Advantages of Saliva Sampling in Bioanalysis Using Microextraction by Packed Sorbent and Dried Saliva Spot with LC–MS–MS

Saliva offers a fast and non-invasive sampling matrix for determining drug concentration levels, making it a suitable alternative to plasma and blood. During the analysis of biological samples attention is focused on sample pre-treatment. In addition, liquid chromatography coupled to tandem mass spectrometry (LC–MS–MS) is often the method of choice in bioanalysis because of the good selectivity and good sensitivity of the technique. In this article, two sample handling and sample preparation methods for saliva samples are presented and discussed. The first method is microextraction by packed sorbent (MEPS), and the second method is dried saliva spot (DSS). Both methods were applied for determining the presence of lidocaine in saliva.

Liquid chromatography coupled to tandem mass spectrometry (LC–MS–MS) is a key technique in bioanalysis and drug discovery. In the past decade, major developments have been achieved in LC–MS–MS instrumentation to keep up with the requirements of clinical and environmental laboratories. LC–MS–MS has revolutionized drug development because of its sensitivity, selectivity, and high-throughput capabilities (1), and LC–MS is widely used in bioanalytical and clinical laboratories.

Despite these advances in LC–MS–MS, a complex matrix cannot be injected directly into the analytical instrument. Ion suppression effects in MS can be a significant problem when biological fluids are used. Sample pre-treatment is required to remove the matrix components interfering with the separation and/or detection and to pre-concentrate the analytes of interest from the target matrices.

Microextraction by packed sorbent (MEPS) is a miniaturized type of solid-phase extraction (SPE) in which the solid sorbent (1–4 mg) is integrated in a micro-syringe (2). The same syringe can be used for sample extraction and injection on-line. The technique offers several advantages when compared to other sample preparation techniques (3).

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The MEPS technique was first developed to meet the demands of high-throughput performance in laboratories; the technique is automated, reducing handling time and expense. MEPS retains the main effective properties of SPE, namely its broad applications, but also its simplicity and improvement of the subsequent analytical performance that can be obtained with several analytes and matrices (2–4). MEPS has been discussed in a number of publications, and several reviews demonstrate its usefulness for improved sample preparation, particularly to enhance the speed, sensitivity, and selectivity provided by LC–MS–MS (3). MEPS has been applied to extract several drugs and metabolites from biological samples such as urine, plasma, and blood samples (4–17). MEPS also has less environmental impact than conventional SPE, because it requires less sample volume and fewer solvents. It is also more robust than solid-phase microextraction (SPME) (18,19).

There is a push to find alternatives to blood and plasma samples for pharmacological and pharmacokinetics studies. It is well-known that drugs and metabolites can be detected in human saliva, and the use of saliva as a diagnostic tool has been established in many laboratories for systemic diseases. Saliva collection eases the diagnostic process in specific population groups, such as children and elderly patients, for whom blood removal is usually difficult and/or traumatic. Saliva collection is non-invasive and offers unique advantages such as easy sampling, management, and transport compared to plasma or blood. Another important advantage is the lower concentration of proteins in saliva than is found in plasma, which decreases potential drug binding to proteins. Human saliva consists mainly of water (>99%), while the enzymes, glycoproteins, and antibacterial components make up less than 1%.

As mentioned previously, the detection and monitoring of metabolites and drugs has traditionally been performed using blood and plasma matrices. The use of saliva as an alternative matrix has been advocated because it offers both fast and easy sampling. In recent years, several publications studying the drugs, metabolites, and biomarkers in oral fluid have been produced (20–26). The reasons for potentially using saliva should be researched when looking at disease markers and are underscored in a proteomic comparative study of human saliva and plasma (23). It was stated that local oral diseases can be detected by studying saliva alone; however, the study argues that systemic non-proximal diseases can be detected and monitored through oral fluids. When looking at proposed biomarkers related to diseases such as cancer and cardiovascular disease, approximately 40% of such markers was found to be present in saliva; comparing the proteomes of human saliva and plasma, only a 27% overlap was found (23). Many biomarkers present in plasma might be absent in saliva, which could also point towards saliva as an unharnessed pool of potential biomarkers for systemic non-proximal diseases that cannot be found in plasma.

Several other articles have shown that various drugs and metabolites can be detected in saliva with high correlation to blood plasma levels (24). One research group studied the course of the organophosphate pesticide cloropyrifos (CPF) in rats (25). CPF is processed into 3,5,6 - tricholro-2-pyridinol (TCPy), which can then be detected in saliva. Once formed, it was shown that the saliva TCPy levels correlated with those in the blood regardless of the blood concentration, pH of the saliva, or the flow rate of saliva. Another study showed how three biomarkers for non-small-cell lung cancer (NSCLC) could be detected in saliva. The combination of these three biomarkers allowed determination of NSCLC in a small study group with a sensitivity of up to 88.5% and a 92.3% specificity (26).

However, saliva sample collection and sample pre-treatment were not developed enough during the years. Previously it was difficult to stimulate and collect saliva samples. Moreover it was not easy to handle saliva samples in microlitre volumes.

Experimental

The following work was conducted using a Quattro Micro LC–MS–MS (Waters) equipped with an electrospray ionization source (ESI) and operated in positive ion mode. For data handling and quantification, MassLynx software (version 4.1) (Waters) was used. The LC10Advp LC instrument included two pumps (Shimadzu Corporation). The CTC-Pal autoinjector was from CTC Analytics AG. A 3.0 mm × 100 mm, 3-μm ACE C18 column (Advanced Chromatography Technologies) was used. The MEPS syringe and eVol were obtained from SGE.

Xylocaine 5% (lidocaine ointment) was supplied by AstraZeneca. Acetonitrile, methanol, formic acid, isopropanol (99.8%), and ammonium formate (LC-grade) were bought from Merck.

Discussion

A new method to collect and handle saliva, using an electronically controlled micro-syringe and MEPS needle for quantification of lidocaine in saliva, was recently reported (20). The required saliva sample volume was 100 μL and was slowly drawn up by syringe and then pre-treated by MEPS. Lidocaine could be successfully detected by MEPS in combination with LC–MS–MS. It was reported that the C_max for lidocaine in saliva was achieved at 4 h; this is similar to the published results of lidocaine in human plasma samples (20). The study also showed lower concentrations of lidocaine in saliva in comparison to plasma. This is because the free concentrations of lidocaine were measured in saliva while total concentrations were measured in plasma. This study indicated that MEPS and LC–MS–MS can be a useful tool for extracting drugs from exact volumes of saliva samples.

Saliva matrix should be considered a serious alternative to blood, plasma, and urine in clinical settings, forensic toxicology, and biomarker discovery. In addition, the free concentration (bioavailable) of drugs and hormones can be measured in saliva while total concentrations can be determined in blood or plasma

The Advantages of MEPS in Saliva Sampling: MEPS has numerous advantages, but chiefly the speed and the simplicity of the process. It can be used for both sampling and sample preparation. The extraction and the collection of exact volume of saliva is simplified, and the sample preparation time and the solvents used are reduced. In addition, MEPS can handle smaller sample volumes (10–100 μL), which is an advantage when collecting samples from children. Moreover, MEPS is suitable for automation and the packed syringe can be used more than 50 times, making it cost-effective.

Dried Saliva Spot (DSS): Our group developed dried saliva spot (DSS) as a sampling technique to handle saliva samples. The application of DSS showed excellent results for the determination of drugs in saliva (27), with good stability and selectivity. In DSS the saliva (50–100 μL) was placed on a filter paper and then dried in a few minutes. A fixed diameter was punched from the middle of the DSS filter paper and transferred to a sample vial. The analytes on the punched spot were extracted by 150 μL of acetonitrile containing the internal standard. The contents of the sample vial were then vortexed for 30 s and the organic phase was transferred to the autosampler and injected into the LC–MS–MS system. The DSS technique was successfully applied to determine lidocaine in saliva samples (27). The method showed good selectivity and high recovery. In addition, lidocaine was stable on the filter paper for several days at room temperature (27).

The DSS technique is promising and the sampling and the extraction procedures are very simple. In addition DSS can be used for the screening of drugs, metabolites, or hormones. DSS has potential advantages over other sampling methods because it only needs a micro-sample volume (10–50 μL) and has the potential to overcome difficulties associated with plasma or blood, such as special equipment to handle the samples or fridges and freezers for storing and transport. Furthermore, DSS in combination with direct analysis in real time (DART) mass spectrometry could be useful for testing drugs of abuse. DSS could potentially open up new avenues in drug analysis because it eases the bioanalysis process in terms of sampling, storing, and transport.

Conclusions

Two new sampling methods for saliva samples have been developed. We have demonstrated the use of MEPS and DSS techniques with LC–MS–MS as a tool for the screening and determination of lidocaine in saliva samples. Both techniques provide an easy, robust, and rapid analysis for drugs in saliva. Furthermore, the presented method simplifies the saliva sample collection for exact volumes.

Screening and determination of different groups of drugs using DSS will be carried out in the future. Short- and long-term stability with the DSS method will be investigated. The impact of spot size on the limit of detection (LOD) and method accuracy will be studied. The combination of DSS and DART for the determination of drug of abuse will also be investigated.

Abbi Abdel-Rehim has an M.Sc. Eng. in biotechnology from the Royal Institute of Technology (KTH) (Stockholm, Sweden). Until 2011 he was a visiting researcher at Harvard Medical School, Boston, USA. Currently, he is a research fellow at the University of Manchester, Manchester, UK.

Mohamed Abdel-Rehim is a professor in analytical chemistry at Stockholm University (Stockholm, Sweden). In addition, He has worked as a senior and a principal scientist at AstraZeneca R&D, Sodertalje, Sweden, for more than 15 years. He obtained his PhD in pharmaceutical science from Uppsala University, Sweden. Abdel-Rehim has published more than 250 articles including book and encyclopedia chapters, review articles, research papers, technical reports, and conference presentations. He specializes in drug development, bioanalysis, and sample preparation. He is the inventor of MEPS (MicroExtraction by Packed Sorbent).

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