Recently my youngest daughter asked me, “Why do we have two eyes?”
That was easy to answer, “To figure out the distance and size of objects more easily.”
The next question was a bit more challenging: “Are there creatures with more than two eyes?”
Vaguely remembering my zoology classes at university a couple of decades ago, I answered, “Yes, insects have more than two eyes, although they have a different type of eye.”
I do not want to stretch your patience by completely reproducing my elaboration on human eyes and insect eyes (more information to be found here), because this is an analytical chemistry blog and not a hobby biologist forum.
Because kids never stop asking questions, the next was, “Why do they have more eyes than we do?”
The simple answer is that they need to see the complete picture, almost a 360-degree panorama, in order to survive either as a predator or prey. How is this interesting for an analytical chemist?
Why? The complete picture!
Yes! The complete picture is something the analytical chemist wants to see, too.
Have you ever developed an analytical method and wondered, “Does the method fulfill all my requirements: no co-eluting peaks and no undetectable unknowns with the detector I’m using?” Have you ever been concerned that your compounds may not be correctly quantified?
Detailed knowledge of all impurities that might occur during production or storage is required for a proper risk assessment and compliance in a regulatory environment. Not having the confidence in your method gives the nagging feeling of uncertainty, right?
Therefore, in order to stay calm and “survive” the analytical challenges with more confidence in your results, follow nature’s lead: add another eye. In other words, add additional detection capability to your analytical method for comprehensive sample characterization.
How? 1-2-3! The multi-detector approach
- UV/Vis Detector
The most common detection in HPLC is UV detection for drug quantitation and impurity analysis. Recording a single wavelength is one option; a diode array detector and 3D/contour plots allow you to identify a peak by comparing it with a library and checking the peak purity. But what do you do if the analyte only produces a very small signal due to a low extinction coefficient or the analyte does not have a chromophore at all? And how do you quantify if there is no reference standard at hand?
Like what you are learning?
- Mass spectrometer
OK, adding an MS detector will do the job! This is a great idea to get more information on the structure and it is widely used. But not every analyte is ionizable, thus not every analyte will show up on the MS trace. Also, there is the problem of quantitation without a reference standard at hand. Now what?
- Charged Aerosol Detector
The “third eye” to see the complete picture. Combining the LC-UV-MS system with an additional detector like the Charged Aerosol Detector as a “third eye” allows you to see even more and together they deliver a more complete picture.
HPLC combined with UV and CAD give you complementary information on the compounds. Some analytes, if they lack a chromophore, are only detected by CAD. Some compounds are only detected by UV, because volatile analytes cannot be detected by the CAD. The CAD is a mass-based detector. Therefore, the detector response is independent on the physico-chemical properties of the analytes. This allows good estimation of quantities without a reference standard at hand. This easy-to-use and robust detector is an ideal complementary detector for comprehensive impurity profiling or screening of unknowns.
The challenge in setting up such an instrument platform is to balance the performance of the separation and the compatibility of the combined detectors. Therefore, suitable capillary connections need to be chosen in order to align with hardware requirements, such as flow rate and generated back pressure.
Here’s an example of how an LC system combining UV/Vis, charged aerosol detection, and MS for comprehensive sample analysis can be configured (described in detail in this Application Note).
Using a single quadrupole mass spectrometer (equipped with HESI and APCI sources), the compound identity is confirmed. For most analytes detected by either the UV/Vis or CAD detector, there is also an MS signal. The detected mass of the analyte is then used for identity confirmation. UV 3D/contour plots support peak identification.
The CAD detector supports the quantification of unknown compounds using a single calibrant. The detector delivers a uniform response independent of the analyte even under gradient elution conditions as described in detail here. A simplified method setup to enable this is shown with the Thermo Scientific™ Vanquish™ Duo UHPLC System for Inverse Gradient. Learn more about this setup here.
What for? Multi-detection (UV, CAD, MS) setup applied
Reliable verification and quantification with the multi-detector approach combining UV, CAD and MS is demonstrated in several publications. Please find three application examples listed here:
- Extractables and leachables out of cell culture bags: Application Note
- Study degradation products of formulation additives: Different species are in the polysorbate 20 sample due to incomplete esterification during the synthesis and as degradation product. Knowledge on this is important to characterize the impact of the polysorbates on the stability of therapeutic monoclonal antibody formulation. (reference: Li, Y.; Hewitt, D.; Lentz, Y. K.; Ji, J. A.; Zhang, T. Y.; Zhang, K. Characterization and Stability Study of Polysorbate 20 in Therapeutic Monoclonal Antibody Formulation by Multidimensional Ultrahigh-Performance Liquid Chromatography–charged Aerosol Detection–mass Spectrometry. Anal. Chem. 2014, 86 (10), 5150–5157)
- Impurity profiling of stressed multicomponent drug formulation: In early stage of impurity testing no reference standard is available, therefore peaks are quantified by CAD and compounds above the critical threshold level were identified by MS. (reference: Schiesel, S.; Lämmerhofer, M.; Lindner, W. Comprehensive Impurity Profiling of Nutritional Infusion Solutions by Multidimensional off-Line Reversed-Phase Liquid Chromatography × Hydrophilic Interaction Chromatography–ion Trap Mass-Spectrometry and Charged Aerosol Detection with Universal Calibration. J. Chromatogr. A 2012, 1259, 100–110)
Conclusion: All’s well that ends well
The combination of UV, MS and CAD facilitates the detection of chromophores, organics and inorganics. In addition, it gives mass information for impurity identification and allows quantification without a reference standard at hand.
Quantitation and identification achieved in one run out of one sample! The complete picture!
- Find out more about Thermo Scientific™ ISQ™ EM Single Quadrupole Mass Spectrometer
- One software to control it all: Chromeleon CDS