Analysing Guanidino Compounds in Human Serum and Urine by Gas Chromatography

Guanidino compounds are small polar molecules and are difficult to elute from gas chromatography (GC) column. The work examines derivatising reagents isovaleroylacetone (IVA) and isobutyl chloroformate (IBCF) for the GC determinations of 8 substances guanidine (G), creatinine (CTN), guanidinoacetic acid (GAA), methylguanidine (MG), guanidinopropanic acid (GPA), guanidinobutyric acid (GBA), guanidinosussinic acid (GSA) and arginine (Arg) from HP-5 (30 m x 0.32 mm id) column with thickness of film 0.25μm. All the 8 compounds separated completely within 8 minutes, when eluted for 2 minutes at column temperature 90°C, programmed to 10°C/min up to 220°C with rate of flow of nitrogen 1.5 mL/min. The effect of variables on the derivatisation and elution were optimised and calibration curves were observed within 0.2 μg/mL–40.0 μg/mL and detection limits (LOD) and quantitation limits (LOQ) were within 4 ng/mL–110 ng/mL and 12 ng/mL–330 ng/mL. The identification and quantitation were repeatable (n = 4) with relative standard deviations (RSDs) within 2.1%–3.1%. The additions of pharmaceutical ingredients and amino acids did not interfere the analyses of the guanidino compounds. The procedure was studied for the analyses of guanidino substances from the biological fluids of uremic patients and with disease free volunteers. The quantity established in serum and urine of disease free volunteers were below limit of detection (BLOD) to 9.48 μg/mL and BLOD to 1125 μg/mL with RSD 1.1%–3.1% and 1.5%–3.0%, respectively. Similarly, the amounts observed in serum and urine of patients suffering from uremic were 0.099 μg/mL–42.37 μg/mL and 0.014 μg/mL–1338.0 μg/mL with RSD 1.3%–3.2% and 1.5%–3.2%, respectively. The recovery of guanidino compounds from spiked serum was calculated 93.3%–97.7% and from urine 95.1%–98.8% with RSD within 3.1% and 3.2%, respectively. The results obtained for disease free volunteers were compared with uremic patients and t-test was applied at 95% confidence level and a significant difference was recorded for number of guanidino compounds.