Screening of boldenone and methylboldenone in bovine urine using disposable electrochemical immunosensors

Abstract

Electrochemical based immunosensors for the detection of boldenone and methylboldenone in bovine urine were described in this paper. The immunosensors were fabricated by immobilizing boldenone–bovine serum albumin conjugate on the surface of screen-printed electrodes (SPEs), and followed by the competition between the free analyte and coating conjugate with corresponding antibodies. The use of anti-species IgG–horseradish peroxidase conjugate determined the degree of competition. The electrochemical technique chosen was chronoamperometry, performed at a potential of +100 mV whereby the product of the catalysis of 3,3′,5,5′-tetramethylbenzidine undergoes reduction produced by the enzyme label. The limits of detection of assay were 30.9 ± 4.3 pg ml⁻¹ for boldenone and 120.2 ± 8.2 pg ml⁻¹ for methylboldenone, respectively. Results of repeated analysis of each androgen carried out using three different batches of electrodes indicate suitable repeatability (EC₅₀ = 1.0 ± 0.3 ng ml⁻¹ (n = 3, N = 3), R² = 0.969, R.S.D. = 9.6% for boldenone and 1.5 ± 0.3 ng ml⁻¹, 0.971, 10.5% for methylboldenone, respectively). Urine samples were determined directly after a single dilution step, omitting extraction and hydrolysis. This method offers the advantage to pick up both boldenone and its major metabolites in an efficient manner due to the high cross-reactivity pattern of α-boldenone with this antibody. The concentration of methylboldenone in urine detected by developed methods does indicate methylboldenone administration to heifers. Gas chromatography coupled to mass spectrometry analysis was performed to quantitate the individual metabolites present in urine samples, and results were validated with both ELISA and immunosensor data.

Introduction

Boldenone and methylboldenone are anabolic steroids often illegally used to boost animal growth during the breeding of animals for human consumption, thereby causing potential health risks to consumers. The use of natural and synthetic hormones for growth promoting purposes in animals is banned in the European Union (Directive 88/146/EEC). The determination of residues is scheduled in animals and in fresh meat (Directive 86/849/EEC).

Many methods currently used for the detection of anabolic steroids are based on a chromatographic approach. Gas chromatography coupled to mass spectrometry (GC–MS) has been frequently used in detecting and quantitating the major steroids and their metabolites in complex matrices [1], [2]. A steroid can be identified in terms of its retention characteristics and its unique mass spectrum. The combination of liquid chromatography with mass spectrometry (LC–MS) provides a simplified and specific alternative to GC–MS methods, requiring less time and more simplistic sample preparation [3], [4], [5], [6], [7]. The need for derivatization is removed. Whilst these techniques have been used as confirmatory methods, however they do need expensive instrumentation, specialized personnel and suffer from considerable time delays between sampling and obtaining results. These disadvantages limit their routine use and restrict assaying to extensive laboratory environments.

The ideal method for screening of anabolic steroids should be fast, simple, cost-effective, easy to perform, and enable measurement in a small volume of biological fluid [8]. Immunoassays are based on the molecular recognition of antigens by antibodies to form a stable complex. They are one of the more sensitive assays available because of the high affinity of antibodies and fulfill most of the criteria suitable for routine high throughput screening. A significant advantage of immunological techniques is the ability of antibodies to bind broad classes of metabolites that contain a common substructure. Radioimmunoassay (RIA) was commonly used in the past for screening since this technique was rapid, sensitive and inexpensive [9], [10]. However, its drawbacks due to the use of radioactive compounds and to concern about worker safety led to the development of enzyme linked immunosorbent assay (ELISA) as an alternative method. A study by Hungerford et al. [11] reported a method of ELISA to detect 17α-alkyl anabolic steroid metabolites in equine urine and applied it as a primary screening tool for detection of new and known anabolic steroid metabolites. Another ELISA method was developed to detect the anabolic steroid boldenone in equine blood and urine [12], proving high sensitivity and simple sample pre-treatment. The cumbersome handling procedures that accompany them hinder their use as on-site detection systems. By comparison, immunosensors combine the sensitivity of the antibody–antigen interaction with other advantages including cost-efficient, speed of analysis and portable screening, which allows for decentralization from the lab environment. They are widely applied in the area of human and animal health care, food and fermentation industries, environmental monitoring, agriculture and defense [13], [14]. Disposable screen-printed electrodes (SPEs) used in conjunction with electrochemical immunosensors provide an approach to develop such cost-effective devices and offer a means to miniaturize the analytical system. Electrochemical immunosensor systems based on screen-printed electrodes have been reported and have been shown to be capable of the measurement of progesterone in cow's milk [15], [16] and estradiol in human serum [17]. To our knowledge, there is little information on electrochemical based immunosensors for the detection of boldenone and methylboldenone in biological fluids.

Electrochemical immunosensor systems based on screen-printed electrodes for the rapid detection of residues of boldenone and methylboldenone in bovine urine are presented in this paper. ELISA was primarily used to develop all assay systems and then was method transferred to an electrochemical immunosensor set up. In addition, the developed immunomethods were applied to analyze the urine samples of treated heifers. GC–MS analysis was performed in an inter-laboratory to quantitate the individual metabolites present in urine samples, and results were compared with both ELISA and immunosensor data.