Ods hypersil column

Analysis of MDA, 4-HNE, and 4-HNE concentrations was then conducted according to a previously described method (20 (link)). The preparation of 10 mg/ml-MDA was procured through the hydrolysis of TEP in a 5%-TCA solution. The standard working solutions of (0.02 ~ 10 mg/L for MDA, 4-HNE, and 4-HNE) were prepared using ethanol/water 1:1 (v/v). Then 2 g oil was mixed with 2.5% (w/v) TCA and DNPH [0.05 mol/L in ethanol/HCl 12 M 9:1 (v/v)]. After preparing derivatives, the separation was used with a CBM-20A HPLC system (Shimadzu, Shanghai, China) equipped with an SPD-M20A detector and an ODS HYPERSIL column (4.6 × 250 mm, 5 μm) from Thermo Scientific. The UV wavelength was set at 378 and 310 nm to detect 4-HHE, 4-HNE, and MDA derivatives. The flow rate was set at 1.0 ml/min, whereas the injection volume was set at 20 μl. Furthermore, the mobile phase was acetonitrile and water (solvent A and B). The gradient elution program was 45% solvent A maintained for 0–18 min, then from 45 to 70% solvent A in 5 min, and then 70% solvent A for 15 min.

Urinary DTC content of the pre-dose and 24 hr urines were measured using the cyclocondensation reaction [13 (link)]. Briefly, 100 and 200 μl of urine were incubated in 2.0 ml of 50% methanol, 50% water, 100 mM sodium borate, pH 9.2 and 16.3 mM 1,2-benzenedithiol in a sealed 4 ml vial for 2 hr at 65°C. After reaching room temperature and centrifugation, 200 μl from each vial was injected onto a 100 x 4 mm ODS Hypersil column (Thermo Scientific, Waltham, MA, USA) at 23°C and eluted with 80% methanol: 20% water at 0.5 ml/min for 10 min. The absorption peak area of the cyclocondensation product (1,3-benzodithiole-2-thione), was recorded at 360 nm, and the concentration in the urine samples was determined by comparison with the peak areas of authentic, recrystallized 1,3-benzodithiole-2-thione standards. The DTC contents of the pre-dose urines are expressed as nanomoles/mg of creatinine and that of the 24-hr urines as total μmol (S1 Table).
Subject compliance with the protocol (adherence to the dietary restrictions and complete collection of 24-hr urines) was validated by direct querying of subjects upon delivery of 24-hr urine collections, and measurement of both urine volume and creatinines in the returned 24-hr urine collections and creatinines in the pre-dose urines. Creatinine measurements were performed by Hagerstown Medical Laboratory, (Hagerstown, MD, USA).

The HPLC technique was implemented using Shimadzu CTO-20A (Kyoto, Japan) with UV detector (VWD 1260) and ODS Hypersil column, with an average particle size of 5 μm, 150 mm tall and 4.6 mm internal diameter (Thermo Fisher Scientific, Waltham, MA, USA). Corresponding to a previously reported method [24 (link)] with minor changes modifications, acetonitrile and potassium phosphate buffer (pH 5) were used as a mobile phase with ratio (65:35 v/v), respectively. An isocratic elution was used with flow rate 1.5 mL/min and injection volume of 20 μL. The UV detector was set at 290 nm and 254 nm. The method was validated with reasonable linearity in the range of 1–8 µg/mL with 0.995 correlation coefficient. In addition, all samples were assayed for interday and intraday accuracy and precision.