Saliva Sampling

May 11, 2014

Mohamed Abdel-Rehim, professor of analytical chemistry at Stockholm University in Stockholm, Sweden, spoke to Bethany Degg of The Column about a novel approach to determine drug intake using saliva samples.

Q: What are your main research interests?


(PHOTO CREDIT: AKRIS/GETTY IMAGES)
A. My research interests have now developed into the following areas: The screening and detection of drugs and metabolites in saliva; the screening and identification of biomarkers in saliva and breath for early diagnostic purposes; the formulation of new drugs for easy administration to children and older people; the development of new sampling methods for saliva and breath; and the development of new chromatography sorbent material such as monoliths and molecular imprinted polymers for liquid chromatography (LC) and solid-phase extraction (SPE).

Q: Why did you select saliva as a sampling material to determine drug intake?

A. Saliva is quicker and less invasive to sample from patients than plasma, therefore making it suitable for children and elderly adults. Another important advantage is that there is a lower concentration of proteins in saliva than in plasma, which decreases potential drug binding to proteins. It is widely known that human saliva consists of 99.5% water and the remaining 0.5% consists of electrolytes, glycoproteins, enzymes, and antibacterial compounds. In addition, drugs and their metabolites can be detected at different concentrations within saliva. Our preliminary results indicate that the pharmacokinetic curve of the local anesthetic lidocaine (used as a model analyte) in saliva is similar to those in plasma samples — the only difference is that the concentration levels are lower in saliva than in plasma.1

There have recently been significant developments in the understanding of the target drugs and their pharmacokinetics in oral fluid, but the collection of an exact volume of saliva is not sufficiently accurate. Therefore, the development of new sampling methods for saliva samples is needed.

Q: What novel approach did your group take to sample saliva?


Figure 1: A picture of MEPS–eVol set with catheter used for saliva sampling.
A. In this method we used our innovative microextraction by packed sorbent (MEPS) for sampling and for sample pre-treatment of saliva. Small sample volumes of 100 microlitres or less can be handled. The method is easy, of low cost, and "greener". We used an automated analytical micro syringe and a MEPS to collect and handle saliva samples. The method was performed to screen and determine levels of lidocaine, in human saliva samples. In our method a semi-automated MEPS syringe was attached to a catheter to aspirate saliva. A sample of 100 µL or more can be taken directly from the mouth. The saliva was then dispensed through the MEPS sorbent for extraction (Figure 1).

Sometimes saliva can be difficult to work with. This is because saliva stimulated by sympathetic innervation can be thick. We performed our method in two steps: First, we collected saliva using the syringe with a catheter, and then we pushed the saliva through the MEPS needle for the extraction step (Figure 1). Now we are working with a newly developed syringe with two catheter inputs — one for saliva and one for washing and elution solutions — using a wider catheter and a syringe equipped with a micro-valve.

Q: How did you develop MEPS as a sample preparation method for biological samples? What is the story behind it?

A. There is a long history behind the development of MEPS. From 2000–2002, when we were testing solid-phase microextraction (SPME) in bioanalysis, we saw that the robustness of SPME was not good enough for the handling of plasma samples. The most problematic issue of SPME in quantitative bioanalysis is the fibre lifetime. This can drop from 100 extractions to less than 20 depending on the matrix (plasma or blood) and additives used. In addition, the quality of the fibre can differ from batch to batch.2


Figure 2: Diagram to demonstrate principle of MEPS.
I then considered SPE: It is a more robust technology but could it be miniaturized and used with on-line analytical instruments such as gas chromatography (GC) or LC systems? One week later I came up with an idea. I placed silica sorbent between two frits and fitted it into a syringe barrel (Figure 2). I first tried to perform MEPS manually with lidocaine diluted in a water sample, passing the water sample up and down through the C18 silica sorbent several times via the syringe. After that I eluted it with a few microlitres of acetonitrile directly into the GC–MS system.

After a short time I got a lidocaine peak. I was very pleased because it worked the first time and in just a few seconds. When I presented the MEPS to my colleagues, they were astonished that no one had ever thought about it before. The technique provides speed and simplifies the sample preparation process. Later MEPS was patented through AstraZeneca.3 My group and I have performed many experiments with MEPS using different matrices such as water, plasma, urine, blood, and saliva. MEPS has now been automated and we now have MEPS on-line for multiple uses and for single use. Today, many different researchers use the MEPS technique.4