Review: Identification and Quantification of Synthetic Cathinones in Blood and Urine Using Liquid Chromatography-Quadropole/Time of Flight (LC-Q/TOF) Mass Spectrometry

Emily C. Lennert





liquid chromatography, mass spectrometry, tandem, quadrupole, LC, MS, LC-MS, MS/MS, quadrupole, time-of-flight, time of flight, TOF, Q/TOF, drug, drugs of abuse, illicit, urine, blood, psychoactive, substances, synthetic cathinones, solid phase extraction, SPE

Article Reviewed

  1. Glicksberg, L.; Bryand, K.; Kerrigan, S. Identification and quantification of synthetic cathinones in blood and urine using liquid chromatography-quadropole/time of flight (LC-Q/TOF) mass spectrometry. Journal of Chromatography B. 2016, 1035, 91-103.


The opinions expressed in this review are an interpretation of the research presented in the article. These opinions are those of the summation author and do not necessarily represent the position of the University of Central Florida or of the authors of the original article.


Synthetic cathinones are classified as new psychoactive substances (NPS) that serve as stimulants, known to produce mood enhancing and psychoactive effects. Synthetic cathinones may also produce adverse effects in users, such as panic attacks, tremors, psychosis, hallucinations, violence, and aggression. Existing immunoassay screening methods are insufficient for synthetic cathinone analysis, with many drugs within the class being undetectable or inadequately detected by the methods. While gas chromatography – mass spectrometry (GC-MS) is a common method of drug analysis used in labs, synthetic cathinones are prone to thermal degradation and may be difficult to detect by GC-MS methods. Liquid chromatography – mass spectrometry (LC-MS) can bypass the thermal degradation issue because samples remain in the liquid phase, rather than being heated to produce a gas like what is done in GC-MS. High resolution mass spectrometry (HRMS) methods allow for analysis with high mass accuracy and selectivity. In this study, a LC-MS method using tandem mass spectrometry, i.e. two mass spectrometers, was employed. Quadrupole and time-of-flight (Q/TOF) mass spectrometers were used to allow for tandem mass spectrometry.

A total of 22 synthetic cathinones were analyzed in blood and urine by LC-Q/TOF. Standard solutions of each target compound were prepared and used to spike drug free urine and blood samples. An internal standard mixture containing 9 compounds of known concentration was used to allow for quantification of an unknown concentration of a target compound within the sample. The unknown concentration of the target compound can be estimated by comparison of the signal obtained to the signal from the internal standard of known concentration, using the peak area and a calibration curve. Calibration samples containing known concentrations of each cathinone were prepared ranging in concentration from 5 – 1000 ng/mL. Additionally, 22 common drugs, 10 amphetamine like drugs, 15 designer drugs, 4 other drugs and metabolites, and 4 putrefactive amines were obtained to study potential interferences; these compounds are listed in Table 2 within the study.


Urine samples were prepared for analysis after spiking with the cathinone mixture. Internal standard was added to a 1.0 mL sample of urine to reach an internal standard concentration of 25 ng/mL. The sample was then diluted with 2.0 mL of 0.1 M phosphate buffer, pH 6, and briefly vortex mixed. Samples were then transferred to solid phase extraction (SPE) columns and allowed to flow through the SPE column. After the sample passed through the column, the column was rinsed with 1.0 mL water followed by 1.0 mL of 1M acetic acid. The SPE columns were then dried and washed with 1.0 mL each of hexane, ethyl acetate, and methanol. Cathinones were then eluted from the SPE column with a total of 1.0 mL of elution solvent. 30 μL of acidic methanol was added to the eluted cathinone solution, and the solution was evaporated to dryness. The extract was then reconstituted in 25 μL of a 50:50 mixture of the mobile phase A and B solutions: 0.1% formic acid in water and 0.1 formic acid in acetonitrile. The mobile phase is the liquid that carries the sample through the LC. For further explanation of LC-MS, please refer to Review: Identification and Quantification of 35 Psychotropic Drugs and Metabolites in Hair by LC-MS/MS: Application in Forensic Toxicology.

Blood samples were prepared for analysis after spiking with the cathinone mixture. Internal standard was added to a 2.0 mL blood sample for an internal standard concentration of 25 ng/mL. Proteins were separated within the blood by precipitation. 4.0 mL of cold acetonitrile was added to the blood sample while vortex mixing. The sample was centrifuged, i.e. spun, to separate the protein precipitate and the supernatant, i.e. top layer, was removed and diluted with 6.0 mL of 0.1 M phosphate buffer, pH 6, and vortex mixed briefly. Samples were then transferred to SPE columns and cathinones were extracted in analogous fashion to the urine samples. Extracts were reconstituted in 25 μL of a 50:50 mixture of mobile phase solutions A and B.


Samples were evaluated for extraction efficiency, calibration model, limit of detection (LOD), limit of quantification (LOQ), interferences, and more. Chromatographic separation was achieved for all synthetic cathinones, with each cathinone producing a distinct peak despite some overlap between cathinones. Extraction efficiencies were determined to be 84 – 104% in urine at 25 ng/mL and 81 – 93% in blood at 100 ng/mL, as seen in Table 3 within the study. Calibration curves were successfully generated for each compound and found to be non-linear, resulting in a weighted 1/x quadratic model. A general depiction of the model can be seen below.

The LOD for all cathinones ranged from 0.25 to 5 ng/mL in urine and 1 to 5 ng/mL in blood. The LOQ for all cathinones also ranged from 0.25 to 5 ng/mL in urine and 1 to 5 ng/mL in blood. A summary of the LOD and LOQ for each cathinone in urine and blood can be found in Tables 4 and 5, respectively, within the study. Matrix interferences and interferences from other compounds were investigated. No matrix interferences were noted, and matrix effects such as ion suppression were within tolerable limits. No significant interferences were reported in the additional compounds analyzed.

Scientific Highlights

  • Protocols for the extraction of synthetic cathinones from blood and urine were presented.
  • 22 synthetic cathinones were extracted from blood and urine samples and successfully analyzed and quantified using LC-Q/TOF with negligible matrix interference.
  • The presence of multiple other drugs and related compounds in the samples did not interfere with the detection and analysis of synthetic cathinones by this method.


Detection of drugs in complex matrices, such as blood or urine, may be difficult due to possible matrix interferences, as well as other factors influencing the detection of the target drugs. The method presented allowed for the detection and quantification of 22 synthetic cathinones at low concentrations, both with and without other drugs present, in blood and urine samples.

Potential Conclusions

  • The method presented provides a viable protocol for sample preparation and analysis of suspected synthetic cathinones in blood and urine samples by LC-Q/TOF.
  • The method allows for specific and sensitive analysis of synthetic cathinones, even in a complex sample.