Novel Validated Uhplc-Dad Method for Quantification of Leptosperin in New Zealand Mānuka Honey: A Definitive Chemical Marker for Authentication and Quality Control Incorporating Sustainable Practices with Minimal Sample and Plastic Usage for Cost-Effectiv

Abstract

Leptosperin is a unique floral marker compound found exclusively in the Leptospermum genus, with particularly high concentrations in Leptospermum scoparium (mānuka) honey from New Zealand. Due to mānuka honey’s global demand and valued therapeutic properties, reliable authentication methods are essential to ensure product quality, traceability, and prevent adulteration. This study reports the development and full validation of a sensitive and robust Ultra-Performance Liquid Chromatography with Diode Array Detection (UPLC-DAD) method for the quantification of Leptosperin in honey samples. The method employs a straightforward aqueous extraction followed by reverse-phase chromatography, enabling accurate and selective detection of Leptosperin at 282 nm.

Validation was conducted in accordance with international guidelines, encompassing specificity, linearity, precision, accuracy (expressed as recovery), trueness, limit of detection (LOD), and sample stability. The method exhibited excellent linearity (R² > 0.99) over a concentration range of 5 to 436 mg/kg. Precision studies showed relative standard deviations (%RSD) below 3% for both intra-day and inter-day measurements. Recovery rates ranged between 90% and 94%, demonstrating the method’s accuracy in complex honey matrices. The LOD was established at 10 mg/kg, sufficient to detect typical Leptosperin levels in mānuka honey. Stability testing confirmed that extracted samples remain stable for up to 36 hours’ post-preparation under refrigerated conditions, facilitating flexible laboratory workflows. Method performance was further corroborated through comparative analysis with accredited external laboratories, yielding strong concordance with relative standard deviations below 10.1%. This validated UPLC-DAD method offers a rapid, reliable, and cost-effective analytical tool for mānuka honey authentication and quality control. Its applicability extends to both monofloral and multifloral mānuka honey types, supporting research, regulatory compliance, and commercial quality assurance efforts. In summary, the method provides a scientifically rigorous approach to quantify Leptosperin—a definitive chemical marker of Leptospermum species—thus enabling robust authentication of mānuka honey to maintain consumer confidence and uphold export quality standards.