Urinary pterin spectrum analysis

Pterin spectrum analysis for urine samples was carried out using high-performance liquid chromatography (HPLC). Control reagents: 6-neopterin (6-Neo), 7,8-dihydrobiopterin (BH₂), pterin (Pte), 6-biopterin (6-Bio), 7-biopterin (7-Bio), L-(5-15N)-biopterin (IS-Bio), (5-15N)-7,8-dihydrobiopterin (IS-BH₂) and D-(5-15N)-neopterin (IS-Neo), produced by Schircks Laboratories (Jona, Switzerland).

Urine samples of the control group and the HPA patients were oxidized using dithiothreitol. Oxidized samples were diluted with deionized water according to creatinine concentration (up to 0.1 g/l).

Before mass spectrometry the samples were diluted 10 times with mobile phase containing internal standards in concentrations 20, 20, 5 ng/ml—IS-BH₂, IS-Neo, IS-Bio respectively.

Chromatographic separation was carried out on a Waters XBridge BEH Amide column (130Å, 3.5 μm, 2.1 mmX100mm) with a Waters XBridge BEH Amide precolumn (130Å, 3.5 μm, 2.1 mmX5mm). Mobile phase А – 20 mmol ammonium formate + 0.1% formic acid, mobile phase В –acetonitrile, flow rate– 0.4 ml/min. Gradient program: 0 minutes—6% А, 15 minutes 15% А, 17 minutes 15% А, 17.1 minutes 6% А and 3 minutes for column recovery.

The samples were analyzed using a Nexera HPLC system (Shimadzu, Japan) and a Q-Trap 5500® mass spectrometer (AB/SCIEX, Canada), curtain gas (CUR)– 20 psi, ion spray voltage (IS)– 5500 V, ion source heater temperature (TEM) - 600°C, source gas 1 (GS1)– 50 psi, source gas 1 (GS2)– 65 psi. Experimental MRM (Multiple reaction monitoring) transition parameters were optimized in positive ionization mode for each examined substance (Table 2), declustering potential (DP)– 120 V, entrance potential (EP)– 8 V, cell exit potential (CXP)– 15 V.