Chemometrics-assisted validation of a high-throughput method for quantitative analysis of multiclass phytohormones across plant matrices by UHPLC-QQQ-MS/MS for targeted metabolomics.

The quantification of phytohormones in plant samples is significantly challenging because of their trace concentrations and the complex plant matrix. This study establishes and optimizes a simple and fast method for simultaneous quantification of multiclass phytohormones using ultra high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QQQ-MS) for high-throughput targeted metabolomics analysis. It is characterized by simplicity and operational feasibility, and it involves sequential steps of ultrasound-assisted extraction, centrifugation, and nitrogen flow-drying. The assessment of sample preprocessing method employs the technique for order preference by similarity to an ideal solution (TOPSIS) chemometrics-assisted approach, and methanol containing 1% formic acid was selected as the optimal extraction solvent. Quantitative analysis is performed via a multiple reaction monitoring method. The method was validated satisfactory for 31 phytohormones, the specific concentration levels used to estimate recovery and precision were 0.25-200 ng/mL, and the results showed good linearity (correlation coefficients: 0.9910-0.9999), sensitivity (LOD: 0.01-2.87 ng/g; LOQ: 0.02-8.70 ng/g), accuracy (recovery rates: 64.50-129.67%), precision (relative standard deviations <20%, except for IAA-Trp), and its applicability to a variety species of medicinal plant was verified, including Taxillus chinensis (DC.) Danser, Ophiopogon japonicus (L.f.) Ker-Gawl, Citrus grandis "Tomentosa," Plumeria rubra L. "Acutifolia," and Pogostemon cablin (Blanco) Benth. In the real samples analyzed, auxins concentrations exceeded 4.07 μg/g, salicylic acids concentrations ranged from 0.027 to 79.7 μg/g, and concentrations of other categorize of phytohormones were below 1 μg/g. The established method demonstrated high throughput, accuracy, stability, and reliability. It can be extended to cover additional phytohormone types and diverse application scenarios. This method serves as a potent tool for investigating the function of multiclass phytohormones in the regulation of secondary metabolite accumulation in medicinal plants, elucidating complex signal transduction mechanisms and interaction networks, and laying the groundwork for improving the quality of Chinese medicinal materials.