High-performance liquid chromatography determination of σ1-acid glycoprotein in small volumes of plasma from neonates

In order to investigate how the α1-acid glycoprotein (AGP) concentrations of neonates change in response to surgical stress, a simple high-performance liquid chromatography (HPLC)-assay for the measurement of α1-acid glycoprotein levels was developed. A fraction containing α1-acid glycoprotein was isolated from the bulk of plasma protein by addition of 0.6 M perchloric acid and was then analysed directly on a short PLRP-S 4000 Å reversed phase column column. The method was validated by analysis of pooled plasma from healthy adults both in comparison with a calibration curve and by standard additions. The procedure was able to isolate α1-acid glycoprotein rapidly (<30 min) and required only 50 μl of plasma. The mean extraction recovery was 79.1% (CV 6.4%). The within-run precision for the analysis of three replicates of quality control sample ranged from ±1.2 to ±3.8% and the between-run precision was ±6.1%. The method was linear (r2 = 0.988) over a concentration range from 6 to 100.0 mg/100 ml. The AGP levels in neonatal samples ranged from 25 to 93 mg/100 ml.