Dispersive liquid—liquid microextraction for the determination of three cytokinin compounds in fruits and vegetables by liquid chromatography with time-of-flight mass spectrometry

Highlights

• Three cytokinin compounds are preconcentrated using dispersive liquid—liquid microextraction.

• The combination LC—TOFMS permits the unequivocal identification of the three analytes.

• Detection limits in the 0.02—0.05 ng g⁻¹ range have been attained.

Abstract

The paper presents a novel approach for the determination of three cytokinin compounds, thidiazuron (TDZ), 1,3-diphenylurea (1,3-DPU) and forchlorfenuron (CPPU), in fruit and vegetables samples using liquid chromatography with electrospray ionization and time-of-flight mass spectrometry (LC—ESI—TOFMS). Analytes were extracted from the sample matrix with ethanol, and the extract, after dilution with water, was submitted to dispersive liquid—liquid microextraction (DLLME). Once acetonitrile and 1,2-dichloroethane had been selected as extraction and disperser solvents, respectively, the influence of the following experimental parameters was studied using a Plackett—Burman design: volume of extraction and disperser solvents, sample mass and time and speed of centrifugation. The best analytical conditions were 250 µL 1,2-dichloroethane, 1.5 mL acetonitrile, 5 g sample mass, and centrifugation at 3000 rpm for 3 min. The optimized method provided DLs in the range 0.02—0.05 ng g⁻¹, depending on the compound. Satisfactory recovery values between 89 and 106% were obtained for spiked samples (kiwifruit, watermelon, grape and tomato) in the 0.2—1.0 ng g⁻¹ concentration range, depending on the compound. None of the target analytes was detected in any of the samples analyzed.

Introduction

Plant growth regulators (PGRs) are classified into five main classes: cytokinins (CKs), auxins, gibberellins, ethylene and abscisic acid [1]. Most of these compounds are naturally produced in plants, where they regulate the growth or destroy undesired parts of plants. Synthetic PGRs are widely sold for use in agriculture because of they are cheaper and show greater stability than the natural homologs [2], although their use in the European Union is governed by the Council Directive 91/414/EEC [3]. CKs, which were discovered during the 1950s, play an important role in several phases of plant development and growth, and are used to enhance fruit set, fruit size and the yield of different crops. The functions of CK receptors in plants have been reviewed recently [4]. The CK group comprises two types of compound: adenine and phenylurea derivatives [5]. The phenylureas comprise a group of synthetic compounds, the first one identified being 1,3-diphenylurea (1,3-DPU), whereas forchlorfenuron (CPPU) and thidiazuron (TDZ) were synthetized later and show higher activity than other natural PGRs, such as the adenine-type cytokinin zeatin [5].

The main effect of CPPU is to increase fruit size (e.g. kiwifruits, grapes and apples among others), and to promote the development of the ovary into fruit without fertilization or seed formation. CPPU acts synergistically with auxins. The correct application of this compound does not involve human risk and, moreover, CPPU has been classified as not likely to be a human carcinogen or endocrine disrupter [6]. In 2006, CPPU was included in Annex I of Directive 91/414/EEC of the European Union as an authorized PGR for kiwifruit, with a maximum residue limit of 50 ng g⁻¹ [7]. TDZ, also classified as not likely to be a human carcinogen, has been used for chemical defoliation before the mechanical harvesting of cotton and for promoting plant growth [8], [9]. The effect of 1,3-DPU on callus induction on the surface of certain plants has been described [10], as well as its stimulatory effect on plants [11], although its cytokinin activity is 10,000 times lower than that of CPPU.

CPPU in fruit and vegetables can be determined by immunoassay procedures [12], [13], using liquid chromatography (LC) with UV detection [14], [15], [16], tandem mass spectrometry (MS/MS) [17], [18], [19], [20], [21] and time-of-flight mass spectrometry (TOFMS) [22], [23]. However, the literature only shows two analytical methods for the determination of TDZ in fruit and vegetables: LC—UV detection [24] and LC—MS/MS [17], the latter also including the determination of CPPU. On the other hand, TDZ has been quantified in waters [8], [25], [26] and fertilizers [2]. Sample treatments in the cited references include extraction of the analytes into an organic solvent, and clean-up by liquid—liquid extraction (LLE) [14], solid-phase extraction (SPE) [15], [16], [21], [22] and dispersive solid-phase extraction (DSPE) [20], [23]. The simultaneous extraction and clean-up using QuEChERS methodology has also proved satisfactory [17], [19], [22]. In this study, we propose the simultaneous determination of CPPU, TDZ and 1,3-DPU in fruit and vegetables by solvent extraction of the analytes, with low consumption of the organic solvent and preconcentration of the extract by means of a miniaturized technique whose main advantages are its rapidity, efficacy, low cost and lack of memory effects: dispersive liquid—liquid microextraction (DLLME) [27], [28], [29], [30]. DLLME has only been applied for the determination of TDZ in water samples [26]. To the best of our knowledge this is the first time that the simultaneous determination of the three phenylurea cytokinin compounds has been approached. Moreover, in this case, the same friendly sample treatment (DLLME) is proposed for the analysis of fruit and vegetables by LC—ESI—TOFMS.