An Introduction to Testing by LC-MS/MS 

 

  1. What is liquid chromatography/tandem mass spectrometry?

  2. What is the difference between a screening and confirmatory test?

  3. What is the clinical significance of the limits of detection?

  4. Why should negative samples be confirmed?

  5. What additional services will we provide?

 

What is liquid chromatography/tandem mass spectrometry?

Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is a confirmatory technique that allows a laboratory to confidently identify and quantify a range of compounds. Unlike point of care urine cups or immunoassay screening techniques, the LC-MS/MS allows for determination of a specific compound. The extensive validation prior to patient sample analysis ensures that interferences do not occur and the identification of individual drug analytes can be completed with certainty. The LC is a technique in which a small volume of the urine sample is injected through a column which allows for separation of each individual analyte present in the patient sample from one another. This occurs based on the interaction between each compound with the polarities of both the solvent and chromatographic columns. The MS/MS portion allows for the identification by fragmenting the compounds and detecting their mass to charge ratios. The individualization is structure dependent and because every analyte has a unique structure, these compounds can be identified. The determination is made by comparing the unique fingerprint of an analyte against that of a reference standard. The combination of the LC with the MS/MS allows for highly sensitive and specific determination of drugs and their metabolites.

What is the difference between a screening and confirmatory test?

Screening is used as a first step in the identification of drugs and their metabolites. The purpose of the screening test is to direct confirmatory testing. The goal is to cast a wide net to catch as many positives as possible and send those samples for confirmation tests. Although these tests are rapid and are useful in directing certain confirmatory tests, they have limitations including false negative and false positive results. Immunoassays tend to produce false positive results due to cross-reactivity as is seen with the MDMA and Amphetamine immunoassay kits (described in detail below). False negative results can be caused by high thresholds or low specificity for certain drug/metabolites including fentanyl and the nor-metabolites of drugs like oxycodone, fentanyl and meperidine. Great care must be taken in the interpretation of immunoassay screening results. Confirmatory testing is typically performed using either gas chromatography or LC-MS/MS as described above. These techniques are essential in the identification and quantitation of compounds with a high degree of certainty.

Our laboratory currently uses Syva EMIT Ecstasy reagent to perform the MDMA screen on our immunoassay analyzer. In the package insert, there is a published list of compounds that have cross-reactivity with this kit. Amongst these compounds is Bupropion or Wellbutrin. We have found that a lot of patients are screening positive for MDMA and this is only triggered as positive because of the presence of Bupropion in their urine. This is simply a false positive on the screening test and that further indicates the necessity for confirmation testing. The confirmation testing completed on the liquid chromatograph tandem mass spectrometer produced no positive results for MDMA, indicating that the positive screen is most likely due to the patient using Wellbutrin as prescribed.

What is the clinical significance of the limits of detection?

The limits of detection used in clinical testing such as pain management or drug treatment testing must be lower than the limits used in workplace drug testing. This is particularly true for opiates where the workplace threshold of 2000 ng/mL would be much too high for monitoring opiates in drug treatment facilities. For most drugs, the limits of detection using the LC-MS/MS will identify drug use for at least several days after last use. This may be extended further when metabolites are also monitored.

Why should negative samples be confirmed?

Screening tests that look for only a specific drug, such as marijuana or cocaine, may not require confirmation testing if the screening test comes back negative. However, most of the screening tests look for a class of drugs, such as benzodiazepines and opiates. Just because the screening test says that it looks for a class of drugs, it does not mean that all drugs in that class would be detected to the same extent. The screening tests have a variable ability to detect different drugs within a class. An example includes the limited reactivity of benzodiazepine immunoassay kits to commonly prescribed benzodiazepines such as clonazepam or lorazepam. They will also have a variable response to some of the metabolites of the drugs. These metabolites are important because they increase the detection time of drugs in the urine. The only way to ensure that all of the drugs of interest are detected if present is to perform the more extensive confirmation test. There are also drugs such as methylphenidate and carisoprodol that will not be detected by any of the available screening tests. Therefore, confirmation testing is the only way to detect the presence of those drugs.

What additional services will we provide?

Our laboratory is staffed to provide a resource to physicians with questions regarding drug metabolism, results concerning consistency and inconsistency with medications, and detection windows. Below is an example of the descriptions physicians can ask for when using the testing services provided by Lehigh Valley Toxicology. Our laboratory can also provide references to aid in the determination of how often patients should be tested and what compounds should be tested.