Saliva collection system

The principle steps of the sample preparation workflow are summarized in Fig. 1. An equivalent volume of 500 μl neat oral fluid was submitted to SPE after the addition of stable isotope-labeled internal standards (10 μl, buprenorphine-D4 and norbuprenorphine-D3 with 1000 ng/ml all other with 500 ng/ml) and centrifugation (2200×g, 10 min). SPE was accomplished on Strata-X cartridges (33 μm, 200 mg/3 ml, Phenomenex, Torrance, USA). SPE columns were washed with 2 ml MeOH and equilibrated with 2 ml water. Next, the sample was rinsed through the column. After washing with 3 ml water and 2 ml 30% MeOH in water (v/v), columns were dried under vacuum for 10 min to enable elution with two times 750 μl of 2% FA in ACN (v/v). The eluate was evaporated to dryness at 25 °C under a gentle stream of nitrogen. Finally, the dry residue was reconstituted in 50 μl aqueous 0.5% HOAc solution (v/v).Fig. 1

Overview on the principle steps of the sample preparation workflow employed for processing saliva samples collected with the Greiner Bio-One (GBO) saliva collection system

At baseline, a stimulated saliva sample was collected from each participant by using a saliva collection system (Greiner Bio One, Kremsmuenster, Austria) according to the manufacturer's instructions. Specifically, patients were instructed to rinse the oral cavity with the saliva extraction solution (4 ml, citrate buffer pH 4.2) for 2 min; then stimulated whole saliva mixed with the extraction solution was collected in a beaker. For water‐jet sampling, the SAF container was filled with water from a new bottle and 2.5 ml water‐jet samples were collected with the used (test and positive control devices) or a brand‐new nozzle (negative control devices) into a sterile tube. Samples from a newly, and 1‐ and 3‐week opened water bottle were also collected. Immediately after collection and removal of the amount required for culturing, both saliva samples and water‐jet samples were stored at −80°C until polymerase chain reaction (PCR)‐based analysis.