A Simplified High‐Performance Liquid Chromatography–Mass Spectrometry Method for Quantifying Soluble Sugars in Plant Leaves: Comparative Extraction Evaluation With Improved Sensitivity

The development of rapid and reliable methods for soluble sugar analysis is essential for routine applications in plant physiology, ecology, and agriculture. Conventional techniques for sugar quantification often require dedicated detectors, derivatization steps, or lengthy sample preparation, limiting throughput and sensitivity. This study presents a fast, high-performance liquid chromatography–mass spectrometry method for the simultaneous determination of sucrose, glucose, and fructose in plant leaves. Chromatographic separation was achieved on a Rezex column with gradient elution, and detection was performed in negative electrospray ionization mode using selected ion recording. The method demonstrated excellent linearity (R2 ≥ 0.99), high precision (coefficient of variation ≤ 2%), and recoveries of 98%–102%. Limits of detection and quantification were 0.8 and 1.6 µg mL−1, respectively, markedly improving sensitivity compared to conventional ultraviolet- or refractive index-based approaches. Several extraction protocols were evaluated, including water, methanol, and ethanol under thermal or ultrasonic conditions. Ultrasonic extraction with ultrapure water provided the highest efficiency, minimal matrix effects, and simplified sample preparation. The optimized workflow enables the preparation of multiple samples within a single day and overnight chromatographic analysis, offering a practical, cost-effective, and high-throughput solution for routine soluble sugar quantification in plant tissues.