Mestrenova 11

To evaluate the structure of pegfilgrastim in formulation buffer, PBS buffer, or DMEM medium, spectra of 1D ¹H were collected on a Bruker Avance III 850 MHz spectrometer equipped with a QCI cryo-probe. The NMR samples that were analyzed comprised 0.035 mL of pegfilgrastim (Neulasta®), 0.02 mL of D₂O, and 0.295 mL of formulation buffer, PBS buffer, or DMEM medium. The experimental temperature was 37 °C. The 1D ¹H NMR experiments were performed using Bruker pulse program p3919gp with 128 scans averaged. The experimental time for 1D ¹H NMR was 6 minutes. Spectra were continuously collected over a time course of 48 h to monitor structural changes. NMR data were processed and analyzed using MestReNova 11.0 software (Mestrelab Research S.L.).

The markers of oxidative stress, including 2-hydroxybutyric acid, 4-hydroxynonenal (4-HNE), l-tyrosine, pentosidine, and N6-carboxymethyllysine, as well as the reduced and oxidized glutathione contents from blood serum, were conducted using a Bruker Advance Neo 500 MHz (Bruker Daltonics, Billerica, MA, USA) nuclear magnetic resonance spectrometer (NMR) using deuterium oxide (D₂O) as the test solvent and trimethylsilylpropanoic acid (TSP) as the standard internal substance. A 1H-qNMR examination was performed on a 500 MHz spectrometer. The quantitative resonance peaks of each aforementioned molecular marker were at δ2.30 ppm, δ8.09 ppm, δ 7.19 ppm, δ7.44 ppm, δ1.30 ppm, δ2.92 ppm, and δ2.16 ppm, respectively (Figure 1). The data was processed using MestReNova 11.0.4 software (Mestrelab Research S.L., Santiago de Compostela, Spain) to specify peaks, analyze the spectra, and calculate sample concentrations [34 (link)].

A Bruker Avance III spectrometer (Bruker, Rheinstetten, Germany) operating at a frequency of 400.13 MHz and equipped with a BBI probe was used for quantitative ¹H NMR interaction experiments. ¹H NMR experiments for the identification of new chemical degradation products were conducted on a Bruker Avance Neo 500 MHz system (Bruker, Rheinstetten, Germany) equipped with a cryo-TCI probe. All samples were prepared in 5 × 178 mm NMR tubes (Z107374 USC tubes, Bruker, Faellanden, Switzerland) at 298 °K. Data were acquired and processed using TopSpin 3.6.0 and 4.0.7 (Bruker, Rheinstetten, Germany) and MestReNova 11.0.4 (Mestrelab Research, La Coruña, Spain).
The ¹H NMR measurements were acquired using a standard 1D pulse sequence (zg) from the Bruker software library (TopSpin 3.6.0 and 4.0.7). The probe was locked, tuned, matched, and shimmed with the sample in place. The specific settings for each sample, and the processing of the spectra were performed as described recently [21 (link),41 (link)]. An in-house taste compound data base was used for the identification of degradation products in the LMW fractions of chemically treated red wine polymers by means of ¹H NMR spectroscopy [42 (link)].

¹H NMR spectroscopy was performed on a Unity INOVA 600 spectrometer equipped with a cryogenic probe (Agilent Technologies, Ltd., Santa Clara, CA, USA) at 25°C. ¹H NMR spectra were collected on 64 K data points over a spectral width of 8000 Hz, resulting in an acquisition time of 4.096 s. The delay time was set to 30 s, and water resonance was suppressed by presaturation. The free-induction decays (FIDs) were multiplied by an exponential function with a line-broadening factor of 0.3 Hz prior to Fourier transformation. The ¹H signal of the methyl group of DSS-d₆ was set to 0 pm and used as a standard signal for peak identification and quantitation of cerebral metabolites of mice with the help of Biological Magnetic Resonance Bank, MestReNova 11.0 (Mestrelab Research, Santiago de Compostela, Spain), Chenomx NMR Suite Professional 5.0 (Chenomx Inc., Edmonton, AB, Canada) [18 (link)] and some published papers [19 (link),20 (link)]. To prove the difference in metabolites in the brain tissue of each mouse group, clustering analysis and principal component analysis (PCA) were performed using SPSS 22.0 (International Business Machines Corporation, Armonk, NY, USA) and SIMCA-P 13.0.3 (Umetrics, Umeå, Sweden).

The HPLC peak areas were post-processed by mean-centering as the default setting in R Studio (ver. 1.2.5042). HPLC peak area boxplot comparisons were performed in Minitab (ver. 18.1) (Minitab LLC., State College, PA, USA) to compared wild samples with cultivated samples. The NMR data were post-processed by median method normalization and Pareto scaling for a better elucidation of the compounds contributing to the separation of each plot in the score plots, and converted to ASCII files using MestReNova 11.0 (Mestrelab Research, Santiago de Campostela, Spain). Spectra in the 0.00–10.00 ppm range were blinded by the solvent peak (2.55 ppm, DMSO-d₆) and the region of 8.00–10.00 ppm was characterized by chlorophyll constituents. The water peak (3.3 ppm) was removed by pre-saturation. The signal was integrated into bins of 0.02 ppm in width, resulting in 285 variables. The generated ASCII files were first imported into Microsoft Excel (version 2019) for secondary variable labeling, and then into R Studio for PCA, HCA, and OPLS-DA. The whole data set was used without splitting off a test set for cross-validation in OPLS-DA.

1D ¹H NOESY (Nuclear Overhauser Effect Spectroscopy) spectra were recorded through a Bruker Avance 400 MHz spectrometer equipped with a 5 mm inverse probe. The following acquisition parameters were used to record the spectra. 1D ¹H NOESY: pulse program = noesygppr1d; size of fid (TD) = 64 K; spectral width (SW) = 20 ppm; transmitter offset = 4.70 ppm; 90° hard pulse (p1) = 8.16 μs; power level for pre-saturation (pl9) = 62.77 dB; dummy scans (ds) = 4; number of scans (ns) = 128; acquisition time = 4.08 s; mixing time (d8) = 0.01 s; recycle delay (d1) = 7 s.
Each spectrum was acquired using TOPSPIN 2.1 software (Bruker BioSpin GmbH, Rheinstetten, Germany) encompassing sample loading, temperature stabilization for 5 min, tuning, matching, and shimming.
NMR raw data (Free Induction Decays, FIDs) were processed using the software MestReNova 11.0 (Mestrelab Research SL, Santiago de Compostela, Spain). The FIDs were zero-filled to 128 K number of points and then underwent the Fourier transformation by applying an exponential multiplication function with a line broadening of 0.1 Hz. Phase and baseline were automatically corrected. The horizontal scale of the chemical shifts is reported in ppm and the TSP-d₄ singlet signal, set at δ = 0.00 ppm, was used as a chemical shift reference.