Simultaneous determination of six plant growth regulators in fruits using high performance liquid chromatography based on solid-phase extraction and cleanup with a novel mixed-mode functionalized calixarene sorbent

Abstract

In this paper, a rapid and practical high performance liquid chromatography (HPLC) method, using home-made tetraazacalix[2]arene[2]triazine-modified silica gel (NCSi) as a solid-phase extraction (SPE) sorbent, was developed for the simultaneous determination of six plant growth regulators (PGRs) including thidiazuron (TDZ), naphthaleneacetic acid (NAA), p-chlorophenoxyacetic (PCPA), 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (6-BA) and forchlorfenuron (KT-30) in fruits. NCSi is a novel multi-interaction and mixed mode functionalized calixarene sorbent, which can enrich different classes of PGRs. Prior to HPLC separation, the key factors influencing SPE performance were investigated in the sample pretreatment step, including the dosage of the NCSi sorbent, and the flow rate, volume and composition of the eluant. HPLC analysis was carried out on a Waters XBridge C18 column (250 × 4.6 mm i.d., 5 μm) with a mobile phase of methanol/0.1% H₃PO₄ (55 : 45,v/v). With a detection wavelength of 220 nm, the good linearities of the six PGRs were obtained in the concentration range of 0.03 to 200 mg kg⁻¹ (R² ≥ 0.999). The lower detection limits (4.3–20.4 μg kg⁻¹) and quantitation limits (12.9–64.0 μg kg⁻¹) of the six PGRs were also obtained, which can meet the trace analysis requirements. In addition, overall recoveries through the extraction and purification steps ranged from 72.4 to 94.9%, and RSDs were less than 4.84%. The proposed method was successfully used to determine residual PGRs in real samples. It is practical and promising for the study of plant physiology by the determination of trace PGR content in the plant or fruits with a complex matrix.