Prominence hplc

Sample supernatants were thawed at room temperature and filtered with a 0.22 μm PES membrane (Millipore). Lactate concentrations were measured by high-performance liquid chromatography (Prominence HPLC, Shimadzu). One milliliter of each sample supernatant was placed with a syringe in the sampler holder and 20 μL of the sample was manually injected into the column. The column used was a Fast Fruit Juice (Waters) and H₃PO₄ 5 mM at 0.8 mL/min was used as the eluent. The temperature of the column was maintained at 55°C with a water bath and the separated compound was detected with a refractive index detector (IOTA). Lactate known concentrations were analyzed by HPLC to produce a standard calibration curve in the same operated conditions as for sample supernatants. ${\text{NH}}_4^{+}$ concentrations were measured by using the Nessler colorimetric method. One milliliter of each sample supernatant was mixed with 1 ml of arabic gum (0.5% (w/v) and 100 μL of Nessler reagent (potasium iodomercurate and ammonia). Absorbance was read at 400 nm in a spectrophotometer (UviLine9400, SECOMAM, France) after an incubation time of 5 min. ${\text{NH}}_4^{+}$ known concentrations were analyzed as well by the Nessler colorimetric method to produce a standard calibration curve in the same operated conditions as for sample supernatants.

Samples were resuspended in 50 µL of 50 mM ammonium formate and fractionated offline with high pH C18 reversed phase (RP) chromatography [38 (link)] with the following changes. A Shimadzu Prominence HPLC (Shimadzu, Columbia, MD, USA) with a Hot Sleeve-25 L Column Heater (Analytical Sales & Products, Inc., Pompton Plains, NJ, USA) was used with a Security Guard precolumn housing a Gemini NX C18 cartridge (Phenomemex, Torrance, CA, USA) attached to a C18 XBridge column, 150 mm (column length) × 2.1 mm (internal diameter), 5 µm particle size (Waters Corporation, Milford, MA, USA). Buffer A was 20 mM ammonium formate, pH 10 in 98:2 water:acetonitrile, and Buffer B was 20 mM ammonium formate, pH 10 in 10:90 water:acetonitrile. The flow rate was 200 µL/min with a gradient from 2–7% buffer B over 0.5 min, 7–15% buffer B over 7.5 min, 15–35% buffer B over 45 min, and 35–60% buffer B over 15 min. Fractions were collected every 2 min, and UV absorbances were monitored at 215 nm and 280 nm. Peptide containing fractions were divided into two equally numbered groups, “early” and “late”. A volume equal to 15 milli-absorbance units of the first “early” fraction was concatenated with the first “late” fraction, and so on. Concatenated fractions were lyophilized and cleaned with Stop and Go Extraction Protocol (STAGE tip) using Waters Oasis MCX material.

4–27 were purified using a Shimadzu Prominence
HPLC using a
C18 column (Eclipse XDB-C18 5 μm, 4.6 × 150 mm). Two differing
purification methods were used: A1, H₂O + 0.1% TFA and
MeOH from 1% CH₃OH/H₂O + 0.1% TFA to 90% CH₃OH/H₂O + 0.1% TFA in 25 min; A2, H₂O
+ 0.1% TFA and CH₃CN from 1% CH₃CN/H₂O + 0.1% TFA to 70% CH₃CN/H₂O + 0.1% TFA in
15 min.

All chemicals were purchased from Alfa Aesar (Ward Hill, MA) or Aldrich (Milwaukee, WI). ¹H and ¹³C NMR spectra were used for compound identification on a Varian (Palo Alto, CA) 400-MR spectrometer. Purification of reaction products were carried out by silica gel (200–400 mesh) column chromatography monitored by UV at 254 nm. Analytical high performance liquid chromatography (HPLC) was performed on Shimadzu Prominence HPLC with a Zorbax C18 (or C8) column (4.6 x 250 mm) monitored by UV at 254 nm. The purities of the reported compounds were found to be >95%. The synthesis and characterization of compounds 1–40 can be found in Experimental Section.

Vitamin E congeners were quantified in triplicate as previously described (Grebenstein & Frank, 2012 (link)). In brief, about 200 mg flour was weighed into a glass tube and 2 ml of ethanol (containing 1% ascorbic acid (w/v)), 900 µl of H₂O, and 600 µl of KOH were added and samples saponified for 30 min at 70°C. Samples were then extracted three times with 2 ml of hexane and in total 5 of 6 ml added hexane was collected and evaporated. Samples were dissolved in 100 µl of ethanol and analyzed on a Shimadzu (Kyoto, Japan) Prominence HPLC equipped with an LC‐20 AT pump, a DGU‐14A degasser, a CTO‐10AS column oven (set to 40°C), cooled autosampler SIL‐20 AC HT (4℃), and an RF‐20A fluorescence detector. Tocopherols and tocotrienols were separated on a Phenomenex KinetexTM PFP column (2.6 µm, 150 × 4.6 mm; Phenomenex) using methanol/H₂O (85:15, v/v) as eluent at a flow rate of 1.2 ml/min. The fluorescence detector was operated at an excitation wavelength of 296 nm and an emission wavelength of 325 nm. Peaks were recorded and integrated using Lab solution LC software (Shimadzu, Kyoto, Japan) and quantified against external calibration curves.