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    Columns

    Quality Focus: The Laboratory Notebook

    Answering important questions about quality.

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    Robert Green11.01.07

    The Laboratory Notebook



    Answering important questions about quality.



    By Robert Green



    This month’s question about method development provides an opportunity to describe the analytical science of mass spectrometry and how it differs from high performance liquid chromatography (HPLC).

    Q. I have a new product for which I need a laboratory to develop an analytical method. I have received proposals from different labs, and the analytical instrumentation they intend to use varies among them. Some labs propose to develop the method using HPLC alone, while another proposes to include mass spectrometry. Is there an obvious choice as to which would be better for the analysis of my product?

    A. First of all, you should be commended for studying your proposals so closely. It is extremely important to understand how your lab intends to proceed with developing a method for your product, especially if you intend to use it for quality control throughout its production. The more you know, the better the analytical service will be. Besides, there is always the chance that you may need to use another lab in the future so today’s knowledge will avoid tomorrow’s “reinventing the wheel.” Most important, the recently enacted dietary supplement GMPs make the client responsible for the work of its outside lab—now blindly relying on the work of your lab puts you at risk.

    The difference between the two methods mentioned is significant. One method relies on separating the known components in your product and quantifying each one by comparing its UV spectrum with that of a pure standard (HPLC). The other method provides information about the molecular weight and chemical structure of the compound by measuring the masses of individual molecules that have been electrically charged (mass spectrometry).

    The short (and unsatisfying) an-swer is that the best instrument method to use to test your product depends on your product and circumstances. If you are developing a multivitamin for which all of the components are known, the supplier(s) are reliable, and you will be using this method to verify the quantities of these components throughout production, then the HPLC is your instrument. If, however, there are questions as to the identity of your components, or you are concerned about the existence of unexpected materials (such as adulterants and impurities), then mass spectrometry is the way to go. An HPLC analysis can be fooled because two different compounds can have similar HPLC appearances. However, that is extremely unlikely with mass spectrometry analysis. Since we have often discussed HPLC analysis in this column, let’s take a look at mass spectrometry.

    The application of new technology comes in waves. In the old days we used thin layer chromatography. You remember that from high school—you spot the material of interest on the bottom of a plate and watch the spot migrate up. Then we advanced to HPLC, today’s workhorse technology. The theory is the same—chromatography, but its application is very different. The next wave is upon us—mass spectrometry. As the cost of mass spectrometers decrease (although they are still pricey, generally several hundred thousand dollars), the availability of expertise increases, and the technology is applied to a wider variety of applications, its use increases. The pharmaceutical industry is greatly expanding the use of mass spectrometry, and the supplement industry is not that far behind.

    So what is this technology? In short, mass spectrometry is a powerful analytical technique used to confirm the identity of known compounds, identify unknown compounds and determine the structure and chemical properties of molecules. Compounds in very minute quantities can be detected. This means that the sample size can be quite small and the compounds of interest can be of very low concentration in complex mixtures.

    A mass spectrometer measures the masses of individual molecules that have been converted into ions, that is, molecules that have been electrically charged. Since molecules are so small we do not use any traditional unit of measure. Instead, we use a unit of measure called the dalton (Da for short). One dalton equals one-twelfth (1/12) of the mass of a single atom of the isotope of carbon-12. We are talking small!

    So how does this instrument work? There are three essential parts to a mass spectrometer: the ion source, the mass analyzer and the detector. First, the ion source creates the charged particles (ions) from molecules in your sample. There are several ionization methods, some of the most common being Electron Impact (EI), Chemical Ionization (CI), Electrospray (ESI), Fast Atom Bombardment (FAB) and Matrix Assisted Laser Desorption (MALDI). Next the analyzer, of which there are also several to choose from, including: Quadrapole, Sector (Magnetic and/or Electrostatic), Time-of-Flight (TOF) and Ion Cyclotron Resonance (ICR), separates the ions of different masses. The detector is last, detecting the ions that each mass produced. Once the sample has moved through the mass spectrometer, sophisticated software and an experienced analyst then analyze the data.

    Reasonably pure compounds can be introduced directly into the instrument. However, a sample with several compounds present can create an overlapping or mixed spectrum making unequivocal identification impossible. This means that multiple compounds in a mixture must first be separated. This can be accomplished through several methods, the most popular being gas chromatography (GC) and liquid chromatography (LC). Nowadays a GC or LC is coupled directly with the mass spectrometer (creating a GC-MS or LC-MS), allowing compounds to enter the mass spectrometer separated in time, so that the components of mixtures can be detected and analyzed sequentially. Today’s sophisticated instruments also contain on-board libraries that assist in the identification of the compounds present.

    As one can see, mass spectrometers come in many varieties, each for a particular type of sample. In order to accommodate a multitude of sample types, there are six different types of mass spectrometers. Frankly, it is our pharmaceutical work that enables us to support so many mass spectrometers, but that does make them available for supplement work as well.

    Mass spectrometry analyses are really handy when it comes to investigating a product for adulteration. Unfortunately this is a serious problem these days, sometimes accidental and sometimes intentional.

    An increasing number of supplement testing labs are introducing mass spectrometry capabilities. In fact, in order to offer a full range of services, at least some mass spectrometry capability is essential. As with any sophisticated technique, accurate results are dependant on the instrumentation, expertise and experience of your analytical lab. While the HPLC is still the way to go for routine identification and quantification of known compounds, the increasing complexity of our world and our industry requires the mass spectrometer’s sensitivity and specificity.NW
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