Effects of Anticoagulants in Amino Acid Analysis: Comparisons of Heparin, EDTA, and Sodium Citrate in Vacutainer Tubes for Plasma Preparation

Abstract

Measurements of the concentrations of free amino acids in physiological fluids such as blood, cerebrospinal fluid (CSF), and urine are used mainly as biochemical indicators of inborn errors of metabolism (aminoacidopathies), nutritional status, and as monitors of therapy (1). Amino acids can be measured by thin-layer chromatography, electrophoresis, mass spectrometry, and ion-exchange chromatography (2). The last method has many advantages: it provides good resolution, is very accurate, can be automated, and is easy to perform. The long analytic time is the only drawback in this method. The steps in plasma free amino acid analysis, including sample pretreatment, deproteinization, and instrument operation, are easily performed. However, the use of different anticoagulants for plasma sample preparation can increase the potential for analytic errors (2)(3). Currently, heparin and EDTA are widely used as anticoagulants, but sodium citrate-treated plasma and serum samples are occasionally used. We investigated the analytic variation among samples treated with these anticoagulants. We used a System 6300 high performance amino acid analyzer (Beckman Instruments), which uses a single-column, ion-exchange method that uses four lithium buffers (Li-ADEF method), a ninhydrin reagent, and a 12-cm lithium high performance column. Four different types of Vacutainer Tubes were purchased from Becton Dickinson, including a plasma tube with heparin (sodium heparin), a whole blood tube with liquid EDTA (K3, 15%), a whole blood tube with 0.129 mol/L buffered sodium citrate (3.8% sodium citrate), and a serum separation tube. Centrifuge filtration was performed to eliminate the residual protein in sulfosalicylic acid dihydrate (SSA) supernatants. The microspin filter units (0.2 μm) were from Lida Manufacturing. All lithium buffers, ninhydrin reagents, and calibrators were purchased commercially from Beckman Instruments. Four lithium buffers with various pHs were used in this experiment: Li-A buffer (pH 3.0), Li-D buffer (pH 3.0), Li-E buffer (pH 3.7), and …