Electron-capture dissociation (ECD) has been shown to be a valuable tool for peptide analysis and protein identification, as well as for the characterization of protein post-translation modifications. ECD has several inherent advantages over traditional collision-induced dissociation (CID) that make it exceptionally useful for such studies. It tends to restrict cleavage to N-C(alpha) bonds along the peptide backbone, leaving post-translational modifications intact and available for mass spectral evaluation. Also, it performs especially well at fragmenting long peptides. However, traditionally there have been limitations to using ECD in protein and peptide analysis. Historically, it was restricted to expensive, complex, and resource-intensive Fourier-transform ion cyclotron resonance instruments. Additionally, the ECD reaction in these instruments occurred on a relatively long timescale, precluding its use in liquid chromatography–mass spectrometry experiments. Finally, some peptides simply were not amenable to ECD analysis for reasons not well understood. Here we describe the use of a novel ECD device, QuECD, that can be interfaced to a linear ion trap time-of-flight mass spectrometer, which addresses some of these weaknesses. The reaction time of this ECD technique is on par with traditional CID reactions and is compatible with the chromatographic timescale. In addition, we demonstrate "hot" ECD (HECD), a technique in which the energy of the electron beam used in the QuECD fragmentation reaction is raised to roughly an order of magnitude over traditional QuECD, promoting the fragmentation of otherwise difficult samples. We demonstrate both of these extensions to traditional ECD in an investigation of human transferrin.

Protein and peptide analysis via tandem mass spectrometry (MS-MS) has resulted in a wealth of information regarding protein identification, structure, and abundance levels over the past 10 years. Techniques such as neutral loss scanning and collision-induced dissociation (CID) have been especially helpful in facilitating the identification of a multitude of previously unknown sites of protein phosphorylation. However, many of the techniques used to obtain this information are labor intensive and work inconsistently. To address this problem, much effort has been put forth to find alternative methods of fragmenting peptides and proteins that are less difficult and applicable to a wide gamut of peptide classes. Examples of recently developed dissociation techniques include infrared multiphoton dissociation (IRMPD) and electron transfer dissociation (ETD). The implementation of these new techniques has widened the spectrum of peptides amenable to tandem mass spectral analysis. 

Electron-capture dissociation (ECD) is yet an additional peptide fragmentation technique that is gaining in popularity (1). ECD has a number of advantages over traditional fragmentation techniques such as CID. CID tends to cleave the bond between a posttranslation modification (PTM) and its partner peptide before it cleaves the peptide backbone bonds themselves. This premature loss of a PTM during the dissociation process precludes any ability to obtain structural information regarding that PTM. ECD, conversely, tends to leave the bond between a PTM and its partner peptide intact. Instead, it almost universally cleaves the N-Cα bonds of the peptide backbone (except for the N-terminal side of proline). This distinct dissociation pattern makes ECD an extremely useful technique for characterizing PTMs (2).  

In addition, ECD has proven to be effective at fragmenting very large peptides, and even small proteins. Indeed, ECD has been  applied successfully to ubiquitin, a 76-amino acid protein with a mass of 8564.47 Da. The experiment resulted in a sequence coverage of 85% at the amino acid level (65 of the 76 possible fragments were observed) (3).  

While ECD has been an effective method for peptide fragmentation for almost 10 years, historically it has been restricted to costly and complex Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometers, limiting its accessibility to a few specialized laboratories. In addition, the ECD reaction on these instruments occurs on a timescale that is too long to be coupled to a liquid chromatographic separation. Rather, samples must be introduced via direct infusion, prohibiting the study of complex mixtures.  

Recently, both of these issues have been resolved with the advent of QuECD, an ECD system that can be coupled to a time-of-flight mass analyzer (4). The QuECD cell comprises a radio frequency linear ion trap that is preceded by a stand-alone linear ion trap and succeeded by a time-of-flight mass analyzer (Figure 1). The initial ion trap accumulates ions of a specific mass and injects them into the QuECD cell, where the ECD reaction occurs. The ions are subsequently transported to the TOF analyzer for high resolution and mass accuracy analysis.  

 Figure 1: A schematic diagram of the QuECD platform. Specific ions are accumulated and trapped in the initial linear ion trap and directed to the RF-linear ion trap QuECD cell. The resulting fragments are then directed to the time-of-flight tube for high resolution and mass accuracy analysis. 

The QuECD system has two prominent advantages over traditional ECD. First, the QuECD reaction occurs on the chromatographic timescale, and is thus capable of analyzing complex mixtures via LC–ECD analysis. The second advantage of the QuECD system is rooted in the fact that the dissociation reaction is achieved by the action of an electron beam focused on the sample within the QuECD cell. Typically, the energy of this electron beam is <1 eV. However, not all peptides fragment efficiently at this energy level. With QuECD, the energy of the electron beam can be modulated to a level that more effectively fragments a particular peptide. "Hot" ECD (HECD) typically is performed at energy levels of 7–10 eV. Some peptides that are not amenable to fragmentation at lower energy levels exhibit very effective fragmentation under HECD conditions. 

In this work, we demonstrate examples of both of these extensions to conventional ECD in an analysis of human transferrin. First, we demonstrate QuECD's capability to perform an LC–ECD analysis on a Lys-C transferrin digest. Second, we show the effectiveness of HECD in getting optimal ECD fragmentation of specific transferrin glycopeptides. While these extensions to conventional ECD do not solve all peptide fragmentation problems, they add more tools to the biochemist's arsenal of techniques with which to characterize protein structure, function, and identity. 

Human transferrin (Sigma, St. Louis, Missouri) was resuspended in 8 M urea, 50 mM ammonium bicarbonate, pH 8.5, and subsequently incubated with 10 mM DTT for 60 min at 37 °C, then 20 mM iodoacetamide for 60 min at room temperature. The sample was diluted 10-fold in 50 mM ammonium bicarbonate, pH 8.5, Lys-C was added in a 1:100 Lys-C to protein ratio, and the solution was incubated overnight at 37 °C. 

Figure 2: Total ion chromatograms of the (a) LCâCID and (b) LCâECD analyses of a human transferrin Lys-C digest. 

The resulting peptides were separated on a 150 mm × 0.05 mm MonoCap C18 column (GL Sciences, Tokyo, Japan) using a 2–35% acetonitrile gradient over 120 min. Figure 2 shows total ion chromatograms for both a traditional LC–MS experiment (utilizing standard CID) and for an LC–ECD experiment. The similarity in the traces indicates that the fundamental change in the tandem MS mode does not alter the basic ability of the instrument to analyze peptides as they are eluted from the column. Of more interest is the similarity between the base peak intensities of the MS-MS events during the chromatographic run (Figure 3). This similarity attests to the ability of QuECD to work on the chromatographic timescale. In fact, the scan speed observed during ECD analysis is only marginally slower than that observed during traditional CID analysis. Over the course of the 120-min LC–MS run, 2340 MS-MS spectra were taken in CID mode, while 1896 ECD MS-MS spectra were recorded, a difference of only 25%. These data demonstrate not only that ECD data can be taken during an LC–MS run, but also that the amount of data generated almost matches traditional CID. 

Figure 3: Base peak ion chromatograms of the MS-MS spectra obtained during (a) LCâCID and  (b) LCâECD analyses of a human transferrin Lys-C digest. 

There are three N-linked glycosylation sites in human transferrin — Asn-432, 491, and 630 (5). We observed ions corresponding to the glycopeptides AA421-433 (3683 Da), AA490-508 (4465 Da), and AA619-646 (5500 Da) during a standard LC–MS experiment. However, we were unable to generate MS-MS spectra for these peptides using traditional CID. In the LC–ECD experiment, however, we were able to obtain ECD spectra for two of these glycopeptides, AA421-433 (

 1111.0, 4+) (Figures 4a and 4b, respectively). By combining the CID and ECD results, the sequence coverage was increased from 57% to 63%. More importantly, we were able to confirm the location of the glycosylation sites for these two peptides.  

Figure 4: ECD spectra of N-glycopeptides of transferrin: (a) AA421-433, 3683 Da, m/z = 921.5 (4+); (b) AA619-646, 5550 Da, m/z = 1111.0 (5+). 

Although the ability to perform ECD during an LC–MS run is advantageous, not all peptides have proven amenable to ECD fragmentation. One of the advantages provided by the QuECD platform is the ability to change the energy of the electron beam utilized in the fragmentation process. While traditional ECD reactions are performed at energies of < 1 eV, in HECD, the energy of the electron beam is increased to a value of 7–10 eV. Figure 5 shows ECD spectra of the AA619-646 peptide taken under regular ECD (Figure 5a) and HECD (Figure 5b) conditions. For both the c- and z-ion series, the number of observed ions is markedly increased under HECD conditions. While this ion was not identified using the traditional ECD data, it was easily identified using the richer HECD data.  

Figure 5: ECD spectra of transferrin's AA619-646 glycopeptide: (a) traditional ECD and (b) HECD. 

Modern biochemists require as many tools as possible in the effort to characterize protein structure and function. While MS has long been useful in these efforts, such experiments have proven costly and difficult. Advances in dissociation techniques have made progress in addressing these shortcomings. ECD is a powerful technique because of its utility in characterizing posttranslational modification as well as long peptides and small proteins. Because it can be implemented on a time-of-flight mass spectrometer, the QuECD platform has been successful in increasing the accessibility of ECD to nonspecialized laboratories. Further, QuECD allows for the analysis of complex mixtures via LC–ECD experiments. HECD increases the population of peptides that are amenable to ECD analysis. These advantages have brought QuECD to the forefront of structural protein characterization via MS. 

The authors would like to thank Takeshi Sakamoto of the Central Research Laboratory, Hitachi Ltd., for his help. 

 are with Hitachi High-Technologies America, Pleasanton, California.  

 are with Hitachi High-Technologies, Hitachinaka, Ibaraki, Japan.

(2) M.A. Shaw, M. Watanabe, T. Nabetani, Y. Hirabayashi, A. Tsuboyama, and C. Ostrander, 

(3) T. Baba, et al., Hitachi High-Technologies Application Note, NF/TN(E)-002, 2007. 

Articles

The authors discuss the use of electron-capture dissociation coupled with a linear ion trap time-of-flight mass spectrometer to investigate the structure of human transferrin.

Investigation of Transferrin Structure via Novel Electron Capture Dissociation Techniques Using a Hybrid Linear Ion Trap Time-of-Flight Mass Spectrometer

 recent PharmSep Symposium, held in Philadelphia from September 9–11, I heard one piece of advice repeated over and over from various conference-goers: Cover more mass spectrometry next year. I was certainly aware of the prominence of mass spectrometry, both in hyphenated techniques across the disciplines of chromatography and spectroscopy, and also as an independent tool to be used on its own. However, the popularity of this technique was truly brought home to me over the course of this three-day event. 

At a symposium attended almost exclusively by practitioners of chromatography, the one thing they all wanted more of was mass spectrometry information. I think this illustrates just how much a part of the fabric of today's analytical laboratory mass spectrometry has become, possibly even more effectively than any poll or survey could. When you hear firsthand testimonials from the people who do the research in the lab every day, the impact is dramatic, and in this case, those people were some of the best and brightest minds in the industry today. 

   With this in mind, we're happier than ever to bring you this November installment of the 

 supplement series. Inside you'll find articles on the newest and most cutting-edge applications of mass spectrometry from the leading minds in the field. Articles on pesticide analysis in food, consumer beverage analysis, and biomedical advances using mass spectrometry all demonstrate the kind of life-affecting research this technique is enabling every day, research that saves lives and — to use a cliche that is overused but very appropriate — makes our world a better place. 

 We hope you find this issue useful in your own research, and just as at the PharmSep Symposium those many weeks ago, we welcome and value your feedback, so feel free to contact any of our staff members at the e-mail addresses listed in the masthead. 

Deparments: From the Editor

At a symposium attended almost exclusively by practitioners of chromatography, the one thing they all wanted more of was mass spectrometry information.

From the Editor

Laboratories embroiled in the intricacies of drug discovery and development, proteomics, and food and water testing are becoming increasingly dependent upon sophisticated mass spectrometers to overcome the significant and limiting challenges they face. Problems such as sample type, quality, and concentration, ever-increasing throughput demands, and limitations of currently available mass spectrometry workflows are all a matter of course and must be dealt with to push the boundaries of analysis even further.

For drug discovery workflows, the issue of metabolite detection and identification in in vivo systems is a critical challenge. The wide range of complex matrices (such as bile, plasma, urine, and fecal extracts), and the ion suppression effects of these biological fluids, can cause a severe decrease in the ability to detect metabolites. Greater instrument sensitivity is necessary to detect these compounds and, at the same time, helps to minimize sample preparation, simply diluting the negative effects of these complex matrices and avoiding the time- and labor-consuming sample cleanup or concentration steps that otherwise might be required. 

In drug development, microdosing (using less than one hundredth of the normal dose) studies allow earlier, and potentially safer, assessment of the pharmacokinetics of new compounds in humans compared to conventional Phase I trials, as well as to reduce or replace safety and toxicology testing in animals. The high sensitivity of liquid chromatography–mass spectrometry (LC–MS) is a significant factor in such strategies, allowing the detection of much lower concentrations of compounds and metabolites in both quantitative and qualitative work. 

Many of the compounds of interest occur in low levels, a familiar issue in biomarker verification and validation work too, and insufficient instrument sensitivity could lead to the failure to detect key compounds. In the food and environmental industries also, quality control processes constantly demand greater sensitivity to keep apace with legislative regulations that progressively reduce acceptable limits of an increasing number of contaminants. With the large number of compounds that must be detected, less time is available to monitor each compound within a run, resulting in poor sensitivity and often leading to multiple injections, adding considerable time to experiments. Improved instrument sensitivity and scan speed would allow many more analytes to be monitored in the same run at the required sensitivity so that fewer injections would be required per experiment, thereby offering the potential to obtain all the necessary answers in a single injection. The speed of the mass spectrometer is also important in other ways. With the current trend for faster chromatographic analysis times of 5 min or less, a system must be capable of yielding sufficient data points from each chromatographic peak for effective LC–MS analysis. In addition, faster scanning and improved duty cycle enable more efficient automated workflows. 

For the identification of a large number of compounds (for example, in the pharmaceutical discovery phase), a first pass traditionally has been performed with a triple-quadrupole mass spectrometer for the selective scan functions, like precursor ions and neutral loss, helping to filter out unwanted compounds. In general, the selective nature of multiple reaction monitoring (MRM), precursor ion, and neutral loss scans provides the highest level of detection compared to any single MS-based methodology. Then, for confirmation, other instruments like ion traps were used to obtain high quality MS-MS spectra. 

Now, with a drive for greater quality, efficiency, throughput, and confidence in data using as few injections as possible, mass spectrometers are being designed to meet scientists' needs precisely. Instruments with improved sensitivity, speed, and selectivity, coupled with unique combinations of technologies within a single platform to enable more efficient workflows, are being developed by leading manufacturers. Hybrid instruments include Applied Biosystems/MDS Analytical Technologies' AB SCIEX QTRAP 5500 System, which combine a triple quadrupole and a linear accelerator trap on the same platform. Such instruments are changing the way MS analysis, like metabolite identification, is performed (for example, predictive MRM [pMRM], coupled with full-scan MS-MS in the same analysis to confirm that the compound is the expected metabolite). The hybrid nature also allows more than one test to be performed in one run, which is ideal for applications such as testing for pesticides and mycotoxins in grain-derived food products, and screening for pharmaceuticals and personal care products (PPCP) in drinking water. 

With the addition of a newly developed ion guide and improved Q3 LIT, the latest of these systems shows great increases in signal response, particularly in LIT mode. Duty cycle is improved further with trap fill times as short at 50 μs, because of the increase in linear accelerator trap sensitivity. We are also seeing scan rates up to 20,000 Da/s in linear accelerator trap mode and four times faster in quadrupole mode compared to previous systems. This greater cycle rate allows for compatibility with sharp chromatographic peaks, as well as improved information-dependent acquisition (IDA) coverage provided by a greater number of MS-MS-dependent scans, a great advantage when complex matrices, such as bile, contain a notable number of nonmetabolite peaks and may cause extraneous IDA triggers. The improvements in triple-quadrupole scanning rates also allow multiple survey scans, and simultaneously trigger full-scan MS-MS from any peaks that might be present using LIT. The greater speed of the newest instruments, which can detect more than 1000 compounds, means that they are ideal for multitarget analysis, detecting all compounds of interest in just one injection (Figure 1). 

 Figure 1: Analysis of grape samples for more than 1000 pesticides. Methomyl and trifloxystrobin were quantified and identified using scheduled MRM and fast MS-MS library searching. 

With polarity switching as fast as 50 ms, it is possible to combine the most powerful modes of metabolite detection in a single injection workflow with sufficient sensitivity in an overall duty cycle time of around 2 s, for an accurate result in comprehensive detection of metabolites (Figure 2) and pesticides (Figure 3). 

Figure 2: Detection of simvastatin hydroxyl acid in both positive and negative ion. The lactone prodrug simvastatin is hydrolyzed to the active compound (simvastatin hydroxyl acid), and both simvastatin and the hydroxyl acid ionize in positive ion, but the hydroxyl acid is better detected in negative ion. Of the 34 metabolites, five were detected only in positive ion, 28 detected only in negative ion, and one (Mx) was detected in both polarities. 

The improved sensitivity and speed are also important factors for efficient targeted peptide quantitation workflows, including biomarker verification and validation, and the high selectivity for compounds of interest certainly increases the throughput of automated workflows. The newer combined systems enable hypothesis-driven verifications, removing the need for synthetic protein standards in assay development, and the heightened sensitivity enables the linear accelerator trap to match the sensitivity of the MRM, speeding up development and identification confirmation. Scheduled MRM enables much higher multiplexed assays while still maintaining analytical reproducibility; with the latest instruments capable of up to 2500 MRMs in a single run, equating to 100 proteins, a single run is enough to obtain all the data required, eliminating what was previously a potential bottleneck. 

Figure 3: Ultrafast LCâMS-MS analysis of four pesticides in drinking water at 0.1 mg/L using fast polarity switching and fast MRM detection. 

The latest software programs that accompany MS platforms have been developed to bring all the sophisticated, complex methods right to the researcher's fingertips, with just a few clicks of the mouse. User-friendliness is a key feature, helping to make MS technology accessible to many more users. Ready-to-use, preconfigured protocols and experiments are especially helpful to those who are new to LC–MS, and allow them to be running meaningful experiments within weeks, rather than months.  

Data analysis software similarly has been upgraded to handle the huge amounts of data resulting from the greater throughput of new MS instruments, streamlining and automating the analysis process to require minimal manual intervention.  

The new systems offer a complete solution for LC–MS-MS workflows in various applications, with the best of both quantitative and qualitative accuracy and reproducibility, as well as generating superior targeted quantitative analysis, validation, and confirmation in a single run. 

 are with Applied Biosystems, Inc., Foster City, California. 

The authors discuss the challenges presented by the many new applications of mass spectrometry.

A New Perspective on the Challenges of Mass Spectrometry

Liquid chromatography coupled with tandem mass spectrometry (LC–MS-MS) is emerging as a complementary method to traditional methodology used for clinical applications. Reduced sample preparation and high-throughput capabilities are providing significant benefits to clinical scientists conducting routine analyses. This technology is expected to expand rapidly as scientists focus on more complicated challenges that can be solved efficiently by adding LC–MS-MS to their arsenal of techniques.

The use of mass spectrometry (MS) in clinical diagnosis goes back to the early 1970s with the application of gas chromatography (GC)–MS to the determination of a variety of biologically significant molecules. Because GC requires a certain level of analyte volatility, and since most biologically active molecules are polar, thermolabile, and involatile, elaborate extraction and derivatization protocols needed to be devised to make GC–MS useful for the analysis of clinically relevant samples. To make sample analysis less difficult by MS there had been a significant amount of R&D invested over several decades aimed at coupling high performance liquid chromatography (HPLC) with MS since HPLC is a much better separation technology than GC for polar thermolabile biologically relevant molecules. This coupling was not without significant challenges; most of the LC–MS coupling techniques that evolved during the 1970s and 1980s were not very successful, and many of those that enjoyed some widespread popularity, such as thermospray, flow-fast atom bombardment (FAB), and particle beam, are now virtually extinct. All of these techniques were quickly displaced by interfaces involving ionization techniques that separated the ionization process from the high vacuum analyzer portion of the instruments, namely atmospheric pressure ionization (API), including electrospray (ESI), ion-spray (nebulizer-assisted electrospray, ISP), atmospheric pressure chemical ionization (APCI) using a heated nebulizer, and atmospheric pressure photoionization (APPI), also using a heated nebulizer. Not until the development of API did there become a much more rapid adoption of LC–MS to the analysis of clinically important samples. 

An exceptionally good review of the development of API techniques was written by Thomson (1). As the name implies, API creates ions at atmospheric pressure, outside the pristine analyzer, thereby making LC–MS very robust and relatively free of analyzer contamination. Coupling of HPLC to API-MS is trivial since the MS vacuum system is not directly involved; this relative ease-of-use permitted those researchers more familiar with biochemistry than with analytical technologies to actually use LC–MS more routinely. This caused an enormous increase in applications for LC–MS across a wide range of biologically significant molecules, including biopolymers such as polysaccharides, DNA, and proteins as well as small molecule analytes such as nucleotides, amino acids, acylcarnitines, sphingolipids, phospholipids, and biogenic amines. The vast majority of LC–MS applications for clinical research today are being run using API.  

One of the features of API techniques is that it is very "soft," creating primarily intact molecular ions, which, unless very high resolution and mass accuracies are employed, are not very analytically specific. This has necessitated the use of tandem mass spectrometry, or MS-MS, to enable the analysis of trace analytes from complex biological matrices typically encountered in clinical research samples. There have been some excellent reviews on this subject (2,3). 

Examples of the most prevalent uses of MS-MS in clinical research include the screening of newborns for congenital metabolic diseases such as aminoacidopathies, organic acidurias, and fatty acid oxidation disorders (4,5); multi-analyte therapeutic drug monitoring (TDM), especially for the administration of cocktail therapies involving immunosuppressants (6–8), oncology drugs (9), and anti-retrovirals (10,11); toxicant and drugs-of-abuse screening, in which samples can be screened and validated in a single run (12,13); the analysis of endogenous peptides, especially where different isoforms exist; and the analysis of steroid hormones (14). With respect to the latter, there has been a recent growing level of interest in the application of LC–MS-MS to clinical research in endocrinology to the point where the American Endocrine Society has issued a statement recommending LC–MS-MS for the determination of endogenous levels of steroid hormones such as testosterone over more traditional technologies such as immunoassays (15). The rationale for this position has been the superiority of analytical results by LC–MS-MS, especially for low levels of these analytes (16,17). The reader is referred to a recent review article on the use of LC–MS-MS for a variety of endocrinology applications (18). Despite these useful applications and the rapid growth of LC–MS-MS in clinical research, the number of LC–MS-MS systems in use in routine diagnostic laboratories is actually relatively small compared to more traditional biochemical or immunological analyzers. 

For clinical research, the major advantages of LC–MS-MS over traditional technologies are the speed of assay development; the same relatively low cost-per-assay whether there is one or many analytes in a single sample; high specificity, significantly higher than immunoassays especially for small molecule analytes; and no costly analyte specific reagents (ASRs) are required. There are also some disadvantages to LC–MS-MS, such as the high capital cost of these instruments, usually with no "reagent-rental" payment options; LC–MS-MS systems are complex instruments and not trivial to learn to operate; LC–MS-MS is not sensitive enough for some important classes of analytes compared to immunoassays (such as proteins); and relatively labor-intensive or complicated sample preparation is sometimes required for trace analytes in biological matrices. Such sample preparation techniques are necessary because of the relatively high levels of proteins, salts, and lipids encountered in biological matrices, which can interfere with the ionization of analytes should they be coeluted. These disadvantages are responsible for the relatively limited uptake of LC–MS-MS into routine clinical diagnostic workflows where technicians operating instruments are normally required only to place samples directly onto the analyzers with little or no pretreatment. 

Recently, significant breakthroughs in sample preparation and multidimensional chromatography have been commercially introduced that are solving many of these issues for LC–MS-MS. Specifically, turbulent flow chromatography (TurboFlow, Thermo Scientific, Franklin, Massachusetts) has enabled rapid highly automated on-line sample extraction before analysis by LC–MS-MS. Instruments such as the Thermo Scientific Aria LC–MS system and Transcend TLX turbulent flow ultrahigh-pressure liquid chromatography (UHPLC) systems (Thermo Fisher Scientific, Waltham, Massachusetts) can accommodate the direct analysis of biological samples by LC–MS-MS, often only requiring sample dilution before placing the sample onto the system. This minimizes time- and resource-consuming off-line sample preparation, which can often lead to delays in assay results or to the introduction of errors associated with manual sample handling, transfer, or aliquoting.  

TurboFlow technology involves the proprietary use of large-diameter particles (~ 30 μm) with a high surface area packed into narrow-bore columns (0.5 to 1 mm i.d.) that are available in a variety of stationary phases. Samples such as dilute serum, cerebrospinal fluid, plasma, urine, and biological matrixes are injected directly onto these columns under high linear velocities, inducing turbulent-flow conditions; this is as opposed to laminar flow, considered ideal for conventional chromatography. Under turbulent flow conditions, small molecule analytes are able to diffuse into the cavities on the surface of the large particles where they are retained, depending upon the stationary phase characteristics and the properties of the analytes of interest. Proteins are too large, and the mobile-phase linear velocities too high (column residence time too low) to allow these compounds to diffuse effectively into the surface pores and be retained. Furthermore, salts are too polar to be retained; consequently turbulent flow chromatography simultaneously eliminates interferences from salts and proteins. Lipids are often retained in turbulent flow chromatography; however, judicious choice of loading and eluting mobile phases as outlined by the manufacturer eliminates interference from this class of compounds in the analysis of a wide variety of analytes including lipophilic compounds such as steroids (Figure 1). Figure 1 demonstrates the ability of turbulent flow chromatography to eliminate ion suppression. In the present experiment a 1 ng/mL solution of nandralone, an anabolic steroid, is continuously infused at 5 μL/min after the analytical HPLC column while a negative control serum sample and a water-blank are processed by a multidimensional LC–MS-MS system. There is no perturbation of the nandralone response during its expected retention time window (shaded area). The result for the negative control serum sample is essentially identical to the water blank, confirming a complete absence of any ion-suppressing matrix components from equine serum during this critical retention period. These data were part of a thorough study on the use of the turbulent flow chromatography system for the determination of the anabolic androgenic steroids trenbolone, boldenone, nandrolone, testosterone, and stanozolol in equine serum (19). Turbulent flow chromatography greatly simplifies sample preparation, allowing LC–MS-MS to fit more effectively into routine workflows in laboratories doing clinical research. 

Besides sample handling, one of the other major issues with LC–MS-MS in clinical research as noted earlier is the high capital cost of instruments. The LC–MS-MS experiment is often quite inefficient as visualized in Figure 2 top trace, where the MS-MS analyzer is acquiring meaningful data only 25% of the time; in this case, most of the run-time is actually involved in waiting for the analytes of interest to emerge from the column or for re-equilibration. The Transcend TLX-4 system can increase duty cycle and throughput by up to 400%, increasing the overall efficiency of the LC–MS-MS system and contributing significantly toward return on investment (Figure 2, bottom). The turbulent flow systems are also capable of utilizing UHPLC; when combined with < 2 μm particle size, analytical columns can further shorten the chromatographic time scale, increasing throughput and making multiplexing that much more efficient. 

Many applications now exist demonstrating the workflow simplification and increased throughput using multidimensional multiplexed LC–LC–MS-MS systems. Laboratories with high sample throughput requirements are especially receptive to this technology if the advantages also include no compromises in data quality. One such method was developed for the therapeutic drug monitoring (TDM) of a prescription analgesic (21). Much of the TDM of analgesic drugs is currently being done using GC–MS, which is the gold standard for such applications. Occasionally, certain pain medications are better analyzed using liquid introduction techniques like LC–MS-MS because their properties, such as thermolability or high polarity, make them incompatible with GC introduction, or they may be dosed at low levels, making the higher sensitivity of LC–MS-MS more suitable. The TLX-4 system used for this study was configured with a TSQ Quantum Access triple-quadrupole MS-MS system (Thermo Fisher Scientific). 

Fentanyl is a potent opioid analgesic that is 80 times stronger than morphine used to treat chronic pain. Because of its potency, it is a candidate for TDM to ensure compliance and that proper dosing regimes are adhered to.  

The method involved the dilution of urine samples with the addition of the internal standards fentanyl-

, followed by vortex and centrifuge before analysis. Norfentanyl is one of the major metabolites of fentanyl and both are excreted in urine. The two analytes and internal standards were detected by LC–MS-MS using multiple reaction monitoring (MRM), in which the MS-MS system selects molecular ions of each analyte with the first MS analyzer and monitors one or more product ions with the second MS. The MRM channels were 337→188 and 337→105 for fentanyl, 342→188 and 342→105 for fentanyl-

, 233→177 and 233→84 for norfentanyl and 238→155 and 238→84 for norfentanyl-

. Ionization was by heated electrospray ion source (H-ESI). Calibrators ranging from 0.5 to 200 ng/mL were created in negative control urine and multiple calibration curves generated per sample batch. Two MRM channels per analyte were used, and their ratios had to be maintained within stringent intensity ratio criteria to ensure specificity as defined by WADA. 

The total HPLC run time per sample was 4.3 min; a typical MRM trace is shown in Figure 3. Each turbulent flow cycle requires less time than the HPLC method. After each cycle, a plug of cleaned-up analyte is sent to its respective HPLC column for analysis; however, only the critical 1.3-min interval, when the analytes of interest emerge from the HPLC, is actually sampled by the MS-MS. S/N values were acceptably high as indicated, allowing quantification within what was considered to be the therapeutic range. The Aria software automatically uses all four turbulent flow channels with their respective HPLC columns in a staggered manner, increasing throughput and duty cycle by over 300%. Hundreds of samples can be run on a single four-channel turbulent flow system each day. 

Calibration curves for each of the separate turbulent flow channels were essentially superimposable, typically demonstrating correlation coefficients (

 ) of > 0.99 and CVs, of between 5% and 15% for large sample sets over lengthy time periods (up to 2 weeks). Besides the increased productivity of the analyzer, sample preparation was greatly simplified compared with the GC–MS method — no extraction was required; no derivatization was required; no evaporation or re-extraction steps were needed; a 15-step method for GC–MS was replaced by a greatly simplified five-step method for LC–MS-MS; tubes needed to be labeled only once for the turbulent flow method compared with four times per sample for GC–MS; one four-channel TurboFlow–MS system was equivalent in output to four or five GC–MS systems for this analyte; but most importantly, the higher sensitivity and specificity afforded by LC–MS-MS for fentanyl increased confidence in the analytical results. 

Testosterone is one of the most challenging analytes in endocrinology clinical research, especially when evaluating the low testosterone levels found in hypogonadal males, in women, and in prepubertal children. Testosterone is a major androgenic hormone crucial to the development of male reproductive organs and masculine characteristics in adults. In females, its main role is as an estrogen precursor. Testosterone measurements are frequently used for the diagnosis and management of male hypogonadism and pubertal delay. In adult women, excess testosterone production results in varying degrees of virilization, including hirsutism or infertility. Mild-to-moderate testosterone elevations are usually asymptomatic in males, but can cause distressing symptoms in females.  

For adults, the normal values for testosterone are about 300–1000 ng/dL for males and 20–80 ng/dL for females. Problems occur with immunoassays below the sub-100 ng/dL level; in recognition of this, the Endocrine Society and the CDC have recently started an initiative, recommending that "in the absence of other information, direct immunoassays (those performed on whole serum or plasma) perform poorly at low testosterone concentrations (i.e., women, children, and hypogonadal men) and should be avoided" (15,22). 

The requirements for increased precision, accuracy, and sensitivity have led many clinical researchers to use isotope dilution (ID) LC–MS-MS for the determination of reference ranges for subjects exhibiting symptoms indicative of these lower testosterone concentrations. Traditionally, steroid hormones such as testosterone were extracted from plasma or serum using liquid–liquid extraction (LLE) methods. Even after LLE, extracts contain considerable matrix (such as phospholipids), requiring relatively long LC–MS-MS run times. Compared with immunoassays, LC–MS-MS methods using LLE impose punishing time and resource requirements on labs wishing to run many samples.  

Scientists at the larger reference laboratories (23) have recently begun to use the Aria and Transcend multidimensional LC–MS-MS systems to eliminate complicated off-line sample preparation methods like LLE and replace them with on-line extraction methods (Table I). These researchers have observed that on-line sample cleanup typically reduces prep time by about a factor of 6 over LLE and reduces sample preparation costs by 70–80%. Furthermore, interference from proteins, salts, and phospholipids can be effectively eliminated and LC–MS-MS run times reduced significantly with multidimensional LC–MS-MS. 

Table I: Comparison of LLE sample handling steps with online sample cleanup for testosterone LCâMS-MS analysis from serum or plasma 

In the present example, a 0.1-mL aliquot of serum was diluted with 0.25 mL acetonitrile containing the internal standard, mixed, centrifuged, and placed onto the Aria TLX system for analysis. The MS-MS instrument used was a Thermo Scientific TSQ Quantum Ultra with IonMax APCI ion source and run in the positive ionization mode where the vaporizer temperature was 525 °C and the capillary temperature 350 °C. Calibration standards were prepared in 5% BSA in 0.01 M PBS (pH 7.4) at the following concentrations: 5, 10, 20, 50, 100, 200, 500, 1000, and 2000 ng/dL.  

The sample injection volume was 100 μL. The cleanup column was a 4 mm × 2 mm i.d. C18 column; the sample was loaded with 100% water, then washed with 10% methanol and eluted onto the analytical column with 80% methanol at 150 μL/min. On its way to the analytical column, the cleanup column effluent was diluted with 600 μL/min of 20% acetonitrile and loaded onto the column (Hypersil Gold C18; 33 × 4.6 mm 3 μm). The sample was finally eluted into the MS-MS with 1.0 mL/min of 50% acetonitrile. The total analytical LC–MS-MS run time was just over 4 min where the testosterone is eluted at around 

 = 2.6 min. The total analysis time per channel, including on-line extraction, was 6.02 min where the MS-MS system monitors each channel's effluent for about 1.2 min centered around testosterone's retention time (Figure 4). 

The testosterone MRM transitions were 289.2→97.1 and 289.2→109.1; the internal standard was testosterone-

 with MRM transitions 292.2→97.1 and 292.2→109.1 using a dwell of 100 ms per channel. The method precision was evaluated by analyzing subject sample pools at concentrations of 20, 50, 140, and 1000 ng/dL. Intra-assay variability was determined by processing and analyzing 20 replicates of the lowest two QC sample pools. Interassay variability was determined by processing and analyzing two replicates of each QC sample pool in six different batches. Intra-assay and interassay precision results are displayed in Table II as % CV. 

Table II: Intra- and interassay precision 

Multiplexed on-line cleanup and analysis with the TLX-4 system permitted the continuous staggered injection of a diluted serum sample every 1.3 min, providing a throughput of over several hundred samples per instrument-day. Calibration curves for each channel were similar to that of Figure 5, showing good linearity from 5 ng/dL to 2000 ng/dL and typical correlation coefficients of 

This testosterone method provided a fully automated system capable of analyzing dilute serum samples with no manual sample preparation steps. Multiplexing using the four-channel TLX-4 system increased throughput, allowing for duty cycle and efficiencies of about 400% over single channel operation and very high throughput for this challenging analyte.  

Traditionally, clinical laboratories have used systems that required extensive sample preparation and derivatization. By adding an LC-LC–MS-MS technique for these applications, scientists doing clinical research can not only benefit from the sensitivity and confidence of MS-MS data, but also reduce the sample preparation steps such as extraction, derivatization, and evaporation. This is particularly true for lipophilic analytes such as steroids and vitamin D and its hydroxylated metabolites, described below. 

Thermo Fisher Scientific developed a quantitative method for analyzing 25-hydroxy-vitamin D

 using the TLX-4 TurboFlow system (24). The method enables an online extraction of biological samples. As demonstrated earlier, online sample extraction has the potential to greatly reduce the sample preparation procedures and therefore a potential source of human error and cost. The combination of turbulent flow chromatography and LC–MS-MS enables clinical research laboratories to provide even faster turnaround times as well as more accurate and reliable analytical results.  

The level of vitamin D in serum is measured by analyzing its major metabolites. Among the vitamin D metabolites, 25-OH-D has the highest concentration and longest half-life in blood. It is considered to be the best indicator of vitamin D concentration in the blood. The MS analysis for this method was carried out on a TSQ Quantum Ultra MS-MS system. Table III lists the SRM transitions and their parameters. 

Table III: MS-MS method - SRM transitions for 25-hydroxy-vitamin D2  and D3  and the internal standard, [2H6]25-hydroxy-vitamin D3 

Turbulent flow methods reduce matrix effects and allow users to inject biological fluids directly into the LC–MS-MS system without further sample handling. Before injection into the turbulent flow system, the 25-hydroxy-vitamin D samples require protein precipitation due to the strong protein binding, thus releasing the analyte for analysis. Injecting the precipitated biological samples into the four-channel turbulent flow system ensures the removal of the interferences, or potential interferences, which may affect method performance when injecting thousands of biological samples, including salts, phospholipids, and residual proteins. This is a significant advantage in laboratories where sample volumes are high, making extensive manual sample preparation impractical. Figures 6 and 7 show the linear calibration curves, both with 

 values better than 0.99 for 25-OH vitamin D3 and D2 in bovine plasma, respectively. Table IV shows a data summary. 

Table IV: Data summary for 25-hydroxy-vitamin 2 and 25-hydroxy-vitamin 3 assays  

Figure 8 shows the representative lower limit of quantitation (LLOQ) chromatogram of 4 ng/mL 25-OH-D3, internal standard, and 25-OH-D2 in bovine plasma.  

The four-channel turbulent flow–MS system delivered fast and efficient quantitative results with negligible ion suppression and low chemical noise. Throughput was as high as a sample approximately every 1.2 min when analyzed in a multiplexed fashion. The mass spectrometer has available a unique H-SRM mode (high resolution precursor ion mass selection, 0.2 u FWHH), allowing for much higher analyte specificity, and as a result, both LLOQ and LLOD were improved. Wide dynamic range of 4–1000 ng/mL (10–2500 nmol/L) was reported. Similar to the above studies, the % CV values for both neat samples and plasma samples meet the bioanalytical method validation criteria for GLP laboratories, which require less than 20% for LLOQ and less than 15% for all the other points on the calibration curve. 

As shown, turbulent flow chromatography can provide a fast, robust, and sensitive analytical method for quantitative analysis with minimal sample pretreatment for a wide variety of endogenous biologically important compounds. This method can be multiplexed using a four-channel turbulent flow system, which will provide a result about every 1.0 to 1.5 min, or over 40 per hour. One mass spectrometer with a four-channel turbulent flow system in multiplexing mode can analyze four times as many samples as a single LC-LC–MS-MS system. These benefits offer significant advantages to clinical research and reference laboratories looking to improve efficiencies and reduce operational costs. For example, data recently presented indicated a cost reduction of over 70% was realized for this assay by switching from radio immunoassay to the four-channel turbulent flow LC–MS-MS system (25). 

LC–MS-MS is emerging as a complementary method to traditional methodologies used for clinical applications. Reduced sample preparation and high-throughput capabilities as demonstrated here can provide significant benefits to clinical scientists conducting routine analyses. Although the initial capital investment for LC–MS-MS instrumentation is substantial compared to traditional techniques, the operational costs are low and the increased flexibility, sensitivity, and specificity are valuable. The addition of turbulent flow and multiplexing capabilities enables a high degree of automation and simplicity in sample handling as well as increased efficiency. As such, the combination of these technologies is expected to expand rapidly in the future development of clinical laboratory services by overcoming the main obstacles to implementation, namely cost and ease-of-use. As scientists focus on more complicated challenges that can be solved efficiently by adding LC–MS-MS to their arsenal of techniques, turbulent flow and multiplexing on-line sample preparation systems will undoubtedly play an increasing role. 

, Director Clinical and Toxicology LC/MS, Thermo Fisher Scientific, Franklin, Massachusetts and 

, Ph.D., CSO, Clinical Mass Spec Consultants, Toronto, Ontario.

The authors discuss the emergence of liquid chromatography coupled with tandem mass spectrometry as a complementary method to traditional methodology used for clinical applications.

Liquid Chromatography Coupled with Tandem Mass Spectrometry for Clinical Applications

Recently, many analytical chemistry groups have begun to investigate and put into practice a number of fast chromatography methods. These methods are being implemented both to increase the resolution of the separation technique and to accelerate and improve the throughput of chromatographic analyses. The problem becomes that as fast high performance liquid chromatography (HPLC) methods are implemented, many traditional mass spectrometers, often used for analysis of unknowns and molecular formula determinations, lose significant resolution. This causes a significant downgrading of the analytical capabilities of the mass spectrometer (impairing its ability to perform accurate mass determination) and precludes effective deployment of many of the current generation of instruments in fast chromatography methods. The net result of the loss of performance of high-resolution mass spectrometry (MS) with increased chromatography speeds will result in an inability to identify some compounds accurately and definitively, making it more difficult to effectively perform many analyses currently done in HPLC conditions with fast chromatography. This conundrum has the potential to impact the utility of both MS and fast chromatography.

For many years, and after several notable failures, many researchers were convinced that it was impossible to design a quadrupole time-of-flight (qTOF) mass spectrometer that was able to retain its ability to perform the high-resolution measurements necessary for definitive molecular formula determination of unknowns. Conventional wisdom indicated that there were many reasons (for example, temperature stability, ion diffusion, and ion loss on grids of reflectrons) that would make it impossible to improve resolution of these types of instruments. Figure 1 shows a schematic of an instrument designed for high-resolution measurements with fast chromatography (Maxis UHR-TOF mass spectrometer, Bruker Daltonics, Billerica, Massachusetts). The instrument includes an ion chiller, a series of ion refocusing operations, a single reflectron, and temperature control of the overall flight tube of the instrument.  

Figure 1: Schematic of a high-resolution TOF mass spectrometer (Maxis UHR-TOF mass spectrometer, Bruker Daltonics). 

Evaluating Method Performance with Fast Chromatography of Small Molecules

To evaluate the performance of the fast chromatography–MS method for use in molecular formula determinations, a series of five unrelated drug compounds was studied. These compounds were chromatographed using a reversed-phase column over a 30-s elution gradient. The results of the initial studies are presented in Figure 2.   

Figure 2: Separation and measurement of drug compounds with fast chromatography. 

As can be seen, the method was able to achieve baseline separation of the five compounds in 30 s. In addition, the peak widths, when measured at half height, were all less than 1 s. 

Figure 3 is a closer examination of the fourth peak in the compound series, oxybutynin. The left half of the figure shows the extracted ion chromatogram, demonstrating that even with a 1-s peak, over 20 data points were taken. This leads to the high levels of both mass accuracy (0.8 ppm) and resolution (>40,000) shown in the right half of the figure. Other peaks showed similar results.  

Figure 3: Close examination of a single fast chromatography peak. 

Evaluating Method Performance with Fast Chromatography of Peptides and Proteins

As many research groups are focusing more and more on protein and peptide analysis, the method was also tested for its ability to analyze proteins and peptides effectively. Figure 4 illustrates the data obtained when measuring isobaric peptides. The data shown here indicate that even with isobaric peptides, the method is able to differentiate these peptides very clearly, even when the molecular weight difference is only 50 mDa. Finally, a profile of a whole cell lysate was obtained to determine the number of proteins that could be identified. The results of this experiment are shown in Figure 5.  

Figure 4: Resolution of isobaric peptides. 

The high-resolution MS method enabled identification, with a high degree of certainty, of a very large number of proteins, even in a complex sample such as a cell lysate. Not only were the number of proteins identified very large, but the overall accuracy of identification was also very good. The high-resolution capability demonstrated here would be extremely useful for rapid profiling of a series of proteins or peptides in a biomarker study, for instance.  

Figure 5: MS analysis of E. coli whole cell lysate. 

Today's analytical chemistry environment demands the deployment of more sophisticated methods and instrumentation to keep pace with the profound changes in separation techniques being adopted by many laboratories. Two of the most important tools for the future will be fast chromatography and MS. The method described in this article represents a successful combination of high-resolution MS with fast chromatography. 

 is marketing manager with Bruker Daltonics, Billerica, Massachusetts. 

The author discusses the impact of fast chromatography methods on the performance of high-resolution mass spectrometry.

The Impact of Fast Chromatography and Its Effects on Mass Spectrometry

Obtaining a complete profile analysis of low molecular mass organic acids (LMMOA) is essential because these compounds play crucial roles in a broad range of processes. Previously reported methods focus primarily on a limited number of LMMOAs, and thus lack the capability to provide a significant LMMOA profile. This article describes an ion chromatography–mass spectrometry (IC-MS) method for profiling LMMOAs in consumer beverages and biomass used in biofuel production. The factors that influence the optimization of coupling MS to IC are discussed, and results from both MS and MS-MS detection of LMMOAs are compared. Method performance is evaluated with respect to linearity and range, and detection limits and reproducibility, and LMMOA profiles of several beverages and biomass are compared.

Organic acids are present in many media and play various crucial roles. Low molecular mass organic acids (LMMOAs) have been researched extensively in many areas, such as food chemistry, as these acids affect taste, shelf-life, and food safety (1–3); agriculture, ecosystems, and environmental science, as these acids act as key components in mechanisms that some plants use to cope with environmental stress (4) and fertilizer release (5); biotechnology, to better understand and optimize fermentation processes (6,7); and biomedical research (8–11). Recently, LMMOAs were found to have inhibitory effects on the bioconversion efficiencies of lignocelluloses to ethanol (12). 

Numerous studies have been performed to develop analytical methods for LMMOAs, including various chromatographic techniques such as ion chromatography (IC), gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE). Detection methods include ultraviolet (UV), refractive index (RI), conductivity–suppressed conductivity detection (CD), and mass spectrometric detection (MS or MS-MS). Many reported methods focus primarily on a limited number of LMMOAs and are incapable of providing a complete LMMOA profile. 

 Figure 1: Adding acetonitrile through dedicated C-channel to maintain chromatographic efficiency. 

This article describes an IC–MS method for profiling analyses of LMMOAs in beverages and biomass. Ion chromatography was applied to separate LMMOAs due to its superior selectivity. Chromatographic resolution was carefully tuned to ensure desirable separation for critical pairs, such as isomeric analytes. MS detection was utilized to provide sensitive and selective quantification. 

LMMOA standards were purchased from Sigma-Aldrich (St. Louis, Missouri). Deionized water (18.2 MΩ-cm) was generated in house from a Millipore Milli-Q water station (Billerica, Massachusetts). Acetonitrile (HPLC grade) was purchased from Burdick & Jackson (Morristown, New Jersey). Isotope-labeled internal standards (valeric-

 acid) were purchased from C/D/N Isotopes (Pointe-Claire, Quebec, Canada). Beverage samples were purchased from local grocery outlets and refrigerated until analysis. Microalgae biomass samples were collected in the growth phase and delivered to the analytical laboratory and refrigerated before analysis. 

ICS-2000 IC system (all instruments and materials were obtained from Dionex, Sunnyvale, California, except as noted) was operated in external water mode with deionized water regenerant delivered at a flow rate of 1.0 mL/min by an AXP-MS auxiliary pump. Back-pressure tubing was applied to generate a back pressure of ~1000 psi. 

The IC columns were IonPac AS11-HC 2 mm analytical columns with AG11-HC guard columns and a CR-ATC continuously regenerated anion trap column (all from Dionex). The eluent source was an EGC II KOH system (Dionex). A hydroxide mobile phase gradient was used: -10 min, 1.0 mM; 0 min, 1.0 mM; 8 min, 1.0 mM; 18 min, 15 mM; 28 min, 30 mM; 40 min; 1.0 mM. The flow rate was 0.38 mL/min, the injection volume was 25 μL, and the oven temperature was 30 °C. Three types of detection were used: Suppressed conductivity detection with an ASRS 300 suppressor (2 mm) (Dionex); MSQ Plus (MS) selected ion monitoring (SIM) (Thermo Fisher Scientific, San Jose, California); and TSQ Quantum Access (MS-MS) (Thermo Fisher Scientific) selected reaction monitoring (SRM). 

Acetonitrile was delivered at 0.20 mL/min by a second AXP-MS auxiliary pump and mixed with eluent by a static mixing tee before entering the electrospray ionization (ESI) probe of the mass spectrometer to assist desolvation. Back-pressure tubing was applied to generate a back pressure of ~1000 psi. The nebulizer gas was nitrogen at 80 psi, the probe temperature was 550 °C, the needle voltage was 3000 V, and the scan mode was negative SIM. See Table I for scan details. 

As with the SIM detection, acetonitrile was used to assist desolvation. However, a static mixing tee was not used — acetonitrile was introduced through the sheath liquid channel and mixed with IC effluent at the tip of the ESI needle. The needle voltage was 4000 V, the sheath gas level was 50 (arbitrary units), the auxiliary gas level was 5 (arbitrary units), the sweep gas level was 2 (arbitrary units), the capillary temperature was 270 °C, and the scan mode was negative SRM. See Table I for scan details. 

Table II: Calibration range and %RSD of MS SIM and MS-MS SRM detections 

Primary stock solutions were prepared by dissolving appropriate amounts of individual neat chemicals in deionized water. Calibration standards were prepared by series dilution to 10 levels: 1.00, 5.00, 10.0, 20.0, 50.0, 100, 200, 500, 1000 and 2000 ppb (ng/mL) with a consistent 200-ppb concentration of each isotope-labeled internal standard in each level. 

Beverage samples were homogenized by vigorously shaking, and degassed using vacuum filtration through a 0.45-μm GHP membrane. Three 60-μL aliquots of each filtered sample were spiked with internal standards at 200 ppb and diluted with deionized water to a final volume of 6.0 mL. 

Biomass samples were centrifuged at 6000 rpm for 15 min. A 600-μL clear aliquot of each sample (

 = 3) was spiked with internal standards at 200 ppb and then diluted with deionized water to a final volume of 6.0 mL. 

When using an MS detector for IC applications, an organic solvent is usually teed in to the IC effluent before entering MS probe to assist desolvation and to improve ionization efficiency. The MSQ Plus system was coupled to IC by this means. However, when the premixed flow (0.38 + 0.20 = 0.58 mL/min) entered the TSQ ESI probe, a system back-pressure increase (>120 psi) was observed. The additional pressure applied to the IC suppressor adversely affected chromatographic efficiency (that is, it changed retention times, decreased resolution, and distorted peak shapes). As an alternative, the sheath liquid–calibrant channel (C-channel) was used to introduce organic solvent to the IC effluent. The capillary transferring tubing was minimized in length and attached to a customized grounding unit. The system back-pressure increase dropped to 40 psi, which is approximately the optimum operating back pressure for the ASRS 300 suppressor. Figure 1 shows the SRM chromatograms obtained through both approaches. By using the C-channel to add acetonitrile, the observed chromatographic efficiency of the mass spectrometer was very much improved.  

Figure 2a shows the CD and SIM chromatograms of the standards. Figure 2b shows the SRM chromatograms of the standards at 100 ppb. All 32 LMMOAs were separated and detected. Critical pairs, which cannot be differentiated by MS and MS-MS, such as valeric and isovaleric acids, and succinic and methylmalonic acids, were separated chromatographically.  

Linearity and calibration were evaluated by analyzing calibration standards from 1 ppb to 2000 ppb. Calibration range, coefficient of determination (

 ), and detection limits are listed in Table II. Different fitting types were used for the calibration curve to generate better 

 values. For MS SIM detection, linear, quadratic, and cubic fitting were used to generate a calibration curve with 

 > 0.99. For MS-MS SRM detection, only linear and quadratic fitting were used. Detection limits were determined as the lowest calibration standard that consistently gave a signal-to-noise ratio (S/N) greater than 3 (S/N > 3) and were used as the lower end of calibration range. Low ppb (< 10 ppb) detection limits were achieved for each analyte, with exceptions listed in Table II. Method instrument reproducibility was shown as %RSD calculated from seven replicate injections of a standard at 20 ppb (MS SIM) or 100 ppb (MS-MS SRM). Excellent reproducibility was observed for each analyte by MS SIM detection with %RSD ranging from 2.15% to 8.57%. The %RSD for MS-MS SRM detection ranged from 1.19% to 25.8%. 

Comparison of MS and MS-MS Detection Methods

The single-quadrupole instrument provided a wider calibration range, up to 5000 ppb, while triple-quadrupole MS showed an upper limit of 2000 ppb. Comparable coefficients of determination (

Both instruments showed good detection limits at low parts-per-billion levels. It is notable that the SIM detection of the MSQ Plus system showed excellent detection for LMMOAs with very low molecular weight, such as formic acid and acetic acid. On the other hand, the SRM detection of these analytes was not sensitive even at high concentrations (above 1000 ppb). MSQ SIM detection also showed better detection limits for LMMOAs with molecular weight < 100 amu (propionic acid, glycolic acid, butyric acid, pyruvic acid, and valeric acid). This difference can be attributed to the enhanced low mass ion optics the MSQ Plus system was equipped with to improve ion transmission efficiency. It is also worth noting that the calibration range of lactic acid started from 100 ppb using MS SIM detection. This is because a significant peak was observed in lactic acid's SIM channel at lower concentration, which was not correlated to the specified amount. This abnormal observation might indicate interference for lactic acid at low concentration. As a more specific detection, SRM showed no interference for lactic acid's specific ion transmission 89.2 → 43.6; therefore, the linear range was extended at the lower end to 5 ppb.  

Several LMMOAs showed instability in standard solutions. The previously reported conversion of oxalacetic acid to pyruvic acid (13,14) was observed in our experiment. Thus, oxalacetic acid was not included in calibration standards due to its potential interference with pyruvic acid. Other LMMOAs also demonstrated instability, such as fumaric acid, succinic acid, butyric acid, and pyruvic acid, showing a change in relative peak area ratio over time. Stability evaluation will be required for full method validation. For the real sample quantification performed in this study, a fresh set of calibration standards was prepared along with each set of real samples. All solutions were analyzed promptly after preparation. 

Application for Beverage and Biomass Analysis

This method was successfully applied to profiling LMMOAs in beverages and biomass. The results are shown in Table III (beverages) and Table IV (biomass). Citric acid was observed as the prominent LMMOA in green tea, with a concentration greater than 200 ppm, and a total of 27 LMMOAs were detected in one or both green tea beverages. Gluconic acid, galacturonic acid, malic acid, tartaric acid, citric acid, and isocitric acid were prominent (> 200 ppm) in grape juice, and 28 LMMOAs were detected. The prominent acids found in grape juice were also observed in wine samples, except citric acid, which had a lower concentration. Lactic acid, acetic acid, and succinic acid were also observed in amounts greater than 200 ppm in wine, and 28 LMMOAs in total were detected. Their concentrations are reported in Table III.  

The presence of 17 LMMOAs was confirmed in biomass. Succinic acid was the most prominent LMMOA, with a concentration greater than 200 ppm. Mucic acid, α-ketoglutaric acid, oxalic acid, and citric acid were also present in large amounts (>5 ppm). 

Profiling analysis of LMMOAs was described using IC–MS (MS SIM or MS-MS SRM). This sensitive and selective method ensured the detection of LMMOAs from low parts per billion to 2000 ppb (MS-MS SRM detection) or 5000 ppb (MS SIM detection). Linearity was achieved throughout these ranges with coefficients of determination (

 ) greater than 0.99. Comparable detection limits were observed using both detection techniques, with MS SIM detection providing better sensitivity for low-mass LMMOAs, while MS-MS SRM offered more specific detection. LMMOA profiles of green tea, grape juice, white and red wine, and biomass were reported. The observed prominent LMMOAs agreed with previously reported results (15). 

The authors would like to thank Rodney Corpus for providing biomass samples for the method evaluation. 

, the senior manager, are with the Mass Spectrometric Technical Center, Dionex Corporation, Sunnyvale, California. 

This article describes an ion chromatography–mass spectrometry (IC-MS) method for profiling low molecular mass organic acids in consumer beverages and biomass used in biofuel production.

Ion Chromatography–Mass Spectrometry for Profiling Low Molecular Mass Organic Acids in Beverages and Biomass

Agilent Technologies' Bio-Monolith HPLC columns are designed for high resolution, rapid separations of large proteins, antibodies, plasmid DNA, viruses, and other macro biomolecules. The columns contain a short, polymer-based monolith disk that reportedly allows for flow-rate independent separations. Strong cation exchange, strong and weak anion exchange, and protein A phases are available. 

The SampliQ SPE solid-phase extraction products from Agilent Technologies are designed for fast purification of extracts to improve the accuracy and reproducibility of analytical results. The products include polymer, silica, carbon, and Florisil PR sorbents in formats ranging from cartridges to 96-well plates. 

ProteoIQ software from BioInquire provides support for proteomic data analysis from statistical validation to quantification. According to the company, the software can be used for comparative proteomic analyses in which spectral counting-based quantification is combined with data mining functions to find differences between multiple proteomic data sets. 

Eksigent's NanoLC-Ultra high-pressure nano-LC system is designed for high-resolution separation of low-abundance peptides and proteins in proteomics applications. The system reportedly provides precise gradients at pressures as high as 10,000 psi. 

Prosolia's Omni SprayR ion sources use the desorption electrospray ionization method. The sources are designed to allow for direct and rapid sampling and imaging of surfaces with minimal or no sample preparation. 

The AXIMA Performance mass spectrometer from Shimadzu is designed to analyze high-mass species. In reflectron mode, the instrument reportedly generates high-resolution MS data for peptide mass fingerprinting and complex mixture analysis. Other features include full automation, calibration algorithms, data-dependent workflows, and application software. 

Proteome Dynamics proteomics equipment and software from Thermo Fisher Scientific is designed for the identification, quantitation, and characterization of proteins. The Automated Phosphopeptide Workflow includes a phosphopeptide enrichment kit, a magnetic bead purification system, and a hybrid mass spectrometer. Software includes SIEVE 1.2, for label-free differential analysis, and ProSightPC 2.0, for identifying and characterizing proteins in tandem MS experiments. 

The AB SCIEX QTRAP 5500 mass spectrometer from Applied Biosystems/MDS Analytical Technologies reportedly integrates triple-quadrupole capabilities with a linear accelerator ion trap for quantitative and qualitative analysis. According to the company, the system supports multiple experiments on the same instrument using a feature that allows scientists to move from triple-quadrupole scan modes to full-scan ion-trap mode in less than 1 ms. 

The maXis mass spectrometer from Bruker Daltonics is designed for high-speed chromatography, small-molecule identification, trace analysis, metabolomics, and quantitative proteomics applications. According to the company, the instrument provides resolution in excess of 40,000 FWHM and mass accuracy of 600–800 ppb at speeds of up to 20 full spectra/s in both MS and MS-MS modes. 

The Waters Xevo TQ MS system is a tandem quadrupole mass spectrometer designed for high sensitivity, selectivity, robustness, speed, and accuracy in HPLC– and UHPLC–MS-MS methods. According to the company, the system's collision cell provides enhanced LC–MS-MS data acquisition capabilities. Applications include quantitative and qualitative bioanalysis, ADME screening, food safety, environmental monitoring, and forensics. 

ACQUITY UPLC v 1.31 instrument control software from Waters is designed for operational integration and connectivity between the company's ACQUITY UPLC system and ABI/MDS SCIEX mass spectrometers on Analyst and Analyst QS MS software. According to the company, the software promotes source efficiencies and improvements in MS sensitivity and spectral quality. 

The TSI 3980C MacroIMS ion mobility spectrometer is designed for fast separation of macromoles. According to the company, the instrument's software allows overlay of spectra, raw data smoothing, and molecular weight–based controls and statistics. TSI Incorporated, Shoreview, MN;  

				Picarro's model L1102-I water isotope analyzer is designed for simultaneous measurement of D/H and 				

				O isotopic ratios. According to the company, the analyzer provides precision of less than 0.1% for δ				

				  O and less than 0.5% for δD. The analyzer reportedly is fully automated and is not susceptible to drift. 

The CUSTODION solid-phase microextraction (SPME) syringe from Torion Technologies is designed to sample analytes in air, water, or dissolved solids. According to the company,  target analytes are quickly trapped onto the syringe's SPME fiber and can be analyzed on any SPME-compatible GC or GC–MS system. 

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Researchers and practitioners from various disciplines and subdisciplines within chemistry, biochemistry, and physics regularly depend on mass spectrometric analysis. Pharmaceutical industry workers involved in drug discovery and development rely upon the specificity, dynamic range, and sensitivity of mass spectrometry (MS). Particularly in drug discovery, where compound identification and purity from synthesis and early pharmacokinetics are determined, MS has proved indispensable. Biochemists expand the use of MS to protein, peptide, and oligonucleotide analysis. Using mass spectrometers, they monitor enzyme reactions, confirm amino acid sequences, and identify large proteins from databases that include samples derived from proteolytic fragments. They also monitor protein folding, carried out by means of hydrogen-deuterium exchange studies, and important protein-ligand complex formation under physiological conditions. Clinical chemists, too, are adopting MS, replacing the less-certain results of immunoassays for drug testing and neonatal screening. So are food safety and environmental researchers. They and their allied industrial counterparts have turned to MS for some of the same reasons: PAH and PCB analysis, water quality studies, and to measure pesticide residues in foods. Determining oil composition, a complex and costly prospect, fueled the development of some of the earliest mass spectrometers and continues to drive significant advances in the technology. Today, MS practitioners can choose among a range of ionization techniques that have become robust and trustworthy on a variety of instruments with demonstrated capabilities.

Two decades ago, MS was the preserve of experts and skilled technicians as the instrumentation required constant attention and adjustment. At that time, liquid chromatography (LC)–MS was in its infancy and atmospheric pressure ionization (API) source interfacing was just beginning. Samples requiring analysis were passed from the requesting scientist to these "experts for analysis." The samples would be analyzed, processed, and interpreted, and the results returned via a written report. Two decades later, the users and capabilities of LC–MS have changed significantly. Now mass spectrometers and LC–MS systems are ubiquitous in the analytical laboratory, especially in the pharmaceutical industry. These instruments are used by a wide variety of scientists for a diverse range of tasks, from purity screening in medicinal chemistry, to the quantification of drugs in blood and the identification of proteins for biomarker discovery. The usability of the current MS platforms has improved dramatically, with scientists able to operate the systems remotely via the internet, carry out complex data-dependent tasks, such as purification and peptide fragmentation to open-access systems where nonanalytical chemists can queue their samples for analysis and have the results e-mailed to them without ever having to know or concern themselves about the LC–MS process. 

 Figure 1: Daughter ion scans obtained with (top) and without (bottom) the ScanWave function. 

Recent reports put the number of LC–MS systems sold per year in excess of 2500 units. This large number of units sold each year is also reflected in the increased number of users. In 1980, the number of scientists attending ASMS was around 1250. By 2002, this number had risen to greater than 4000, with a growth rate of 10% per year. This growth in LC–MS users has occurred because of the increase in the number of samples analyzed each year per user, creating larger and larger amounts of high-quality data. More and more, this data is being turned directly into information or knowledge so that decisions are made in real time. Many of these new users have little interest in becoming expert mass spectroscopists and are instead looking for the instrumentation itself to decide the appropriate experiments to be performed as well as to interpret the data automatically and recommend a course of action (for example, pass/fail, pure/impure).  

Figure 2: Mass spectra of alprazolam and a plasma sample.  

The interfacing of LC with MS allowed analytical chemists access to about 80% of the chemical universe unreachable by gas chromatography (GC). It is also responsible for the phenomenal growth and interest in mass spectrometry in recent decades. A few individuals are singled out for coupling LC with MS. Beginning arguably in the 1970s, LC–MS as we know it today reached maturation in the early 1990s. Many of the devices and techniques we use today in practice are drawn directly from that time. 

In its simplest form, LC relies upon the ability to predict and reproduce with great precision competing interactions between analytes in solution (the mobile or condensed phase) being passed over a bed of packed particles (the stationary phase). Development of columns packed with a variety of functional moieties in recent years and the solvent delivery systems able to precisely deliver the mobile phase has enabled LC to become the analytical backbone for many industries. Continued advances in performance since then, including development of smaller particles and greater selectivity, also saw the meaning of the acronym change to high-performance liquid chromatography. In 2004, further advances in instrumentation and column technology achieved significant increases in resolution, speed, and sensitivity in liquid chromatography. Columns packed with smaller particles (1.7 μm) and instrumentation with specialized capabilities designed to deliver the mobile phase at pressures up to 15,000 psi (1000 bar) came to be known as ultrahigh-pressure liquid chromatography (UHPLC). Much of what is embodied in this current technology was predicted by investigators such as Prof. John Knox in the 1970s. 

Mass spectrometers can be smaller than a coin, or they can fill very large rooms. Although the various instrument types serve in vastly different applications, they nevertheless share certain operating fundamentals. The unit of measure has become the dalton (Da), displacing other terms such as amu. 1 Da = 1/12 of the mass of a single atom of the isotope of carbon-12 (

 C). Once employed strictly as qualitative devices — adjuncts in determining compound identity — mass spectrometers were once considered incapable of rigorous quantitation. But in more recent times, they have proved themselves as both qualitative and quantitative instruments. A mass spectrometer can measure the mass of a molecule only after it converts the molecule to a gas-phase ion. To do so, it imparts an electrical charge to molecules and converts the resultant flux of electrically charged ions into a proportional electrical current that a data system then reads. The data system converts the current to digital information, displaying it as a mass spectrum. 

The ions required in MS can be created in a number of ways suited to the target analyte in question. These methods include laser ablation of a compound dissolved in a matrix on a planar surface, such as by matrix-assisted laser desorption ionization (MALDI); by interaction with an energized particle or electron, such as in electron ionization (EI); or as part of the transport process itself, as we have come to know electrospray ionization (ESI), where the eluent from a liquid chromatograph receives a high voltage, resulting in ions from an aerosol. The ions are separated, detected, and measured according to their mass-to-charge ratios (

). Relative ion current (signal) is plotted versus 

, producing a mass spectrum. Small molecules typically exhibit only a single charge. The 

 is therefore some mass (m) over 1, with the "1" being a proton added in the ionization process (represented by M+H+ or M-H+ if formed by the loss of a proton), or if the ion is formed by loss of an electron, it is represented as the radical cation [M+.]. Larger molecules can capture charges in more than one location within their structure. Small peptides typically might have two charges (M+2H+), while very large molecules have numerous sites, allowing simple algorithms to deduce the mass of the ion represented in the spectrum. 

 includes the most notable technique, ESI, which itself provides the basis for various related techniques capable of creating ions at atmospheric pressure rather than in a vacuum. The sample is dissolved in a polar solvent (typically less volatile than that used with GC) and pumped through a stainless steel capillary, which carries between 500 and 4000 V. The liquid forms an aerosol as it exits the capillary at atmospheric pressure, and the desolvating droplets shed ions that flow into the mass spectrometer, induced by the combined effects of electrostatic attraction and vacuum. The mechanism by which potential transfers from the liquid to the analyte, creating ions, remains a topic of controversy. In 1968, Malcolm Dole first proposed the charge residue mechanism, in which he hypothesized that as a droplet evaporates, its charge remains unchanged. The droplet's surface tension, ultimately unable to oppose the repulsive forces from the imposed charge, explodes into many smaller droplets. These coulombic fissions occur until droplets containing a single analyte ion remain. As the solvent evaporates from the last droplet in the reduction series, a gas-phase ion forms. In 1976, Iribarne and Thomson proposed a different model, the ion evaporation mechanism, in which small droplets form by coulombic fission, similar to the way they form in Dole's model. It is possible that the two mechanisms might actually work in concert, with the charge residue mechanism dominant for masses higher than 3000 Da, and ion evaporation dominant for lower masses.  

The mass analyzer is the heart of the instrument and is a means of separating or differentiating introduced ions. Both positive and negative ions (as well as uncharged, neutral species) form in the ion source. However, only one polarity is recorded at a given moment. Modern instruments can switch polarities in milliseconds, yielding high-fidelity records. As well as separating the ions, modern mass spectrometers can trap and fragment ions (MS-MS or MS

 ) to produce a wealth of information about the molecule's structure. Other instruments, such as magnetic sector instruments, hybrid quadrupole–time of flight (Q-ToF), and ion cyclotron (ICR) mass spectrometers, can record the mass of a compound to 1 ppm, allowing for the elemental composition of a molecular ion or fragment ion to be deduced. The increased sensitivity afforded by modern MS over other forms of detection, such as UV and fluorescence, comes from the selectivity and specificity of the MS and MS-MS process. During these experiments, specific ions are allowed to pass through the analyzer and reach the detector. During a multiple reaction monitoring (MRM) MS-MS experiment, only ions that undergo a specific fragmentation are allowed to reach the detector. While this reduces the number of ions reaching the detector, it all but eliminates the noise, resulting in a superior signal-to-noise ratio (S/N). This improves assay sensitivity and specificity dramatically. The vast majority of quantitative experiments are performed on quadrupole-based instruments, whereas ion traps and accurate mass instruments are preferred for structural elucidation experiments.  

Single-quadrupole mass spectrometers require a clean matrix to avoid the interference of unwanted ions, and they exhibit very good sensitivity. Triple quadrupoles, or tandem mass spectrometers (MS-MS), add to a single-quadrupole instrument an additional quadrupole, which can act in various ways. One way is simply to separate and detect the ions of interest in a complex mixture by the ions' unique 

. Another way that an additional quadrupole proves useful is when used in conjunction with controlled fragmentation experiments. Such experiments involve colliding ions of interest with another molecule (typically a gas such as argon). In such an application, a precursor ion fragments into product ions, and the MS-MS instrument identifies the compound of interest by its unique constituent parts. 

An ion-trap instrument operates on principles similar to those of a quadrupole instrument. Unlike the quadrupole instrument, however, which filters streaming ions, both the ion-trap and more capable ICR instrument store ions in a three-dimensional space. Before saturation occurs, the trap or cyclotron allows selected ions to be ejected, according to their masses, for detection. A series of experiments can be performed within the confines of the trap, fragmenting an ion of interest to better define the precursor by its fragments. Dynamic range is sometimes limited in ion-trap instruments, and the finite volume or capacity for ions limits the instrument's range, especially for samples in complex matrices. 

Tandem quadrupole and ion-trap instruments have become the workhorses of modern analytical LC–MS(MS). The two capabilities have been incorporated into one instrument platform to produce a linear ion-trap instrument that had all the structural characterization benefits of ion-trap mass spectrometers with the quantitative capabilities of tandem MS instrumentation. These instruments have become popular with scientists who are required to perform more than one type of experiment (quantitative and qualitative) during the course of their work and require the flexibility to perform this on the same analytical platform. These tasks include impurity identification and quantification, and discovery DMPK, where both dosed parent concentration and metabolite characterization are required. Although they seem ideal, these instruments have some drawbacks, especially when using modern high-resolution chromatography. Here the chromatographic peak widths are so narrow (1–2 s) that there is not sufficient time for these ion trap mass spectrometers to select the ions for trapping, fragment the ions, and measure them to produce enough data points to define the peak accurately. Although the collection of MS-MS spectra can be performed with a standard tandem quadrupole MS instrument while still correctly defining the LC peak, the sensitivity is compromised due to the low duty cycle of the instrument.  

Along with this need to improve the usage and flexibility of the instrumentation is a requirement to improve the data processing and monitoring capability. The recent introduction of a new iteration of the tandem quadrupole mass spectrometer, using a traveling wave technology (as described by Kircher in U.S. patent 5,206,506), holds the potential to resolve many of these issues. The Waters Xevo TQ mass spectrometer employs a traveling wave technology to improve MS capabilities, allowing simultaneous multifunctional data acquisition such as MRM and product ion acquisition, all within a timescale compatible with sub-2 micron LC. The instrument is equipped with a modern tool-free source, which simplifies the process of routine maintenance and cleaning; workflow is also simplified with tuning, method generation wizards, as well as real time data checking functionality to prevent sample wastage if the analytical run fails for any reason. The instrument's software also features an interactive LC-MS method database that ensures the correct method parameters are selected and used. 

The mass spectrometer's T-Wave collision cell allows functionality such as rapid MRM switching, 20-ms positive ion–negative ion switching, and minimized cross-talk. This functionality makes the instrument ideal for rapid method development or use in a drug discovery environment for development of multicomponent assays.  

In these drug discovery environments, mass spectrometers often serve a dual purpose, as both a quantitative and a qualitative instrument in DMPK departments. The mass spectrometer provides the flexibility to perform both of these tasks in the same analytical run, or even the same eluted peak. This functionality is called ScanWave and is achieved by maximizing the duty cycle of the instrument. In conventional mass spectrometers, the selected ions enter from the first quadrupole (Q1) into the collision cell, where they are fragmented. The resulting fragmented ions exit the collision cell and are transferred through the third quadrupole (Q3) to the detector. This quadrupole (Q3) can act either as a selective filter, as in MRM mode, only allowing ions with a specific 

 value to pass through to the detector, or it can scan across the entire 

 value range, providing a full spectrum. This full scan mode is particularly useful when performing structural analysis; unfortunately, in this mode, conventional instrumentation suffers from poor duty cycle. This is because the ions exit the collision cell simultaneously regardless of their 

 value. However, as the third quadrupole scans, it can only measure or detect one 

 value at a time. Therefore, for a scan speed of 1000 

 per sec over a mass range of 1000 Da and a 2-s wide peak, the instrument will only spend 2 × 1/1000 s measuring each 

 value. This mass spectrometer's collision cell is designed to improve full scan sensitivity. In the last third of the collision cell, the fragmented ions are accumulated behind a DC barrier to effect ion enrichment. These ions are then released and contained between the DC barrier and an RF barrier at the end of the collision cell. The RF barrier is gradually reduced, ejecting the ions from the collision cell to the third resolving quadrupole. These ions are ejected according to their 

 ratio, with the heavier ions being ejected first. To improve the duty cycle of the instrumentation, the final quadrupole (Q3) is scanned in synchronization with the ejection of the ions from the collision cell, thus increasing the number of ions reaching the detector and hence the sensitivity. 

The authors discuss the changes that have taken place in the last two decades regarding the users and capabilities of LC–MS.

Special Issues-11-01-2008