625 lc system

In Inter99 Glycosylated Haemoglobin, type A1C (HbA1c) was assayed by ion-exchange high-performance liquid chromatography technique (HPLC) (Bio-Rad variant). In Health2006, HbA1c was measured using HPLC on a Tosoh G8 Analyzer. In DanFunD, HbA1c was measured using the VITROS Chemistry Products d%A1c Reagent Kit utilising a quantitative turbidimetric inhibition immunoassay until July 2013 and from July 2013 using the HPLC technique on a Tosoh G8 Analyzer. Levels of HbA1c were similar in all three cohorts. In the ADDITION study, HbA1c was similarly measured using ion-exchange HPLC (Tosoh Bioscience, Redditch, UK) and in the CAMB cohort using ion-exchange HPLC on a Waters 625 LC system together with a Waters photo-diode-array detector model 996 and WISP 717 autosampler for automatic injection of the samples.
All measurements of HbA1c were DCCT (Diabetes Control and Complications Trial) aligned but were converted to IFCC (International Federation of Clinical Chemistry) units before analysis.

A high performance liquid chromatography (HPLC) method was used for determination of HbA_1c. The HPLC consisted of a Waters 625 LC system together with a Waters photo-diode-array detector model 996 and a WISP 717 auto sampler for automatic injection of the samples. Millennium chromatography software was used for calculation of concentrations (Waters Associates Inc., Milford, United States). A cation exchange column Mono S HR 5/5 from Pharmacia Biotech AB, Uppsala, Sweden was used to separate HbA_1c from other components in the samples [49 (link)]. HbA_1c is a standard indicator of long-term glycaemic control and an HbA_1c level of ≥5.6% corresponds to a fasting glucose level of ≥100 mg/dL, which is a measure of insulin resistance [50 (link)].

DNA samples (5 μg) were denatured by heating to 100 °C and then hydrolyzed for 17 h at 37 °C in a solution containing 5 μl of 10-mM ZnSO₄ and 10 μl of 1.0 U ml⁻¹ P1 nuclease in 30-mM NaOAc (pH 5.4). After nuclease digestion, 10 μl of 0.5-M tris pH 8.3 and 10 μl of 10 U ml⁻¹ alkaline phosphatase in 2.5-M (NH₄)₂SO₄ were added, followed by incubation at 37 °C for 2 h, then centrifugation at 12,000 rpm for 5 min.
Reversed-phase HPLC (RP-HPLC) quantification of overall DNA methylation was performed as described by Johnston et al. (2005 (link)) using a Waters 625 LC system connected to a Millennium 32v 4.0 data processing station. Briefly, a 4u Max-RP C12 (250 9 4.6 mm, Phenomenex) column combined with a 4u Max-RP C12 pre-column was used with a linear gradient of eluent. Two eluents were used: eluent A, 0.5% methanol in 10-mM KH₂PO₄ (v/v); and eluent B, 10% methanol in 10-mM KH₂PO₄. A linear gradient of 10 min 100% A and 100% B, and 15 min 100% B and 100% A, with 5 min of total running time, was used. The percentage of deoxycytidine methylation in relation to the total content of cytidine was calculated according to the following equation: 5mdC% = [5mdC/ (5mdC + dC)] × 100, where 5mdC and dC represent 5-methyldeoxycytidine and deoxycytidine, respectively. Three RP-HPLC technical replicates were conducted for each DNA sample.