Method Validation for the Determination of Cocaine Derivatives in Oral Fluid Using PESI and LC–MS

Oral fluid is the preferred matrix for driving under the influence of drug (DUID) investigations. The collection of oral fluid is simpler than urine: it is non-invasive, requires no medical expertise, preserves patient privacy, is sex‑neutral, and allows direct observation, reducing the risk of adulteration. In France, analyses must be performed using liquid chromatography tandem mass spectrometry (LC–MS/MS). Probe electrospray ionization (PESI) offers a simplified sample preparation process without chromatographic separation, while retaining the specificity and sensitivity of MS. The metal probe repeatedly dips into the sample and acts as both a collector and emitter. Coupling PESI with high-resolution MS allows for better sensitivity and specificity through exact mass detection. The goal was to assess the ability of PESI-quadrupole time‑of‑flight (QTOF) to detect cocaine, benzoylecgonine (BZE), and ecgonine-methylester (EME) in oral fluid and compare its performance to the routine LC–MS/MS method.

Experimental Conditions

A 10 mM ammonium formate solution mixed with ethanol (50:50) was used. Eighty-three DUID cases were analyzed using both PESI-QTOF and the routine LC–MS/MS method (1). All samples were collected with dried FloqSwabs (Copan).

Sample Preparation: Blank oral fluid was obtained from drug-free volunteers. Swabs were dried for at least 12 h, then eluted in 2 mL of ethanol/ammonium formate buffer at 37 °C for 10 min. Solutions were spiked at 5 ng/mL, 10 ng/mL, 25 ng/mL, 50 ng/mL, and 100 ng/mL. A 50 µL measure was then mixed with 450 µL of buffer and 10 µL of internal standard solution (50 ng/mL); 10 µL of this mix was then deposited on the sample plate for PESI analysis.

Analytical Conditions: A full-scan (100–500 m/z) was followed by a targeted MS/MS acquisition for the three analytes and their internal standards. The total run time was 0.45 min.

PESI-QTOF Mass Spectrometry Conditions: PESI: injection volume: 10 µL; take sample position: -46 mm; time spent in the sample: 30 ms; probe voltage: 2.3 Kv; speed of the needle: 400 ms. MS: ionization: positive mode; event time: 0.050 s; interface temp.: 250 °C; heat block temp.: 50 °C; nebulizing gas flow: 3.0 L/min; heating gas flow: 10.0 L/min; drying gas flow: 10.0 L/min.

Synchronization: A crucial parameter was synchronizing ionization and MS acquisition. Optimal needle frequency and MS event timing ensured sharp, reproducible peaks. Parameters such as probe movement frequency and MS event duration were tuned to optimize peak area and shape The best signals were obtained with 0.050 s for both MS1 and MS2 and a needle frequency of 2.3 Hz.

Software: LabSolutions Insight software(Shimadzu).

Results and Discussion

Method validation followed COFRAC guidelines under ISO 15189 (accreditation 8-2607), assessing linearity, precision, lower limits of detection (LLOD), carryover, dilution, and specificity.

Precision Study: Inter- and intra-day precision and accuracy were evaluated at 5 ng/mL, 25 ng/mL, and 100 ng/mL over five days (n = 5/day) and six replicates/day, respectively. Coefficient of variation (CV) and bias remained below 15% (20% at 5 ng/mL).

Linearity Study: The calibration curve covered 5–100 ng/mL. All standards met the 80–120% accuracy criterion. Linearity was confirmed for all compounds.

Lower Limit Of Detection: LLOD was estimated from 30 blank saliva samples (2). Signals within the 0–0.45 min interval were used to calculate standard deviation, with LLOD defined as 3×SD. Values were conservatively rounded up. LLOD for PESI‑QTOF and LC–MS/MS were comparable. The performances of the method are illustrated in Figure 1 and Table I.

Matrix Effects Study: Matrix effects were assessed in six saliva samples spiked at 5 ng/mL, 25 ng/mL, and 100 ng/mL. Signals showed less than 20% variation at 5 ng/mL and less than 15% at higher levels. Signal enhancement was observed for BZE and EME but was corrected by internal standards.

Selectivity Study: Six saliva samples were tested using a solution containing 119 psychotropic drugs (1 mg/L), including antidepressants, benzodiazepines, and drugs of abuse. No interference was observed; bias remained under 15%.

Dilution Test Study: Samples spiked at 150 ng/mL were diluted 1:10 and tested in triplicate. Measured concentrations (15.13 ng/mL, 15.09 ng/mL, and 16.19 ng/mL) confirmed accuracy within 15% bias.

Carryover Study: Carryover was assessed by injecting a blank after a 100 ng/mL sample. No signal above 20% of the lower limit of quantification (LLOQ) was detected for any of the three compounds.

Real Samples: Out of 83 DUID samples, cocaine was detected in 61.6% (51 cases), BZE in 98.5% (50), and EME in 97.2% (49). Full agreement with LC–MS/MS was observed across all cases. The percentage of agreement between the LC–MS/MS method and the PESI-QTOF method were 93.33% for EME, 90.67% for BZE, and 85.33% for cocaine.

Conclusion

This study presents the first application of PESI with high-resolution MS for drug detection in saliva. PESI’s simplicity and speed, combined with QTOF’s selectivity, yielded a robust, ultra-fast, and sensitive method. Key optimizations included synchronizing the probe and acquisition events. Although focused on cocaine derivatives, the method is applicable to other analytes. With a total analysis time of 0.45 min and low LLOD values, PESI-QTOF offers a powerful alternative to conventional LC–MS/MS in forensic toxicology.

Acknowledgment

This validation study was conducted with the help of Limoges University Hospital, France.

References

(1) Dulaurent, S.; El Balkhi, S.; Poncelet, L.; et al. QuEChERS Sample Preparation Prior to LC-MS/MS Determination of Opiates, Amphetamines, and Cocaine Metabolites in Whole Blood. Anal. Bioanal. Chem. 2016, 408 (5), 1467–1474. DOI:10.1007/s00216-015-9248-3

(2) El Balkhi, S.; Saint-Marcoux, F. Chlordecone Determination in Serum by LC-MS/MS and the Importance of Low Limit of Detection. J. Chromatogr. B 2023, 1230, 123915. DOI: 10.1016/j.jchromb.2023.123915

Elisa Jousselin is a PharmD, PhD student. After completing a master’s in analytical chemistry, she began a doctoral thesis focused on developing innovative analytical methods as part of a collaboration between Shimadzu and the Pharmacology-Toxicology Department of Limoges University Hospital.

Elies Zarrouk is a PharmD who earned his doctoral degree in 2024 with a thesis focusing on HRMS (research collaboration with Shimadzu).

Pauline Griffeuille is with the Department of Pharmacology and Toxicology at Limoges University Hospital.

Sylvain Dulaurent is with the Department of Pharmacology and Toxicology at Limoges University Hospital.

Souleiman El Balkhi oversees the clinical, forensic, occupational, and environmental toxicology programs at Limoges University Hospital.

Franck Saint-Marcoux oversees the clinical, forensic, occupational, and environmental toxicology programs at Limoges University Hospital.