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A binary gradient consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) was used as the mobile phase.

Samples were loaded in 0.1% formic acid (buffer A) and separated using a binary gradient consisting of buffer A and buffer B (90% acetonitrile, 0.08% formic acid).

The sample was loaded in H2O + 5 mM NH4OAc + 0.2% acetic acid (buffer A) and separated using a binary gradient consisting of buffer A and buffer B (90/10 acetonitrile/H2O + 5 mM NH4OAc + 0.2% acetic acid).

The best mobile phase combination was a binary gradient consisting of 50 50 methanol/water with 0.05% NH4OH and a 40 40 20 mixture of isopropyl alcohol/chloroform/methanol with 0.05% NH4OH.

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The binary gradient consisted of solution A (water/acetic acid 996:4 v/v) and solution B (acetonitrile).

A binary elution gradient consisting of water and acetonitrile will be used to separate the different PAHs.

The binary solvent gradient consisted of 0.1 % formic acid in water (solvent A) and 100%% methanol (solvent B) in the following gradient: 95 % solvent A for the first 3 min, 50%% solvent A over the next 1 min, 30%% solvent A over next 2 min, and 5%% solvent A over the final 2 min.

The denaturing gradient consisted of 30 80% denaturant (100% denaturant equals 7 M Urea and 37% formamide).

Elution of SGAs was attained by using a binary gradient system consisting of Solvent A (30% acetonitrile, 6 mM Tris-HCl, pH 8.0) and Solvent B (80% acetonitrile, 6 mM Tris-HCl, pH 7.6) at a flow rate of 0.3 mL/min at 25° column temperature.

Midazolam was eluted with a binary gradient mixture consisting of 10 mmol/l ammonium formate containing 1% (v/v) isopropyl alcohol and 0.1% (v/v) formic acid and methanol on a Varian Polaris C18-A 20 cm × 2.0 mm 5 micron column (Agilent, Santa Clara, CA) (flow rate, 0.65 ml/minute).

Separation was performed on a Hypersil Gold PFP column (100 mm × 2.1 mm, 1.9 μm) in 11 minutes with a flow rate of 250 μL/min using a binary gradient mobile phase consisting of acetonitrile (B) and 0.1% formic acid (C) in water according to the following program: gradient from 10%to90%0% C in 6.0 min and held it for 2 min, and gradient back to 10% C in one min and held the gradient for 2 min.

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