Sphereclone

Organic acids were determined following a procedure previously optimized and described by the authors [10 ]. Analysis was performed on a Shimadzu 20A series ultra-fast liquid chromatograph (UFLC, Shimadzu Cooperation, Kyoto, Japan) coupled to photodiode array detector (PDA, Shimadzu), using 215 nm and 245 nm as the preferred wavelengths. Separation was achieved on a SphereClone (Phenomenex, Torrance, CA, USA) reverse phase C18 column (5 μm, 250 mm × 4.6 mm i.d) thermostatted at 35°C. Analytes were eluted with 3.6 mM sulphuric acid at a flow-rate of 0.8 mL/min. The organic acids found were quantified by comparison of the area of their peaks recorded at 215 nm or 245 nm (for ascorbic acid) with calibration curves obtained from commercial standards of each compound: oxalic acid (y = 1x107x + 96178; R² = 0.999); quinic acid (y = 601768x + 8853.2; R² = 1); malic acid (y = 952269x + 17803; R² = 1); shikimic acid (y = 8x107 + 55079; R² = 0.999). The results were expressed in mg per g of lyophilized decoction or infusion.

Optical rotations were measured on a JASCO P-2000 polarimeter (JASCO, Easton, MD, USA). IR data was collected using a Bruker Alpha Fourier transform infrared spectrophotometer (Bruker, Billerica, MA, USA). NMR spectra were measured at ¹H resonance frequency on a Jeol Eclipse + 400 MHz spectrometer (JEOL, Peabody, MA, USA). Chemical shifts were calibrated internally to the residual signal of deuterated chloroform (CDCl₃ δ_H 7.26, δ_C 77.0). For NMR measurements concentration used was in the range of 3–8 mg of compound (depending on the amount of compound available) dissolved in 600 μL of deuterated chloroform. High-resolution mass spectra were obtained on a Bruker micrOTOF-Q III (Bruker Daltonics, Billerica, MA, USA). HPLC separations were performed using an Agilent 1200 HPLC system equipped with a quaternary pump, a diode array detector (Agilent, Santa Clara, CA, USA) and a normal phase silica gel column (Phenomenex Sphereclone^®, 4.6 mm × 100 mm, 5 µm). Reverse phase solid phase extraction (SPE) separation was carried out using SUPELCO Supelclean™ LC-18 (C-18, octadecyl) solid-phase extraction cartridges (Supelco^® Analytical, Bellefonte, PA, USA).

Metaphosphoric acid (4.5%) was added to 1 g of the sample; the mixture was then protected from light and incubated (with agitation) for 20 min at room temperature. After sample filtration, organic acids were determined using a Shimadzu 20A series UFLC (Shimadzu Corporation, Kyoto, Japan) coupled to photodiode array detector (PDA) [39 (link)]. Separation was achieved on a SphereClone (Phenomenex, Torrance, CA, USA) reverse phase C₁₈ column (5 μm, 250 mm × 4.6 mm i.d—internal diameter.) thermostatted at 35 °C. The elution was performed with sulphuric acid (3.6 mM) using a flow rate of 0.8 mL/min. Detection was carried out in a PDA using 215 and 245 nm (for ascorbic acid) as preferred wavelengths. For the quantitative analysis, calibration curves with known concentrations of commercial standards were constructed, and the organic acids present in the two samples were determined by peak area comparison at 215 nm and 245 nm (for ascorbic acid). The results were expressed in g per 100 g dw.

The dehydrated and powdered fruits and stems were investigated for their organic acid composition following the protocol established by the group [37 (link)]; an ultra-fast liquid chromatography coupled to photodiode array detector (UFLC-PDA; Shimadzu Coperation, Kyoto, Japan) was used. The separation of the compounds was performed in a SphereClone (Phenomenex) reverse phase C18 column (5 μm, 250 × 4.6 mm i.d) thermostated at 35 °C, using 3.6 mM sulphuric acid solution as eluent in a flow rate of 0.8 mL/min. The quantification was achieved by comparison of the peak area recorded at 215 nm as the preferred wavelength. For quantitative analysis, a calibration curve with known concentration (10 − 0.0078 mg/mL) for each available compound was built based on the UV signal: oxalic acid (y = 45.973 + 9 × 10 x; R² = 0.9901); quinic acid (y = 46.061 + 610607 x; R² = 0.9995); malic acid (y = 92.665 + 912,441 x; R² = 0.999); citric acid (y = 45.682 + 1 × 10⁶x, R² = 0.9997), and succinic acid (y = 50.689 + 592888 x; R² = 0.9996). The results were expressed in g per 100 g of fruits and stems dry weight.

Organic acids were determined following a procedure previously described [15 (link)]. The analysis was performed using a Shimadzu 20A series UFLC (Shimadzu Corporation, Kyoto, Japan). Separation was achieved on a SphereClone (Phenomenex, Torrance, CA, USA) reverse phase C₁₈ column (5 μm, 250 mm × 4.6 mm i.d.) thermostated at 35°C. The elution was performed with sulfuric acid (3.6 mM) using a flow rate of 0.8 mL/min. Detection was carried out in a PDA (photodiode array detector), using 215 and 245 nm (for ascorbic acid) as preferred wavelengths. The organic acids found were quantified by comparison of the area of their peaks recorded at 215 nm with calibration curves obtained from commercial standards of each compound. The results were expressed in mg per g of dry weight.

L. vannamei samples, chondrotin sulphate A and chondrotin sulphate C (50 µg) were subject to exhaustive digestion by chondroitin ABCase (Sigma, Dorset, UK) in 50 mM Tris-HCl containing 60 mM sodium acetate and 0.02% w/v BSA at pH 8. Samples were incubated at 37 °C for 4 h prior to a subsequent addition of further chondroitin ABCase and incubation at 37 °C overnight. A SphereClone analytical column (4.6 × 250 mm, Phenomenex, Macclesfield UK), was pre-equilibrated in HPLC-grade H₂O, pH 3.5 (1 mL·min⁻¹) prior to the injection of the digested samples. The flow was held isocratic in H₂O for 10 min prior to a linear gradient from 0 to 2 M NaCl, pH 3.5 (HPLC grade; VWR, Lutterworth, UK) over 60 min. Detection of eluted Δ-disaccharides was carried out by monitoring UV absorbance at λ = 232 nm. Retention times were correlated against the 4 most common Δ-disaccharide reference standards for CS according to [40 (link)] (Iduron, Manchester, UK). The SphereClone column was washed with 2 M NaCl pH 3.5 and HPLC-grade H₂O pH 3.5 prior to use and between runs.