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Direct Analysis in Real Time (DART®)-MS Analysis of Fentanyl and Related Analogues from Saliva Using Biocompatible Solid Phase Microextraction (BioSPME)
Author: Emily Barrey, Senior R&D Scientist, MilliporeSigma, Bellefonte, PA, USA
The extraction mechanism for Biocompatible Solid Phase Microextraction (BioSPME) combined with DART-MS was used to rapidly screen for fentanyl and some of its related analogues at ng/mL levels from human saliva. By employing a fast and accurate screening method, the number of samples that require confirmatory analysis can be reduced and laboratories can increase throughput and decrease costs associated with their drug screening programs. Improved sample preparation techniques, along with the use of stable labelled internal standards, allowed for reproducible and accurate quantitation of the compounds. Advantages over current methodologies with respect to the time of preparation, solvent usage and pre-concentration of the analytes to achieve screening detection limits were demonstrated.
Introduction
Fentanyl and its analogues are routinely used for pain management and anaesthesia in the medical field. However, they also have a high rate of abuse in the USA. In recent years, these compounds have been linked to overdose fatalities [1]. Solid phase extraction (SPE) methods are commonly used for the determination of fentanyl and its related analogues in biological matrices like urine [2]. SPE methods typically involve multiple steps (condition, equilibrate, load, wash, elute, evaporate, reconstitute) that can introduce sample preparation errors as well as analyte loss. SPE methods frequently involve an evaporation step prior to analysis to either concentrate the sample or to switch the solvent to one that is more compatible with the analytical technique. This evaporation step can be time consuming as well as introducing the potential for analyte loss of volatile or semi-volatile compounds.
The use of saliva for drug testing is currently on the rise due to less invasive and readiness of sample collection. Using saliva eliminates some of the issues related to urine collection that can be encountered, such as the patient’s ability to produce specimen and also sample adulteration for non-supervised collections. In addition, using saliva can provide benefits over blood collection such as not having to stick the patient with a needle, as well as improving detection for compounds that tend to bind to plasma proteins.
BioSPME is an equilibrium extraction technique in which the analyte of interest partitions between the sample matrix and the extraction coating on a BioSPME device. For this study, the BioSPME device used was a pipette tip format, which consists of a coated fibre housed within a pipette tip (See Figure 1A). This format allows for the device to be easily manipulated via liquid handlers or robotics, and is therefore amenable to high throughput. The extraction coating consists of C18 functionalised silica particles that are embedded within a proprietary biocompatible binder (Figure 1B). The role of this proprietary binder is to reduce or eliminate the co-extraction of matrix
interferences, without reducing analyte extraction. The binder acts as a shield preventing higher molecular weight species like proteins from being absorbed onto the fibre coating. This allows for the isolation of target analytes, while minimising the amount of matrix, resulting in a highly selective and sensitive microextraction technique. Since the binder also allows for the extraction step to be performed via direct immersion into the sample, it provides a sampling platform that can be used for direct analysis into the mass spectrometer.
Figure 1. (A) A commercially available LC tip BioSPME device which consists of a coated fibre housed within a pipette tip. (B) A basic schematic of an extraction performed with a BioSPME fibre. The fibre is coated with functionalised particles that have been embedded within a proprietary binder. The binder allows the fibre to be placed directly within a biological fluid for sampling.
Experimental
SPME LC Tips, C18 (Part No. 57234-U) and LiChrosolv® methanol were purchased from MilliporeSigma (Darmstadt, Germany). LC-MS grade water was obtained from a Milli-Q® Integral water purification system with a LC-Pak® polisher, also purchased from MilliporeSigma (Darmstadt, Germany). Certified analytical reference standards of fentanyl, fentanyl-d5, alfentanil, sufentanil,
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