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This article discusses how chromatographer's toolkits have evolved.
A chromatographer's toolkit traditionally consists of the liquid chromatography system (LC), column, and UV detector; all controlled by the chromatography data system (CDS) software. In the early 1990s, single-quadrupole mass spectrometry (MS) made its way into the chromatographer's toolkit and onto the laboratory bench when advances in MS ionization techniques made it practical to combine LC with MS. Mass detectors are now an everyday tool for many chromatographers. In recent years, MS detectors have become smaller, more affordable, more stable, and more robust. Additional advances in software have made MS as easy to use as any other detector coupled to an LC system. Chromatography data management software has now evolved to become an intelligent intermediary between the chromatographer and the LC–MS system. This article will describe how MS detection benefits chromatographers and how chromatography data software makes both quadrupole and tandem quadrupole MS easily accessible to chromatographers.
MS Aids Method Development
Chromatographic method development represents a large time investment for chromatographers. Adding MS to method development studies offers the opportunity to streamline the method development process. Analytical chemists have long known that analytical detectors do not respond equally well to all analytes. For example, unsaturated functional groups or chromophores do not all absorb UV energy equally well, nor do all molecules contain chromophores; hence, the need for alternative detection methodologies. MS represents one such alternative detection technique because it requires that a gas-phase ion can be generated from a neutral gas-phase analyte.
An example shown in Figure 1 demonstrates the advantage of adding MS detection over using UV detection alone. The chromatographic trace shown in the chromatogram of Figure 1 indicates that there are only two analytes present in the sample. By using MS as an additional detection scheme, we can see that there are actually two coeluted analytes under this one peak represented by the ions of m/z 291 and 307 (the structural analogs).
Figure 1: UV detection alone indicates two analytes represented by the two chromatographic peaks. In contrast, MS indicates that two analytes are present in the first eluted chromatographic peak (structural analogs). Hence, this chromatogram highlights three analytes; not the two indicated by LCâUV alone.
Figure 2 illustrates the chromatographic selectivity change that can occur when mobile phase solvents are changed during method development. Three chromatographic peaks are represented by the black, blue, and red traces. The top two chromatograms highlight the elution order as black, blue, and red when using acetonitrile and ammonium formate as the mobile phases. In contrast, the elution order of the chromatographic peaks represented by the blue and red traces switch positions in the bottom two chromatograms when methanol and ammonium formate are used as the mobile phase.
Figure 2: Analyte selectivity may change with the use of different mobile phases. MS provides a straightforward approach to tracking selectivity changes by monitoring analytes based on their m/z values.
MS is Compatible with Rapid Chromatography
A general rule-of-thumb for quantitating a high performance liquid chromatography (HPLC) peak is for the UV-based detector to take 15–20 data points across each peak. LC systems utilizing sub-2-μm column particles and high backpressures enable rapid separations with exceptional resolution; rapid separations and high resolution result in chromatographic peaks that are 1–3 s wide (traditional HPLC peaks are approximately 10–15 s wide). Any single- or tandem-quadrupole mass detection chosen for the task must be equally as fast at scanning peaks or, in other words, obtaining 15–20 data points across a peak no matter how sharp the peak is. Figure 3 shows a separation of a mixture of small molecule compounds and MS detection in full-scan mode. Although achieving 15–20 points across a peak by using full-scan mode is more difficult for quadrupole instruments versus focused scanning in single ion recording (SIR) or multiple reaction monitoring (MRM) modes, the separation highlighted in Figure 3 demonstrates a separation resulting in 23 data points across the peak in full-scan mode; full-scan mode equally divides scan time (for example, 1 s) over a broad mass range of 900 amu (for example, 100–1000 amu), whereas a focused SIR or MRM scan only looks at a small set of masses of 1–3 amu (for example, 1 amu for the parent mass and 1 or 2 amu for the fragment masses) over a time frame of 1 s. Hence, the number of points across a peak in full-scan mode is often lower because less time is spent at any given amu over the span of a 900 amu mass range.
Figure 3: Rapid MS scan speeds provide greater than 15 points across a peak in full-scan mode.
Qualitative studies generally look for the presence of a parent ion or fragment and have less need for calculating the area of a chromatographic peak. However, in quantitative studies, sufficient data points of ≥15 points across a peak become more crucial where determining accurate and reliable chromatographic peak areas is important to achieve accurate quantitative results. A quantitative study highlighting detection (Figure 4) of approximately 20 pg of sulfamerazine demonstrates 31 points across the peak for a 1.5-s-wide chromatographic peak (base-to-base). Hence, whether conducting qualitative or quantitative studies, single- and tandem-quadrupole mass spectrometers are compatible with the rapid separations achieved by chromatography systems utilizing sub-2-μm column particles.
Figure 4: Mass chromatogram of 20 pg of sulfamerazine injected on column. The tandem-quadrupole detector was operated in MRM mode and obtained 31 points across the 1.5-s-wide chromatographic peak (base-to-base).
Switch-On MS Detection Capabilities
MS detection offers many benefits to chromatography projects, from serving as an alternate analyte detection methodology to streamlining method development. An advanced chromatography data system makes adding MS detection to a chromatographic project a straightforward operation.
For example, a chromatographer wishing to utilize mass spectrometry would simply create a project within the CDS and select the appropriate detection technique. Adding mass spectrometry detection via a "switch-on" approach allows chromatographers to easily add MS detection to a project just as they would add other detection methods such as UV.
For those organizations pursuing a strategy of software standardization, the switch-on approach gives chromatography laboratories the ability to reduce the time and cost associated with validating, integrating, supporting, and providing training for multiple software tools. Additionally, with scalability restrictions removed, the CDS solution can be deployed readily throughout an entire site or multiple sites within a global organization.
Automated Optimization of MS Parameters
One reason that chromatographers unfamiliar with MS fundamentals have avoided MS for quantitative studies is that optimizing the mass spectrometer detection parameters has represented a time-consuming and sometimes challenging proposition. However, recent advances in mass spectrometer acquisition software have automated the optimization of mass spectrometer parameters for quantitation including ionization mode, MRM transition ions, capillary voltage, cone voltage, desolvation gas flow rate and temperature, source temperature, and collision energy. The software systematically guides the chromatographer through the necessary parameter-optimization steps for the MS detector, thereby streamlining the creation of a robust chromatographic quantitative method.
Similar Data Review for MS and UV Chromatography
Reviewing results from quantitative studies that utilize MS as a detection method should not be an obstacle to realizing the benefits of MS in quantitative studies. Data review of LC–UV projects primarily involves two types of data, represented by chromatographic and spectral windows. The chromatographic window shows the separation with respect to time versus absorbance, while the spectral window shows the wavelength absorbance versus total absorbance for a particular chromatographic peak (Figure 5a). Likewise, MS data is organized in a similar fashion with the chromatographic window showing a separation with respect to time versus total ion intensity. The spectral windows show the mass spectrum for a particular chromatographic peak along with the ion intensity (Figure 5b). Hence, conducting UV and mass spectral data review in a similar fashion both streamlines the data review process and serves to minimize the learning curve.
Figure 5: (a) UV review: The center window represents the overall LCâUV chromatogram and the smaller right-hand windows indicate UV absorbance of analytes at indicated wavelengths. (b) MS review: The center window represents the overall LCâMS chromatogram and the smaller right-hand windows show the mass spectra for selected analytes.
Simplified Results Reporting for Regulatory Compliance
Advanced CDSs utilize a relational database to store, catalog, and retrieve data. The benefits of utilizing a relational database include data traceability for compliance with regulations and streamlined result reporting. Relational databases streamline report generation because they facilitate the use of configurable report templates whereby report objects such as tables, spectra, chromatograms, titles, etc. are positioned onto the report template and then automatically filled with data retrieved from the relational database during report generation. A configurable report template filled directly from the database renders many types of data and is agnostic towards the data type. For example, the configurable template-based reporting capability displays text-based information (for example, titles and tables), as well as chromatograms and spectra. Quantitative studies might require thorough documentation, particularly while adhering to regulatory regulations. A CDS solution based upon a relational database simplifies quantitative reporting because all necessary information can be defined to appear in the report template.
Analytes that lack a UV-detectable chromophore require alternative chromatographic-based detection methodologies such as MS. Challenging sample matrices necessitate the need for tandem stage mass spectrometers to provide greater analyte selectivity and sensitivity (for example, discerning analyte peaks from contaminant peaks when analyzing pesticide residues in food). Operating tandem mass spectrometers, however, has traditionally meant that analysts possess specialized skills. In the past, many chromatographers felt that the increase in selectivity and sensitivity that MS detection gives was not worth the additional effort required to operate a mass detector.
In recent years, increasing numbers of bench-top single- and tandem-quadrupole mass spectrometers have become available. These systems are not only easier to use but come equipped with acquisition software that affords an increase in automation. Utilizing MS detectors (particularly tandem-quadrupole systems) controlled by a CDS provides a number of benefits to chromatographers, including:
With the addition of single- and tandem-quadrupole MS and advanced chromatography software to the traditional LC laboratory, chromatographers have the ability to significantly increase their investigatory and development capabilities without suffering the operational inefficiencies often associated with the utilization of advanced detection techniques.
Chris Stumpf, John Van Antwerp, and Steven F. Eaton
Waters Corporation, Milford, MA
Please direct correspondence to Chris_Stumpf@waters.com