A thorough description of MS is outside the scope of this course, but a simple explanation may be useful. To analyze specimens with mass spectrometry, drugs first need to be extracted from urine samples using a series of organic solvents. The elutions are then injected into a chromatography system. Chromatography refers to a filtration type of process in which samples are passed over a stationary phase that contains some chemical substrate, which will retain molecules in the sample to varying degrees. In the case of gas chromatography-mass spectrometry (GC-MS), the sample is vaporized into a gas and carried through a long thin chromatography column. Different drugs in the sample will pass through the column at different speeds, depending on their affinity for the column (how polar or non-polar they are relative to the column's stationary phase). There are many different types of columns that can be used to separate compounds.
In liquid chromatography with tandem mass spectrometry (LC-MS/MS) methods the sample is carried by a solvent and through a column that contains a gel-like liquid, which retains molecules in the samples to various degrees. The purpose of chromatography is to get the molecules (in our case drugs) in the sample to come through the column one by one.
Imagine that you are asked to name and count all the different kinds of candy present in a giant bin containing many different types and pieces of candy. It would be very hard to analyze all the different types of candy in the bin by just looking into the bin. But if we could get each piece of candy to pass by our eye one at a time, in single file, we could easily analyze and count each piece. This is the purpose of the initial chromatography step; it allows a myriad of compounds to be injected but will retain compounds in various degrees and they will (if the method is designed well), elute off the column and enter the MS instrument one by one.
The drug molecules that are slowed or retained by the column will eventually continue through to the mass spectrometer. This device fragments the molecules into charged ions. The ions are then pulled through a vacuum based on their charge. Their trajectory through the vacuum can be controlled using magnetic and radiofrequency adjustments that will allow only ions of a certain mass to hit the detector. The number of ions that hit the detector is directly proportional to the amount of drug in the sample. A technologist then must review and interpret the results from the instrument.
Chromatography with MS is highly specific and can tell us which drugs are present and at what concentrations. Laboratories can develop and validate methods that can detect a given drug or metabolite with a specificity of >99.99%. The reason for this high degree of specificity is that a compound must have very specific qualities to be detected. If we are looking for morphine, for example, we know that our GC-MS instrument will only identify morphine if:
- The compound has the exact retention time as morphine on our chromatography column.
- The compound fragments into the specific ions with the exact mass/charge found for morphine.
- The ratios of those specific ion fragments to each other must match those found with morphine.
The odds that any drug other than morphine will meet these criteria is very low.
One disadvantage to MS methods is they are not highly automated and are require longer run times than immunoassays.