Milli q

Volumetric polyethylene material and micropipettes with plastic tips were used to prepare collected shells for trace elements analysis33 . Plastic bottles, ceramic coated blades and tweezers kept in 2–5% solution of DECON 90 over 2 h were washed with running water, immersed in 10% of HNO₃ for 24 h, washed with Milli - Q (Millipore) water and dried in a laminar flow hood. The preparation for ICP-MS analysis was performed in a class 100 (ISO class 5) clean room. The valves were separated and the organic tissues were removed using ceramic coated blades and tweezers. The right valve was transferred to a previously acid-washed plastic bottle and the left valve discarded.
Samples were soaked in 20 mL high-purity H₂O₂ (30% w/v) (AnalaR NORMAPUR, VWR Scientific Products) overnight (14–16 h) to remove organic matter from the shell including the periostracum. After organic matter removal, the valve was rinsed in Milli – Q (Millipore) water three times. Digestion of entire valves was performed with addition of 20 mL of high-purity concentrated (70% w/v) HNO₃ (Trace metals; Sigma-Aldrich). To avoid having Ca masking the concentrations of the remaining elements34 35 , the resulting solution was diluted with Milli – Q (Millipore) water to a final acid concentration of 2% HNO₃.

Olanzapine (OZ, M_W 312.43 g/mol) was purchased from Merck KGaA (Darmstadt, Germany). Ethanol, Dulbecco’s phosphate-buffered saline (DPBS), anhydrous dimethyl sulfoxide (DMSO), sodium chloride, and sodium hydroxide were obtained from Merck KGaA (Darmstadt, Germany). The water used was produced with Milli-Q ^® (Millipore Corporation, Billerica, MA, USA).
Aliquots of HA-FA-HEG-OA (30 mg) and HA-FA-HEG-SA (30 mg) were dissolved in 3.0 mL of Milli-Q water and left, under vigorous stirring, at room temperature overnight. The day after, OZ (15 mg) was added to polymer dispersions, to get a drug concentration of 5 mg/mL, and the mixture was left under vigorous stirring, at room temperature overnight. After this time, the dispersions were centrifuged at 8000 rpm and 25 °C for 5 min to remove the drug, which was not incorporated in the self-assembling nanoparticles (NPs). Supernatant was recovered and then dried by freeze-drying, thereby obtaining a spongy solid.

One disk (1.5 mm) was cut from DBS samples and placed into a 96 well plate with 10 μL of deionized water (MilliQ, Millipore, Burlingto, MA, USA), 10 μL of 50 mM formate buffer (pH 2.8) and 7 μL of 2 mM 4-methyllumbelliferyl-α-L-iduronide substrate diluted in MilliQ water. In the blank wells, 240 μL of 0.5 M glycine-NaOH buffer (pH 10.3) were added. After mixing, the microplates were sealed and incubated per 20 h at 37 °C on a shaker (400 rpm). After incubation, the reactions were stopped by addition of 240 μL of 0.5 M glycine-NaOH buffer (pH 10.3). Samples were centrifuged at 2,500 rpm for 10 min at room temperature.