Development of a UPLC-MS/MS Method for Tracking Polymyxin B Dynamics in Soil Inoculated with Paenibacillus polymyxa

Polymyxins, including polymyxin B (PMB), are last-resort antibiotics against multidrug-resistant Gram-negative infections in humans and livestock. Residual polymyxins from wastewater and manure can accumulate in soil, facilitating the emergence and spread of polymyxin resistance. <i>Paenibacillus polymyxa</i>, a natural polymyxin producer used in crop cultivation, may increase soil polymyxin burden. Since PMB strongly adsorbs to soil, its reliable quantification has been challenging. To address this, the extraction solvent and solid-phase extraction procedure were optimized to improve recovery and reduce matrix effects. We developed and validated a UPLC-MS/MS method to quantify PMB in soil. The method showed linearity (10-1000 ng/g), with a limit of detection of 0.86 ng/g and a limit of quantification of 2.12 ng/g. Method validation confirmed acceptable analytical performance. A 28-day monitoring of PMB in soil inoculated with varying <i>P. polymyxa</i> doses revealed a dose-dependent increase over the first 14 days, followed by a decline; PMB remained detectable on day 28. Ecological risk assessment using the risk quotient (RQ) indicated that PMB levels in the high-dose group (2 × 10⁸ CFU/100 g) approached the high-risk threshold (RQ ≥ 1) on day 14, while lower doses posed low to medium risk. This work provides a soil PMB quantification method and insight into the ecological risk of <i>P. polymyxa</i> application.