Magnetic dispersive solid-phase micro-extraction combined with high-performance liquid chromatography for determining nucleotides in Anoectochilus roxburghii (Wall.) Lindl.

A novel, simple, and efficacious analytical method for determining of nucleotides in Anoectochilus roxburghii (Wall.) Lindl (A. roxburghii) was developed. Magnetic dispersive solid-phase micro-extraction (MDSPME) combined with high-performance liquid chromatography was applied for extraction and determination of three nucleotides, such as adenosine-5'-monophosphate (AMP), uridine-5'-monophosphate (UMP) and guanosine-5'-monophosphate (GMP) in A. roxburghii from different sources. The structure and morphology of magnetic nanoparticles, Fe₃O₄@GO, were illustrated by Fourier-transform infrared (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), and Thermagravimetric analysis (TGA) techniques. The effects of different extraction conditions on extraction efficiency were investigated and optimized. The optimum extraction conditions were performed as follows: 40.0 mg Fe₃O₄@GO were dispersed in 30 mL adsorption solution (pH 3.50, 2 μg/mL), 50 mM NaOH was employed for elution with 12 min of ultra-sonication at 40 °C. Under the aforementioned extraction conditions, the Fe₃O₄@GO nano-adsorbent obtained an excellent adsorption property. The corresponding linearity range for all three analytes exhibited a good linearity (r² ≥ 0.9982) and notable added recoveries ranging from 88.4% to 109.8%, whereas the limit of quantitation was between 0.8-8 ng/mL. The enrichment factors (EF) were between 174 and 255. The proposed method showed the advantages of full purification, high EF, simplicity, and good recovery. The method was also successfully applied to nucleotides extraction and determination in A. roxburghii, showing superior reproducibility and high sensitivity. Based on this, the method could be expected to provide a novel experimental means and developmental direction for improving pretreatment and purification of nucleotides, reducing matrix effects as much as possible, in traditional Chinese medicinal materials or biological specimens.