Vibramax 110

The reactions were performed using 2 mL glass bottles (vials), containing 1 mg of the immobilized PcPAL-biocatalysts with the optimal enzyme loading and 3 M NH₂CO₂NH₄ at pH 9.6 as the ammonia source. The reaction mixtures were incubated at 40 °C at 750 rpm in a Heidolph Vibramax 110 platform shaker for the specified reaction times.

The enzyme-linked immunoassays (ELISA) used for the quantification of testosterone (T), 11-ketotestosterone (11-KT) and 17,20β-dihydroxypren-4-en-3-one (17,20β-P) in the plasma and gonads of fish were performed according to [11 (link),12 (link),13 (link)], respectively, with some modifications and using reagents from Cayman Chemical Company (Ann Arbor, MI, USA). For steroid extraction, 200 μL of plasma were extracted twice with 2 mL of diethyl ether. Extraction was done by vigorous vortexing (Vibramax 110, Heidolph, Germany) for 3 min. After decanting the organic phase (supernatant), drying twas done under a stream of nitrogen (Reacti-vap III, Pierce, Germany), and the samples were reconstituted in 250 μL of reaction buffer for running in the ELISA.
Before steroid extraction, testes were homogenized in 1 mL NaOH (0.5 M) with an electric tissue homogenizer (Yellow line DI25, IKA-Works, Germany) and sonicated for 30 s, at 30% amplitude (UP200S, Dr. Hielscher GmbH, Teltow, Germany). Steroid extraction was performed twice with 8 mL of diethyl ether. The procedure that followed was as described above for plasma. Samples were finally reconstituted in 500 μL of reaction buffer. The results were presented as ng per mL of plasma or g of tissue.

The ammonia addition reactions were performed using 2 mL glass bottles (vials), containing the immobilized PcPAL-biocatalysts, SWCNT_NH2-SS-PALs or SWCNT_COOH-SS-PALs, prepared as described above. For the biotransformations, 1 mg immobilized biocatalyst was suspended in 1 mL of ammonia containing buffer, 2, 4, and 6 M NH₄OH at pH 10 (adjusted with CO₂) or 1, 2, 3, and 4 M NH₂CO₂NH₄ (pH 9.6–10 without adjustment), with a 2 mM trans-cinnamic acid concentration. The reaction mixtures were incubated at room temperature at 750 rpm in a Heidolph Vibramax 110 platform shaker for the specified reaction times. For determination of conversion values, samples of 50 μL were removed from the reaction mixture, quenched by adding an equal volume of MeOH, vortexed and centrifuged (13 300 rpm, 17 000g, 1 min). The supernatant was filtered through a 0.2 μm modified nylon membrane filter and analyzed by high performance liquid chromatography (HPLC).

The extraction procedure is depicted in Fig. 1. The pb-WTs were conditioned in Milli-Q water under magnetic agitation. Subsequently, pb-WTs were individually transferred into a glass vial containing 5 mL of the 1:4 diluted sample. The length of the sorbents allows for their entire immersion in the sample to maximize the practical surface area of the materials. The samples (up to 25, although the system allows the simultaneous extraction of 50) were simultaneously stirred in an orbital stirrer (Vibramax 110, Heidolph, Schwabach, Germany) for 1 h at 500 rpm. After the extraction step, each pb-WTs was washed with 2 mL of Milli-Q water in an Eppendorf tube (10 min, 500 rpm) to remove potential matrix interferences. The pb-WTs enriched with the analytes were dried with a tissue and transferred into an HPLC vial. The analytes were finally eluted with 200 μL of methanol for 30 min at 500 rpm. 5 µL of the eluate was analyzed by LC–MS/MS and DI-MS/MS for the identification and quantitation of the analytes.Fig. 1

Extraction procedure showing the main steps: conditioning, extraction, washing, and elution

The optimum pre-cleaning cycles depend on the final analysis strategy. Two cycles are used in LC–MS/MS, while 5 cycles are needed in DI-MS/MS.