SFC–HRMS: Application to the Detection of Drugs of Abuse in Oral Fluid

The main objective of this study was to develop a method for measuring drugs of abuse commonly investigated in the context of driving under the influence of drugs (DUID) in oral fluid, using supercritical fluid chromatography coupled with quadrupole time-of-flight mass spectrometry (SFC–QTOF-MS). The method targeted 10 compounds belonging to the main classes of opiates, amphetamine-type stimulants, and cocaine-related substances.

Coupling supercritical fluid chromatography (SFC) with mass spectrometry (MS) was investigated to reduce analysis time and solvent consumption while benefiting from the specificity and sensitivity of high-resolution mass spectrometry (HRMS). A supercritical fluid is a fluid that, at a specific temperature (Tc) and pressure (Pc), known as the critical point, exhibits properties of both gases and liquids. Carbon dioxide (CO₂) is the main mobile phase used in SFC because its critical point is readily attainable and because it is non-explosive, non-toxic, non-flammable, odorless, and environmentally friendly. Its lack of a dipole moment accounts for its low polarity, an important property in SFC. Adding a co-solvent (methanol, ethanol, isopropanol, acetonitrile) extends the range of compounds that can be analyzed, from lipophilic to moderately polar compounds. The schematic representation of an SFC system is shown in Figure 1.

In the context of driving under the influence of drugs (DUID), oral fluid is commonly used, and in France, analyses must be performed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The use of HRMS for drug detection may provide improved sensitivity and specificity through exact-mass measurement.

In this study, we sought to evaluate the ability of SFC–QTOF-MS to detect drugs of abuse (cocaine, opiates, amphetamines, and their derivatives) in authentic oral fluid samples collected in the context of DUID and to compare the analytical performance of this new method with that of the routine LC–MS/MS method.

Experimental Conditions

Ten analytes and their corresponding deuterated internal standards (IS) were considered: 6-acetylmorphine (6-AM), morphine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), 3,4-methylenedioxymethamphetamine (MDMA), cocaine, benzoylecgonine, and ecgonine methyl ester. All analytical components were supplied by Shimadzu. The experiments were conducted using a Nexera SFC/ultrahigh-performance liquid chromatography (UHPLC) switching system, composed of a supercritical carbon dioxide (scCO₂) pump (LC-30ADSF), modifier and makeup pumps (LC-40DX3) with degassers (DGU-405), a back-pressure regulator (SFC-30A), an autosampler (SIL-40CX3), a column oven (CTO-40C), and a system controller (CBM-40). Analytes were detected using a high-resolution mass spectrometer (LCMS-9050) equipped with an electrospray ionization (ESI) source, quadrupole (Q) and time-of-flight (TOF) mass analyzers, and a collision cell placed between the Q and TOF stages.

Sample Preparation: The samples used to evaluate method performance were prepared using blank serum samples collected from patients with no evidence of illicit drug exposure. Authentic oral fluid samples were also analyzed in the context of DUID. Oral fluid was collected using a FloqSwabs kit (Copan), a device consisting of a swab designed for oral mucosa sampling and used by the French authorities.¹ Sample preparation was performed using 100 μL of saliva or serum and Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) salts to purify the matrix. The samples were then spiked at concentrations of 5, 10, 50, 100, 200, and 500 ng/mL to evaluate method performance, and acetonitrile was used as the injection solvent.

SFC Conditions: Methanol containing 5 mM ammonium formate and 0.1% formic acid was used as the co-solvent and mixed with supercritical carbon dioxide during the gradient. The post-column make-up solution was methanol without additives. The make-up solvent was introduced isocratically at 0.100 mL/min. SFC separation of cocaine, amphetamines, opiates, and their derivatives was performed on a DIOL II column (Shimpack) (Shimadzu).

The oven temperature was set at 25 °C. The back-pressure regulator (BPR) pressure and temperature were set at 150 bar and 50 °C, respectively. The total flow rate was maintained at 1.3 mL/min, and the injection volume was 2 μL. The gradient used during the analysis is shown in Figure 2.

Mass Spectrometry Conditions: Compound identification was based on a full-scan acquisition followed by targeted tandem mass spectrometry (MS/MS) acquisition. More precisely, full-scan data were acquired from a mass-to-charge ratio (m/z) of 100 to 500 to detect potential compounds (that is, an untargeted approach; MS1), while scheduled targeted MS/MS acquisition (MS2) focused on the precursor ions of the 10 analytes and their 10 IS, based on their exact masses. The resulting product ions were also analyzed based on their exact masses. The following identification criteria were applied to each compound: detection of two ions, including the precursor ion and one product ion, each with a mass accuracy of ≤5 parts per million (ppm) (or <1 millidalton (mDa) for m/z <200).

Results and Discussion

Method validation followed guidelines of the French Accreditation Committee (COFRAC) under the International Organization for Standardization (ISO) 15189 standard and included assessment of linearity, precision, the lower limit of detection (LLOD), carryover, and selectivity.

Linearity: Correlation coefficients were greater than 0.99 for all calibration curves over the concentration range of 5–500 ng/mL. Representative calibration curves obtained for cocaine and its derivatives are shown in Figure 3.

Precision Study: Inter- and intraday precision and accuracy were evaluated at 5, 25, and 100 ng/mL over five days (n = 5/day) and using six replicates per day, respectively. Inter- and intraday precision and bias values were consistently lower than 15% (or 20% at 5 ng/mL) for all analytes.

Lower Limit of Detection (LLOD): The LLOD was estimated using 20 blank saliva samples. Signals recorded within the 0–7 min interval were used to calculate the standard deviation (SD), and the LLOD was defined as 3 × SD.² LLODs obtained by SFC-QTOF-MS and LC–MS/MS were comparable. The LLOD and lower limit of quantification (LLOQ) values for cocaine, amphetamines, opiates, and their derivatives are presented in Table 1.

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

Carryover Study: Carryover was assessed by injecting a blank after a 100 ng/mL sample. No signal above 20% of the LLOQ was detected for any analyte.

Real Samples: For the present study, 83 DUID cases sent to our lab for the determination of illicit drugs were analyzed using both the SFC–QTOF-MS method and an LC–MS/MS method routinely used in the lab.³ Briefly, this LC–MS/MS method was entirely validated and accredited according to the ISO 15189 standard. The SFC–QTOF-MS method detected drugs of abuse in 51 of the 83 authentic samples (61.6%). Complete agreement with the reference LC–MS/MS method was observed for all 10 analytes, in both positive and negative samples.

Method Sustainability: The chromatographic separation developed in this study had a relatively short run time of 7 min, compared with 12 min for LC–MS/MS, allowing a substantial increase in sample throughput. At most, 40% organic solvent was used in this method. The average consumption of organic modifier was approximately 1.8 mL, compared with 9.6 mL for LC–MS/MS. This represents an approximately 81% reduction, resulting in lower solvent costs and less hazardous waste. Moreover, the carbon dioxide used in analytical SFC is generally recovered as a by-product of existing industrial processes rather than produced specifically for chromatographic applications. Consequently, its use contributes little to additional greenhouse gas emissions. The lower volume of organic waste requiring treatment or incineration further improves the environmental profile of the method.

Conclusion

The SFC–QTOF-MS method demonstrated excellent analytical performance for detecting drugs of abuse in oral fluid, with complete agreement with a routine LC–MS/MS method. In addition to providing reliable identification through high-resolution mass spectrometry, SFC substantially reduced analysis time and organic solvent consumption, making it a rapid, robust, and environmentally friendly alternative for toxicological screening in DUID cases.

Acknowledgment

This study was supported by Limoges University Hospital.

References

1. Fabresse, N.; Hassan, A.; Knapp, A. Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Detection of 10 Illicit Drugs in Oral Fluid Collected With FLOQSwabs™ and Application to Real Samples. Drug Test Anal 2019, 11 (6), 824–832. DOI: 10.1002/dta.2563
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
3. 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