Determination of Pesticide Residues in Black Peppercorns Using LC-MS/MS After Extraction and Clean up Using QuEChERS and Pass-Through SPE

Significance of the Topic

Black peppercorns are a high-value spice widely traded and consumed worldwide. Pesticide application during cultivation and storage poses risks of residue levels exceeding regulatory limits. The complex composition of peppercorns—rich in fats, phospholipids, pigments, and phytochemicals—creates analytical challenges due to matrix effects and interference in mass spectrometric detection. Reliable multiresidue methods are essential to ensure consumer safety and maintain compliance with maximum residue levels (MRLs) in global trade.

Objectives and Study Overview

This work aimed to develop and evaluate a robust multiresidue method for over 200 pesticide compounds in black peppercorns. The study compared a generic QuEChERS extraction followed by conventional dispersive SPE cleanup against a streamlined pass-through SPE protocol using an Oasis PRiME HLB cartridge. Performance metrics included sensitivity, selectivity, recovery, and compliance with SANTE guidelines for MRL enforcement.

Methodology and Instrumentation

Sample Preparation and Cleanup
Ground black peppercorns were extracted by a modified CEN QuEChERS protocol. Two cleanup approaches were compared:

• Dispersive SPE (dSPE) using MgSO4, PSA, C18, and GCB sorbents.
• Pass-through SPE on an Oasis PRiME HLB cartridge, discarding the first 0.25 mL eluate and collecting the remaining 0.75 mL.

UPLC-MS/MS Analysis
The cleaned extracts were analyzed on an ACQUITY UPLC I Class PLUS system equipped with a Premier HSS T3 column (2.1 × 100 mm, 1.8 μm) and a 50 μL post-injection mixing loop. Mobile phases consisted of 10 mM ammonium formate with 0.1% formic acid in water (A) and methanol (B) under a 15-minute gradient. Detection was performed on a Xevo TQ-S micro tandem quadrupole mass spectrometer operating in electrospray positive and negative modes with fast polarity switching. Data acquisition and quantitation used waters_connect software with predefined MRM transitions.

Main Results and Discussion

Cleanup Performance
The pass-through SPE approach retained more matrix co-extractives and yielded higher recoveries (86% of analytes within 70–120% range) compared to dSPE (67%). Both methods reduced matrix effects for approximately half of the compounds.
Sensitivity and Calibration
Lowest calibrated levels of 0.005 mg/kg were achieved for 84% of pesticides, with a further 10% at 0.01 mg/kg. Calibration curves (0.005–1.0 mg/kg) showed R2 ≥ 0.99 for all but two analytes, and residuals < 20%, demonstrating reliable quantification.
Recovery and Selectivity
Across three spike levels (0.01, 0.05, 0.2 mg/kg), 85% of detected analytes met SANTE ion-ratio and retention time criteria with mean recoveries between 70 and 120%.
Peak Shape Optimization
Early eluting polar compounds exhibited improved peak shapes and sensitivity when a 50 μL post-injection mixing loop was employed to dilute the strong injection solvent prior to chromatography.

Benefits and Practical Applications

• Rapid and effective removal of fats, phospholipids, and pigments while maintaining high pesticide recovery.
• High sensitivity compatible with default MRLs (0.01–0.1 mg/kg) in the EU and Codex standards.
• Streamlined workflow suitable for routine MRL compliance screening in spice laboratories.

Future Trends and Potential Applications

• Extension of pass-through SPE cleanup to other complex matrices such as herbs and dried fruits.
• Integration with high-resolution mass spectrometry for non-targeted screening of emerging contaminants.
• Development of automated, high-throughput platforms combining QuEChERS and SPE modules.
• Use of greener solvents and sorbent materials to reduce analytical environmental footprint.

Conclusion

The combination of QuEChERS extraction with an Oasis PRiME HLB pass-through SPE cleanup and UPLC-MS/MS detection delivers a sensitive, selective, and robust multiresidue method for pesticide analysis in black peppercorns. Method validation against SANTE guidelines confirmed reliable quantification down to 0.005 mg/kg and acceptable recoveries for most analytes, making this approach suitable for regulatory compliance and quality control.

References

1. FSSAI Order on Maximum Residue Limits for Spices and Culinary Herbs, 2024.
2. Hird S., Lee J.X., Zhang W. A Smart Clean-up Approach to Reduce Isobaric Interference for Multi-Residue Pesticide Analysis of Black Tea and Cocoa Beans, Waters Application Brief 720008039, 2023.
3. European Committee for Standardisation EN 15662:2018, Foods of Plant Origin – QuEChERS Method.
4. Zhu Y. et al. Evaluation of Clean-up Procedures and Sample Dilution in Multi-Residue Pesticide Analysis of Spices and Herbs by UPLC-MS/MS, Food Addit. Contam. Part A, 38(2):326–338, 2021.
5. European Commission Document SANTE/11312/2021 v2 Guidance on Analytical Quality Control and Method Validation for Pesticide Residues in Food and Feed, 2023.