Avance 3 hd 500 nmr

The samples were analyzed using a validated, dog-specific ¹H NMR metabolomics platform quantifying 123 measurands in absolute units (24 (link)). The quantified measurands are presented in Figure 1. Details about the method and its validation is provided elsewhere (24 (link)–27 (link)).
Briefly, the method utilizes a Bruker AVANCE III HD 500 NMR spectrometer equipped with a 5 mm triple-channel (¹H, ¹³C, ¹⁵N) z-gradient Prodigy probe head and a cooled high-throughput sample changer SampleJet (Bruker Corp., Billerica, Massachusetts, USA). Sample preparation begins with light mixing of the sample and centrifugation to remove possible precipitate (25 (link)). The highly automated process then continues by transfer of each sample into individual NMR tubes and mixing with sodium phosphate buffer using a PerkinElmer JANUS Automated Workstation equipped with an 8-tip dispense arm with Varispan (PerkinElmer Inc., Waltham, Massachusetts, USA) (26 (link)). The NMR spectra are acquired automatically from each sample using standardized parameters and are automatically processed using in-house scripts optimized for dogs (24 (link)–27 (link)). Result generation is based on regression modeling and includes integrated quality control (27 (link)).

NMR spectra were acquired using a Bruker Avance III HD 500 NMR spectrometer (University of Aveiro, Portuguese NMR Network) operating at 500.13 MHz for ¹H observation and equipped with a 5 mm TXI probe.

For NMR analysis, dried aqueous and lipid extracts were dissolved, respectively, in 600 μL of deuterated phosphate buffer (PBS 100 mM, pH 7.4) containing 0.1 mM 3-(trimethylsilyl) propionic acid (TSP-d₄), and 600 μL of deuterated chloroform containing 0.03% tetramethylsilane (TMS). After transferring 550 μL of each sample to 5 mm NMR tubes, analysis was performed on a Bruker Avance III HD 500 NMR spectrometer (University of Aveiro, Portuguese NMR Network) operating at 500.13 MHz for ¹H observation, at 298 K. Standard 1D ¹H spectra (pulse programs ‘noesypr1d’ and ‘zg’ for aqueous and lipid samples, respectively) were recorded with 32 k points, 7002.80 Hz spectral width, a 2 s relaxation delay and 512 scans. Spectral processing in TopSpin 4.0.3 (Bruker BioSpin, Rheinstetten, Germany) comprised cosine multiplication (ssb 2), zero-filling to 64 k data points, manual phasing, baseline correction and calibration to TSP-d₄/TMS signals (δ 0 ppm). Two-dimensional NMR spectra, namely, ¹H-¹H TOCSY, J-resolved and ¹H-¹³C HSQC spectra, were also recorded for selected samples to aid metabolite identification. Signal assignment was based on matching 1D and 2D spectral information to reference spectra available in Chenomx 9.0 (Edmonton, AB, Canada), BBIOREFCODE-2–0–0 (Bruker Biospin, Rheinstetten, Germany) and HMDB.