How do we profile active ingredients?

The method we use to profile active ingredients in a sample is called high-performance liquid chromatography (HPLC). We combine HPLC with mass spectrometry (MS) in order to be able to determine each concentration very precisely. In this article you will learn more about how HPLC-MS works, what information it provides us with and why we prefer HPLC-MS over other methods of chromatography.


What is chromatography?

Chromatography is a separation technique in which mixtures of substances are separated into their respective components. There are various types of chromatography, including gas chromatography, paper chromatography, and thin layer chromatography. The technique we choose depends on what we are trying to find out. For the profiling of active ingredients HPLC is superior, because it is very precise. The results of chromatography are displayed in a chromatogram, a visual representation of the profiling. This chromatogram is similar to a simple graph. The peaks and patterns are indications for the presence of various components (in this case, active ingredients).


How does HPLC (-MS) work?

HPLC-MS is carried out with a highly advanced device that, among other things, consists of an injector, a column and a detector (the mass spectrometer, or MS for short). Using the injector, the sample is brought into the device. In addition, a solution of various liquids, the so-called eluent (also referred to as the mobile phase) is pumped through the device under very high pressure. This eluent ensures that the sample can move through the column. We change the composition of the eluent during the process, which we refer to as changing the gradient. By changing the gradient we can effecuate a first separation of components. Then, the various components are guided through a (very thin) metal column which slows down the components. This process ensures that the components do not exit the column all at once, but at different moments in time. After the components exit the column, they move into the detector where they are bombarded with electrically-charged particles (ions). As a result of this bombing the components break down into pieces, which are called fragments. The mass spectrometer then measures the masses of these fragments. Because components always break down in the same fragments we can identify these components  by looking at the masses of its fragments (which are observed using the mass spectrometer). The collected information is then sent to a computer that processes this information into a graph. We call this graph the chromatogram. By analyzing the chromatogram (among other things) we are able to tell what active ingredients are present in a sample and in what concentrations.


What does HPLC-MS bring us?

HPLC-MS is a technique with a very high resolution, which means that also components that are very similar can be well-separated from each other. When HPLC-MS is used for the profiling of active ingredients in plants and herbs this will give a very precise and reliable result. So when you need to know exactly which active ingredients are in your product, HPLC-MS is the way to go. Using the acquired information you can determine whether your product is suitable for the purpose you have in mind. It also helps you in determining the proper dosage.



In addition to HPLC-MS, there are other methods for the profiling of active ingredients. Other laboratories sometimes use gas chromatography (GC). GC is a cheaper method, partly because it requires less sophisticated equipment. The principle of GC is very similar to that of HPLC, the main difference being that instead of a liquid, a carrier gas is used. GC is less precise than HPLC-MS for the profiling of active ingredients. Distinguishing between two substances that are very much alike is not possible with GC, while this is possible with HPLC-MS. In addition to this, the use of GC poses yet another problem. Because the sample is heated, reactions may occur that alter the composition of the sample. This may cause a component to change into another component when it is heated. Consequently the results no longer reflect the composition of the original sample . Although it is possible to do corrections on the gathered data, the end result is much less accurate than it is when HPLC-MS is used.

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