Introduction: Oral fluid is becoming an increasingly popular matrix due to its ease of collection. While oral fluid is a relatively simple matrix to collect, the collection kits can cause issues downstream. Most kits consist of an applicator with attached sponge, a tube filled with 3 mL of buffer solution, and a cap. The sponge is used to collect 1 mL of oral fluid and is then placed in the buffer solution to ensure analyte stability and inhibit bacterial growth. The total volume of the solution for testing should be 4 mL (1 mL of oral fluid and 3 mL of buffer); however, it is often very difficult to recover the full 4 mL of solution. There are different techniques to manipulate the sponge and improve sample recovery. Some popular techniques include manual compression and centrifugation. The goal is to remove the full volume of liquid from the sponge to improve analyte recovery. In this work, these two techniques will be compared by examining volume recovery and analyte recovery.
Objectives: The primary objective of this work is to demonstrate the advantages of incorporating a centrifugation step to sample preparation and its impacts on volume and analyte recovery when performing analysis of drugs of abuse and (DoA) in oral fluids by LC-MS/MS.
Methods: An LC-MS/MS method was developed using a biphenyl analytical column. Mobile phase A consisted of 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in methanol. A total of 31 commonly abused drugs were separated under gradient conditions with a total cycle time of seven minutes. Samples were prepared in synthetic oral fluid and combined with Quantisal buffer. Two sample recovery techniques were compared and tested in triplicate: manual compression and centrifugation. Samples were prepared using a salt-assisted liquid-liquid extraction (SALLE) with a saturated sodium chloride solution. Samples were then dried down under nitrogen and reconstituted in 90:10 mobile phase A: mobile phase B, before moving to the instrument for analysis.
Results: The two techniques were compared by examining total volume removed and analyte recovery. Volume recovery was tested by pouring the solution into a graduated cylinder and recording the total volume recovered using each technique. When using centrifugation, on average, an additional 200 μL were collected compared to the manual compression. Analyte recovery was compared by spiking a known concentration, 50 ng/mL, into each of the samples. These samples were evaluated using a calibration curve prepared by only using 1 mL of fortified synthetic oral fluid and 3 mL of buffer. The recovery and peak area of all analytes improved when using the centrifuge. Accuracy and precision of the techniques were also compared. At ±15% of target value, only four analytes fell withing this range when using the manual compression technique, compared to centrifugation, which had 21 analytes within range. This improved to 25 analytes when the acceptance criteria was increased to ±20% of the target where the manual compression was only at 12. When assessing precision, the centrifugation technique yielded lower %RSD values for 24 analytes as compared to manual compression. The use of extraction aides was also investigated and compared to these accuracy and precision results.
Discussion: In this work, two different techniques were compared to remove liquid from the sponge of the oral fluid collection kits. Through examining volume recovery and analyte recovery, it was clear that the centrifugation technique showed the best results for total volume recovered as well as increased analyte recovery when analyzed quantitatively. Centrifugation can add time to the method, but this work highlights the necessity of centrifugation when analyzing DoA in oral fluid.
