Rapid and sensitive determination of catecholamines and the metabolite 3-methoxy-4-hydroxyphen-ethyleneglycol using HPLC following novel extraction procedures

Abstract

In the present study assays were improved for the determination of free catecholamines and 3-methoxy-4-hydroxyphenethyleneglycol (MHPG), the major metabolite of peripheral and central noradrenaline. The compounds were extracted by a fluid phase extraction: a diphenyl boric acid method for the purification of catecholamines and an ethyl acetate extraction for MHPG were used, respectively. High-performance liquid chromatography with electrochemical detection was employed for quantitative analysis. In previous studies, significant differences between plasma concentrations of these substances in normal volunteers and hospital patients were demonstrated. Therefore, we established valid reference values for a hospital population. Blood and urine samples of 59 patients without disorders and medication affecting catecholamine synthesis and metabolism or the activity of the sympatho-adrenal system were collected and analyzed for free and total (free plus conjugated) MHPG, noradrenaline (NA), adrenaline (A) and dopamine (DA); total MHPG was assayed after enzymatic hydrolysis of conjugates. Our data clearly demonstrate that these methods are sensitive, specific, rapid, and can easily be standardized. The intra- and inter-assay precision were high (CV 2.6–5.3% and 4.3–6.9% for plasma, CV 3.8–4.9% and 5.1–8.2% for urine, respectively). For plasma, the mean concentrations ± SD were determined to be 20.82 ± 4.70 pmol/ml for free MHPG, 68.43 ± 16.21 pmol/ml for total MHPG, 2.11 ± 0.24 pmol/ml for NA and 0.31 ± 0.08 pmol/ml for A. For 24h-urine the mean concentrations ± SD were determined to be 0.44 ± 0.13 mol/24h for free MHPG, 8.79 +- 2.13 mmol/24h for total MHPG, 169.8 ± 58.25 nmol/24h for NA, 62.19 ± 21.79 nmol/24h for A and 757.2 ± 382.6 nmol/24h for DA. In summary, these novel and rapid methods can clearly be employed in a routine clinical setting.