AGILENT BOND ELUT CERTIFY AND CERTIFY II METHOD MANUAL

Importance of the Topic

Forensic and clinical laboratories must reliably identify and quantify a wide range of drugs of abuse, pharmaceuticals, and their metabolites in complex biological matrices such as urine, blood, plasma and meconium. Sample preparation is often the limiting factor in achieving accurate results, high throughput, and consistent recoveries. Mixed-mode solid phase extraction (SPE) using Agilent’s Bond Elut Certify and Certify II sorbents addresses these challenges by combining hydrophobic, polar, and ion-exchange interactions in a single cartridge. This approach streamlines cleanup, enhances selectivity, and reduces matrix effects prior to GC, GC/MS, HPLC, or LC/MS analysis.

Objectives and Study Overview

This manual provides standardized workflows for more than thirty drug classes, spanning basic, acidic, and neutral compounds. Key goals include:

• Describing cartridge conditioning, sample loading, washing and elution steps for each class.
• Defining pH control, buffer composition, and drying practices to maximize reproducibility.
• Outlining derivatization strategies compatible with common GC and GC/MS detectors.
• Demonstrating adaptability to automated platforms, 96-well plates, and high-throughput manifolds.

By following these protocols, laboratories can develop validated SPE methods tailored to their specific analyte panels and detection technologies.

Methodology and Instrumentation

The core sample preparation method involves:

1. Conditioning the mixed-mode SPE cartridge with organic solvent (methanol) and aqueous buffer to equilibrate both sorbent phases.
2. Adjusting sample pH and ionic strength via phosphate, acetate or TRIS buffers to optimize retention of target analytes.
3. Loading the diluted biological sample at controlled flow rates (1–2 mL/min) under low vacuum or gravity to prevent sorbent drying.
4. Sequentially washing away polar and nonpolar interferences using aqueous acids, organic solvents, or their mixtures.
5. Eluting analytes with alkaline organic solvents to disrupt ionic and hydrophobic interactions, followed by evaporation and reconstitution.
6. Derivatizing extracts for GC or GC/MS when necessary (e.g., BSTFA, PFPA, or TFAI for silylation or acylation).

Instrumentation commonly employed includes:

• Gas chromatography (GC, GC-FID) and gas chromatography–mass spectrometry (GC/MS)
• Liquid chromatography (HPLC, UHPLC) with UV or diode-array detection
• Triple quadrupole LC/MS/MS with Agilent Jet Stream electrospray source
• Automated SPE platforms (Gilson ASPEC series), vacuum manifolds for 12, 20, and 96-well formats
• Mass spectrometer-compatible inert liners, ferrules, and J&W Ultra Inert columns

Main Results and Discussion

The manual demonstrates that Bond Elut Certify effectively retains basic drugs (amphetamines, opiates, benzodiazepines) through mixed cation-exchange and reversed-phase interactions, while acidic analytes (barbiturates, NSAIDs, THC-COOH) are optimally extracted on Certify II with anion-exchange plus hydrophobic retention. Recoveries routinely exceed 80–90 %, with limits of detection in the low ng/mL range when combined with MS detectors. Detailed wash and elution conditions prevent co-extraction of matrix lipids, phospholipids, and endogenous interferences, ensuring stable ion ratios and sharp chromatographic peaks. The flexibility to switch between GC and LC workflows, plus adaptability to 96-well high-throughput SPE, addresses diverse laboratory throughput and sensitivity needs.

Benefits and Practical Applications of the Method

Key advantages include:

• Universal SPE platform: One mixed-mode sorbent accommodates multiple drug classes, reducing inventory complexity.
• High selectivity: Sequential pH and solvent washes eliminate interferences for cleaner extracts.
• Consistent recoveries: Precise pH control and validated buffer recipes yield reproducible results.
• Scalable throughput: Manual cartridges, automated SPE, and 96-well plate formats support small-scale and high-volume testing.
• Compatibility: Methods can be seamlessly integrated with existing GC, LC, and MS systems, preserving established chromatographic conditions.

Practical uses span workplace drug screening, legal forensic toxicology, clinical monitoring, doping control, and pharmaceutical research.

Future Trends and Opportunities

Emerging directions include:

• Integration of micro-SPE disks and microfluidic SPE cartridges for ultra-low sample volumes.
• Hybrid automated platforms coupling SPE with direct online LC/MS for real-time analysis.
• Enhanced sorbent chemistries targeting novel psychoactive substances and designer opioids.
• Machine learning-driven method optimization to predict ideal pH, buffer, and elution conditions for new analyte classes.
• Green sample preparation: Reduced solvent consumption and waste management in line with sustainability goals.

Conclusion

Agilent’s Bond Elut Certify and Certify II mixed-mode SPE methods deliver robust, versatile, and high-throughput sample preparation workflows for forensic and clinical toxicology. By harmonizing hydrophobic, polar, and ion-exchange mechanisms in a single sorbent, laboratories can achieve consistent recoveries, cleaner extracts, and lower detection limits across a wide array of drug classes. The comprehensive manual and accessory offerings—including 96-well plates, vacuum manifolds, and compatible instrumentation—ensure that labs can rapidly implement and customize SPE protocols to meet evolving analytical demands.

Reference

• Agilent Bond Elut Certify and Certify II Methods Manual, Agilent Technologies, 2014.
• United Nations International Drug Control Program, Recommended Methods for the Detection and Assay of Drugs in Biological Specimens, 1993.
• Chen X.-H. et al., Mixed-Mode SPE for Systematic Toxicological Analysis, J. Forensic Sci., 1992, 37(1), 61–71.
• NIDA Research Monograph 73: Urine Testing for Drugs of Abuse, U.S. Dept. of Health and Human Services, 1986.