Determination of catecholamines in urine using hydrophilic interaction chromatography with electrochemical detection

Abstract

The determination of catecholamines in urine was investigated using hydrophilic interaction chromatography (HILIC) as an alternative to the commonly used reversed-phase (RP) method. A number of different approaches were explored, including per-aqueous liquid chromatography (PALC), and HILIC using bare silica, bonded amide and zwitterionic phases. The bonded phases gave superior results in terms of both peak shape and selectivity. The mechanism of the HILIC separation was investigated particularly with respect to the contribution of ion exchange to retention. The electrochemical detection of catecholamines was studied and optimised in typical HILIC mobile phases that contain high concentrations of acetonitrile. HILIC offered a number of advantages over the conventional RP approach, giving good retention of the solutes without use of ion pair reagents, the absence of which also would facilitate detection by mass spectrometry. HILIC used in conjunction with solid phase extraction based on RP also gives orthogonal separation mechanisms in the cleanup and analysis steps. Furthermore, good recoveries from the cleanup stage were obtained, as high concentrations of acetonitrile can be used as eluting solvent that are fully compatible with HILIC, and lipophilic impurities are eluted close to the void volume of the HILIC column.

Introduction

Catecholamines are biological amines released mainly from the adrenal glands in response to stress. They act as neurotransmitters and hormones, playing an important role in maintaining normal physical activity of the body including heart rate, blood pressure and the reactions of the sympathetic nervous system [1]. The most abundant catecholamines are dopamine (D), epinephrine (E) and norepinephrine (NE). Very high levels of catecholamines in plasma and/or urine indicate tumours of the adrenal gland such as pheochromocytoma [3] or neural tumours such as neuroblastoma [4], while levels of catecholamines are also elevated during stress. Therefore there is a need for their quantitative determination in body fluids in order to identify any endocrine disorders and other physiological and pathological abnormality in the body at an early stage [2]. The most common method for analysis of catecholamines in biological fluids is RPLC in conjunction with various detection systems like mass spectrometry (MS), electrochemical detection (ECD) and fluorescence detection [2], [5], [6], [7], [8], [9].

The analysis of some amines in RPLC gives rise to considerable problems due to their hydrophilic nature, which can result in low retention necessitating the use of ion pair agents and/or very low concentrations of organic modifier that can cause phase de-wetting. Ion pair reagents can cause interference with MS detection. Furthermore, the presence of ionised silanol groups on the stationary phase may give rise to peak tailing and overloading effects that can result in poor separation [10], [11]. Recent methods for catecholamine analysis typically use a C18 or C8 column at acid pH, with a low concentration of methanol (2–2.5%) and an ion pair reagent such as octanesulfonic acid [2], [6]. Good resolution of standards was obtained in the latter report [6], but this RP method with ECD failed to separate NE from plasma constituents completely. Another study using a RP column with methanol and acetate buffer at higher pH (4.66) gave broad and tailing peaks for the catecholamines [8]. Thus, alternative methods for the analysis of catecholamines have been sought. Hydrophilic interaction chromatography (HILIC) has been shown to be a complementary method to RP for the analysis of ionised basic compounds [12], [13]. Typically, HILIC uses a bare silica or polar bonded phase in conjunction with a mobile phase containing a high proportion of acetonitrile (typically >60%) together with a significant concentration of water or aqueous buffer (typically >2.5%). The mechanism of separation appears to be complex [14], but may consist of partition of hydrophilic solutes into a water layer held close to the silica column surface and/or associated with polar bonded groups, adsorption onto silanols or polar bonded groups, ion exchange and even some RP behaviour when the concentration of organic solvent is very low. The advantages of HILIC over RP include good retention of polar, hydrophilic and ionised solutes, alternative selectivity to RP methods with elution of solutes broadly in line with increasing polarity (the opposite to that in RP) and the low viscosity of mobile phases that leads to lower operating pressures and faster diffusion of solutes. Thus, longer columns can be used, or columns can be operated at higher flow rates without serious loss in efficiency (smaller van Deemter C terms than in RP). A further significant advantage of the use of high concentrations of organic solvent is the more efficient spraying and desolvation of eluents in ESI mass spectrometry, leading to higher sensitivity than can be obtained in RPLC–MS. Elfakir and co-workers presented a thorough survey of 12 different HILIC columns for their suitability to separate a test mixture containing 12 standard neurotransmitters, using MS or UV as detection methods [15]. However, comparative peak shape data was not reported, and the study did not present quantitative data or application of the various columns to the separation of these compounds in biological fluids. Sandra and co-workers [16] coined the term “per-aqueous liquid chromatography” (PALC) to describe a technique using a bare silica column with a mobile phase containing either 100% aqueous ammonium formate buffer pH 5 or similar buffers with very low concentrations of organic solvent. Excellent separations of standard solutions of catecholamines were shown. PALC appears very similar to the much earlier methods of amine separation on bare silica reported by Bidlingmeyer [17] and later by others including Euerby [18], which appear to be based largely on ion exchange and RP effects with the siloxane backbone of the column. However, in contrast to the findings of Sandra it is notable that peak shapes reported for bases using bare silica columns with largely aqueous buffers can be inferior to those normally found in RP separations [12], [18].

The aim of this study was to investigate the potential applicability of a HILIC-based procedure in conjunction with a simple electrochemical detector for the analysis of catecholamines in urine, exploring also if such a method was compatible with current sample preparation procedures that are mainly based on solid phase extraction (SPE) in the RP mode. There are few if any reports of the use of electrochemical detection with HILIC, although Flanagan and Jane showed the analysis of basic drugs using an amperometric detector with high concentrations of methanol in the mobile phase in separations which appear to be based on ion-exchange [19]. Furthermore, the use of HILIC as the final separation method, in conjunction with sample preparation by RP might prove a useful orthogonal pair of separation methods, giving rise to improved selectivity, although problems might also arise in that elution of the SPE cartridge with largely aqueous solutions (as used typically in conventional procedures for catecholamines [20]) is detrimental to HILIC, as water is a strong solvent in this technique. Additionally, we wished also to study the mechanism of separation, in order to contribute to the understanding of the processes that occur in HILIC, which have not been well investigated.