Chromafil xtra pa 20 13

Urine was thawed at 4 °C, vortex-mixed, then withdrawn with 2-mL polypropylene syringes (NORM-JECT®, Henke Sass Wolf, Tuttlingen, Germany) and filtered using 0.2-μm Nylon syringe filters (Chromafil® Xtra PA-20/13, Macherey-Nagel GmbH, Dueren, Germany) into 0.7-mL polypropylene HPLC vials (Bruckner, Linz, Austria) with polyethylene snap-caps (Bruckner, Linz, Austria) before injection into the HPLC-ICPQQQMS system. Freeze-dried serum was reconstituted in 1.0 mL Milli-Q water, transferred to 2.0-mL Eppendorf® tubes, and mixed with 1.0 mL HPLC grade acetonitrile (VWR® international, Leuven, Belgium) in order to precipitate the proteins. The mixture was vortex-mixed and centrifuged at 21,380×g and 4 °C for 20 min with a SciLogex D3024R microcentrifuge (SciLogex®, Rocky Hill, NY, USA). The supernatant was transferred to 0.7-mL polypropylene HPLC vials with polyethylene snap-caps and injected into the HPLC-ICPQQQMS system.

Three different batches of PG3 0.1 eq., PG4 0.1 eq. and PG4 0.3 eq. at 1.5 mg/mL in water were prepared. Samples were filtered over 0.45 µm membrane (CHROMAFIL Xtra PA-20/13, Macherey-Nagel GmbH & Co, KG, Duren, Germany) and lyophilized (Christ alpha 2–4 ld plus, Osterode am Harz, Germany). Recovery (%) and concentration (mg/mL) before and after filtration were investigated.