Hair testing 101: Mass spectrometry and liquid chromatography

Lolita Tsanaclis

Lolita Tsanaclis

on Aug 25, 2017

Hair testing tubes-2

The science, technology and jargon around hair testing can be bewildering for the first-time user.

As well as understanding the benefits and limits of hair testing for drugs and alcohol, organisations, social workers and lawyers requesting hair tests would benefit from an overview of how the hair testing process achieves a fair, accurate and useful test result.

Liquid chromatography and mass spectrometry form two essential steps of hair testing here at Cansford and other laboratories. Here’s what you need to know about both.

Introducing LC-MS/MS

The hair testing method we use at Cansford Labs is liquid chromatography (LC) with mass spectrometry (MS/MS) or LC-MS/MS for short. It’s a technique that can be used to analyse biochemical, organic, and inorganic compounds found in environmental and biological samples. It’s a popular technique used by scientists across a wide range of sectors including biotechnology, environment monitoring, food processing, forensics and pharmaceutical, agrochemical, and cosmetic industries.

The technique is actually two methodologies working in tandem. The liquid chromatography separates mixtures with multiple components whereas the mass spectrometry combines extremely accurate identification of the individual components with high molecular specificity and detection sensitivity. Combined, we have a tool that can identify and quantify any substance present in the hair.

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How does the technique work?

When we receive a hair sample in our lab, it is washed to remove chemical deposits on the outside of the hair. Once washed, the hair is disintegrated via chemical or mechanical processes to form a paste or liquified hair.

The hair sample is purified using a chemical process called solid phase extraction which removes unwanted substances but retains the drugs and metabolites. Then, it’s passed through an absorbent material under pressure; this is the Liquid Chromatography part.

Different substances pass through the material at different rates – helping us separate them into their individual components. Once separated, we can introduce the components to the mass spectrometer that uses an ion source to break the hair components into ions – their smallest possible form.

As a compound is introduced into the MS/MS, the first MS ionizes the compound into a few, very specific pieces. These pieces or molecular fragments are specific to individual substances. They are called parent ions. The parent ions produced by the first MS are filtered to allow specific fragments to enter into the second MS. The second MS transforms the parent ions and fragments them into highly specific, product ions which are the "molecular fingerprint" that identifies the substance being tested without doubt. The compound(s) are therefore identified by two techniques: chromatographic and spectrometric.

It’s a precise and thorough process, but it is also time-consuming and uses a lot of expensive equipment. For this reason, a sample may sometimes be screened for the target substance using an immunoassay test. In an immunoassay, a chemical is added to a sample to cause a reaction. This indicates whether the target substance is present.

When the immunoassay test returns a positive result, the result is a presumptive positive result and the testing laboratory must subject the sample to the LC-MSMS test that provides greater detail and, more critically, information on the identification and quantities of substance present.

Why is this important?

Not all hair tests involve liquid chromatography and mass spectrometry. However, some laboratories have been guilty of not performing the second test and declaring a ‘presumptive positive’ for the substance, which later turned out to be incorrect.

By performing both liquid chromatography and mass spectrometry, Cansford Labs ensures that the target chemical is detected and then identified – so those requiring the test can be confident of the accuracy of their result.

What are the limits of the two tests?

LC-MS/MS can confirm the presence of drugs or alcohol in hair across a huge time period, the tests cannot pinpoint the exact date when the donor used either substance.

A human hair grows around 1cm each month. Testing a 1cm section of hair, therefore, indicates whether the donor used drugs or alcohol within the month window – but not on a weekly or daily basis. Instead, hair testing is useful to detect substance use over many months – for example, by testing 1cm sections of a 4cm strand of hair to indicate use over four months.

Moreover, neither test can confirm the context in which drugs or alcohol were used. A hair drug test can give a highly accurate reading as to whether drugs are in an individual’s system. But for an accurate interpretation of the positive result, we need to know as much as possible about the person’s previous drug use, anything that may affect the test (if they’ve been to a part surrounded by cannabis users, for instance) or other external factors which help build the picture of the individual.  

For this reason, the party conducting the test must inform their laboratory of the context. Previous cases have seen donors being accused and acquitted of drug use because the laboratory performing the test was unaware of the conditions of the hair – for example when the donor had recently bleached their hair or shared a living space with another user. In these cases, laboratory technicians will adapt their test methods to account for these conditions.

Liquid chromatography with mass spectrometry is a powerful, accurate technique for detecting drug and alcohol use. But getting the most from both methods requires co-operation from both the donor and the laboratory performing the test. Take the time to understand the test methods used by your hair test laboratory, and inform your lab of all necessary details about your sample. Doing so could mean the difference between a fair or false test result.

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Lolita Tsanaclis

Lolita Tsanaclis

Dr. Lolita Tsanaclis, Chief Scientific Officer of Cansford Laboratories Limited, has been developing methods for the analysis of drugs in hair since 1993. She has been involved in drug testing using hair, blood and oral fluid samples for medico-legal and workplace sectors for over three decades. Dr Tsanaclis is published extensively as author and as co-author in highly regarded peer-reviewed publications and scientific presentations.

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