrap mass spectrometry in hormone and veterinary drug residue analysis

The applicability of ultra-performance liquid chromatography (UPLC) combined with full-scan accurate mass time-of-flight (TOF) and Orbitrap mass spectrometry (MS) to the analysis of hormone and veterinary drug residues was evaluated. Extracts from blank bovine hair were fortified with 14 steroid esters. UPLC-Orbitrap MS performed at a resolving power of 60,000 (FWHM) enabled the detection and accurate mass measurement (<3 ppm error) of all 14 steroid esters at low ng/g concentration level, despite the complex matrix background. A 5 ppm mass tolerance window proved to be essential to generate highly selective reconstructed ion chromatograms (RICs) having reduced background from the hair matrix. UPLC-Orbitrap MS at a lower resolving power of 7500 and UPLC-TOFMS at mass resolving power 10,000 failed both to detect all of the steroid esters in hair extracts owing to the inability to mass resolve analyte ions from co-eluting isobaric matrix compounds. In a second application, animal feed extracts were fortified with coccidiostats drugs at levels ranging from 240 to 1900 ng/g. UPLC-Orbitrap MS conducted at a resolving power of 7500 and 60,000 and UPLC-TOFMS detected all of the analytes at the lowest investigated level. Thanks to the higher analyte-to-matrix background ratio, the utilization of very narrow mass tolerance windows in the RIC was not required. This study demonstrates that even when the targeted sample preparation from conventional LC-MS/MS is applied to UPLC with full-scan accurate mass MS, false compliant (false negative) results can be obtained when the mass resolving power of the MS is insufficient to separate analyte ions from isobaric co-eluting sample matrix ions. The current trend towards more generic and less selective sample preparation is expected to aggravate this issue further.