Determination of three plant growth regulators in Dendrobium officinale and Anoectochilus roxburghii by three-phase hollow fiber liquid phase microextraction- high performance liquid chromatography

Abstract

Dendrobium officinale (D. officinale) and Anoectochilus roxburghii (A. roxburghii) are precious raw materials for traditional Chinese medicine. The growing demand for D. officinale and A. roxburghii cannot be met by current production techniques. Hence, the widespread artificial cultivation of D. officinale and A. roxburghii using substantial amounts of plant growth regulators (PGRs) has emerged. The excessive use of PGRs not only affects the quality and efficacy of medicinal materials but also causes a series of safety issues. Therefore, expanding research on residual PGRs in valuable Chinese medicinal materials is important to avoid the health hazards caused by these substances. Unfortunately, the identification of PGRs is challenging because of their trace and complex matrices. High performance liquid chromatography (HPLC) has become one of the mainstream analytical methods for PGR determination. An important consideration in the application of this technique to the detection of trace acidic PGRs is how to improve its accuracy and sensitivity. Three-phase hollow fiber liquid phase microextraction (3P-HF-LPME) has the advantages of a high enrichment factor, complex sample purification ability, low reagent consumption, low cost, and easy integration with chromatographic systems. Thus, the 3P-HF-LPME method overcomes the many shortcomings of traditional sample pretreatment methods. In this study, a novel, simple, and effective analytical method based on 3P-HF-LPME combined with HPLC was developed to extract, purify, enrich, and detect three trace acidic PGRs (indole-3-acetic acid, naphthyl acetic acid and indolebutyric acid) in D. officinale and A. roxburghii. The chromatographic separation conditions and 3P-HF-LPME model parameters were systematically optimized for this purpose. First, the sample solution was prepared by ultrasonication and low-temperature standing, and then adjusted to pH 3.0 using dilute hydrochloric acid. The sample solution (10 mL) and NaCl (1.50 g) were stored in a 15 mL brown extraction bottle with a built-in magnetic stirrer. Next, 30 μL of NaOH solution (pH 11.0) as the inner phase solution was injected into the inner cavity of a hollow fiber tube, which was subsequently sealed at both ends. The hollow fiber tube was soaked in n-octanol for 5 min and dried naturally to remove excess extraction solvent from its surface. Finally, the fiber tube was placed in a brown extraction bottle and stirred using a thermostatic magnetic stirrer at 40 ℃ and 1600 r/min for 2 h. After extraction, the three target analytes were separated on a Welch Ultimate XB-C18 column (250 mm×4.6 mm, 5 μm) under isocratic elution conditions using acetic acid aqueous solution and methanol (45∶55, v/v) as the eluent. The results indicated that the three PGRs showed good linearity in the range of 0.5-100.0 μg/L (coefficients of determination (r2)=0.9999), with limits of detection (LODs) of 0.02-0.15 μg/L. The method recoveries were 88.5-102.2%, with relative standard deviations (RSDs) of less than 3.7% (n=3). The extraction efficiencies and enrichment factors of the three PGRs in 15 batches of fresh D. officinale and A. roxburghii products were found to be 42.0%-86.8% and 140-289. Full-scan mass spectrometry was used to further identify positive samples to avoid false-positive results and enhance the reliability of the experimental method. In summary, the proposed method is sensitive, accurate, reliable, environment friendly, and capable of high enrichment. It could be used to determine the residues of three acidic PGRs in D. officinale and A. roxburghii. Moreover, it can provide technical support for the residue detection of PGRs in other Chinese medicinal materials.