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44 May / June 2016


Capillary flow LC-MS has attracted most attention for bioanalysis applications in pharma and especially biopharma laboratories. Dedicated solutions have been put forth by LC-MS vendors in order to provide robust, easy-to-use applications. The targeted detection of small molecules, such as drugs and their metabolites, and large molecules, such as peptide hormones and drug antibodies, in biological fluids can be a big challenge with analytical flow LC-MS.


Figure 1: Relative sensitivity gain for different column inner diameters and flow rates from 100 nL/min to 450 µL/min. Data is shown for EDLIAYLK peptide of Cytochrome C tryptic digest. Each dot represents results for a particular column inner diameter and flow rate. 75 µm x 15 cm , 0.3 mm x 15 cm, 1.0 mm x 15 cm Thermo Scientific Acclaim PepMap columns with 2 µm particle size, and a 2.1 mm x 10 cm Thermo Scientific Accucore AQ column with 2.6 µm particle size were used.


Often it is believed that the sensitivity increase for nano and capillary flow compared to analytical flow LC-MS is the result of downscaling column dimensions. It is true that reducing column inner diameter can result in an increase in sensitivity [6] However, in LC-MS analysis this effect is only a minor contributor to the overall sensitivity gain. Hence, the theoretical gain in sensitivity can often not be achieved by only downsizing the column.


Compared to capillary LC-MS, nano LC-MS exhibits even greater sensitivity. Thus the question is raised, why is nano LC-MS not more popular given the obvious benefit. The reason for this is the limited throughput and the high level of expertise required to set up the fluidics. The latter problem can be easily resolved by using ready-to- use capillary connections and integrated plug-and-spray consumables but increasing throughput with a nano LC-MS set-up is very difficult. For example, the gradient delay and sample loading volumes contribute to the much longer analysis times in nano flow compared to analytical flow applications. In absolute terms the gradient delay volumes and loading volumes can often be reduced to a 1 µL or less. However, these volumes have a large impact on analysis times in relative terms. A delay volume of 1 µL will already result in 3.3 minutes delay time at 300 nL/min flow rate. As a result, nano LC- MS run times of one hour or longer are the average rather than the exception. Capillary flow solutions result in sample analysis times of 10-15 minutes per sample, which


is comparable to the cycle times usually experienced in analytical flow analyses.


On the MS side, reducing the flow rate is actually beneficial for the system stability. Less system cleaning is required because there is less contamination accumulating on the ion source and in the ion optics of the mass spectrometer. On the LC side nano flow rates can impair robustness. Fluidic leaks which have no impact on data quality in capillary and analytical flow analyses will cause huge effects in nano flow applications. For example, a leak rate of 30 nL/min is 0.006% flow loss at 500 µL/min but 10% flow loss at 300 nL/min. Developments in capillary connections have resulted in the ability to have leak tight connections within seconds without using any tools. Leaks in other components such as pumps and valves are harder to minimise, but modern LC systems can compensate for leakages by active flow control.


Increasing the flow rate to capillary flow further reduces the risk of small leaks impacting on results. Hence, capillary flow LC-MS results in high sensitivity, high sample throughput and good system robustness. Chromatographers using analytical flow rates who require higher sensitivity can move to capillary flow rates without sacrificing sample throughput and instrument robustness. Chromatographers using nano flow rates and who require higher throughput can move to capillary flow rates because it still provides good sensitivity in combination with modern MS detectors.


Ligand binding assays, such as ELISA, were the method of choice for protein quantification. However, antibody specificity has been questioned recently, with several studies finding that antibodies may target a lot more than only the intended target [8,9]. Additionally, polyclonal antibodies cannot be produced with the same specifications repeatedly requiring revalidation of tests after receiving a new batch of antibodies. This consideration has resulted in the search for alternative methods and hence, the adoption of LC-MS analyses and for better sensitivity capillary flow LC-MS analyses. The latter provides sensitivity high enough to detect analytes at pharmacokinetically relevant levels; its selectivity is unambiguous if the right ion targets are selected, and LC and MS instrumentation has matured enough to provide robust methods.


Another great advantage of LC-MS based assays is the fast, straight-forward method development and multiplexing capabilities. For multiple analytes from the same sample type near identical method conditions can be applied differing only in the MS detection settings. Often, multiple analytes can be detected in a single analysis. Most likely, LC-MS will gradually replace most antibody based applications.


LC-MS analysis of peptide hormones or biotherapeutics is mostly done after enzymatic digest of target proteins into smaller peptides. This results in loss of information since post-translational or chemically introduced modifications cannot be evaluated in the context of the respective intact protein species. The first attempts are being made to detect whole proteins in biofluids but major development work is still needed to overcome the technological hurdles [10]. Once the challenges have been mastered whole protein bioanalysis by capillary flow LC-MS will become an important part of the toolkit for biopharma analysis.


Other fields are also likely to adopt capillary flow LC-MS for their applications.


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