Efficient Quantitative Analysis of THC and Metabolites in Human Plasma Using Agilent Captiva EMR—Lipid and LC-MS/MS

Significance of the Topic

The accurate quantification of Δ9-tetrahydrocannabinol (THC) and its primary metabolites, 11-hydroxy-Δ9-THC (THC-OH) and 11-nor-9-carboxy-Δ9-THC (THC-COOH), in human plasma is essential for forensic toxicology. Phospholipids in plasma cause significant matrix effects in electrospray ionization LC-MS/MS, leading to ion suppression, reduced sensitivity, and frequent instrument maintenance. A streamlined sample preparation workflow that efficiently removes phospholipids while preserving analyte recovery enhances data quality and laboratory throughput.

Study Objectives and Overview

This study aimed to:

• Combine in-well protein precipitation (PPT) with Agilent Captiva EMR-Lipid pass-through cleanup for plasma samples.
• Remove >99% of phospholipids (PPLs) while retaining >90% of target analytes.
• Validate linearity, sensitivity (LOQ ≤1 ng/mL), accuracy, and precision over 0.5–100 ng/mL.
• Demonstrate method robustness over multiple days.

Methodology

Sample preparation involved adding acidified acetonitrile to plasma in well, mixing, and applying vacuum through a 1 mL Captiva EMR-Lipid cartridge. The sorbent traps unbranched lipid chains by size exclusion and hydrophobic interactions while passing bulky analytes. Eluates were dried under nitrogen and reconstituted in methanol with 0.1% formic acid. An online dilution feature (10 µL water preceding 5 µL sample) preserved analyte integrity.

Chromatographic separation used a 2.1 × 50 mm, 1.8 µm C18 column at 50 °C, flow 0.5 mL/min, gradient from 35% to 95% organic in 4 minutes. Detection employed multiple reaction monitoring (MRM) in positive ESI on a triple quadrupole MS.

Used Instrumentation

• Agilent Captiva EMR-Lipid 1 mL cartridges
• Agilent 1290 Infinity LC System with inline 0.3 µm filter
• Agilent ZORBAX RRHD Bonus RP column (2.1×50 mm, 1.8 µm)
• Agilent 6490 Triple Quadrupole LC/MS system
• Agilent MassHunter Software for acquisition and processing

Main Results and Discussion

• PPL Removal: EMR-Lipid achieved >99% removal of 11 monitored phospholipids, greatly reducing background and potential system contamination.
• Chromatographic Performance: At 1 ng/mL, THC, THC-OH, and THC-COOH exhibited sharp peaks and S/N >10, with complete separation within 5 minutes.
• Linearity and Sensitivity: Calibration curves from 0.5–100 ng/mL were linear (R² >0.99). Limits of quantitation were ≤1 ng/mL.
• Accuracy and Precision: Recoveries ranged from 97% to 107% with intra- and inter-day RSDs <10% across low (1 ng/mL), mid (10 ng/mL), and high (50 ng/mL) QC levels.

Practical Benefits and Applications

This protocol simplifies forensic workflows by reducing sample handling, minimizing matrix effects, and extending instrument uptime. High recovery and low LOQs support reliable impairment testing and casework analysis in clinical and forensic laboratories.

Future Trends and Applications

Advances may include full automation of in-well cleanup in 96-well formats, integration with high-resolution MS for broadened analyte panels, and adaptation to other challenging biological matrices (e.g., oral fluid, urine). Emerging sorbent chemistries could further enhance selectivity and throughput.

Conclusion

The combined PPT and Captiva EMR-Lipid cleanup delivers a rapid, robust method for THC and metabolite quantification in plasma. It effectively eliminates phospholipid interferences, maintains high analyte recovery, and meets stringent forensic requirements for sensitivity, accuracy, and reproducibility.

References

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