Review: The Role of Ultra High Performance Liquid Chromatography with Time of Flight Detection for the Identification of Synthetic Cannabinoids in Seized Drugs

Emily C. Lennert





synthetic, cannabinoid, spice, controlled, isomers, non-controlled, ultra high performance liquid chromatography, UHPLC, time of flight, TOF, mass spectrometry, MS

Article Reviewed

  1. Marginean, I.; Rowe, W. F.; Lurie, I. S. The role of ultra high performance liquid chromatography with time of flight detection for the identification of synthetic cannabinoids in seized drugs. Forensic Science International. 2015, 249, 83-91.


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.


The authors of this study sought to compare ultra high performance liquid chromatography with time of flight mass spectrometry (UHPLC – TOFMS) and gas chromatography mass spectrometry (GC – MS) for the identification of synthetic cannabinoids. A total of 32 synthetic cannabinoids were examined: 23 controlled synthetic cannabinoids and 9 isomers of the controlled cannabinoid JWH-018. Samples were first analyzed by three UHPLC methods, using varying stationary phases in the chromatographic column.

UHPLC is a high capacity form of LC. LC operates using a stationary phase and a mobile phase. The stationary phase is similar to a filter and consists of a column, or tube, packed with particles. The mobile phase is a high pressure liquid that carries the sample through the stationary phase. Due to interaction of the sample with the mobile and stationary phases, compounds within the sample will separate within the column. Samples then elute from, or leave, the column at different times, called retention times. In a LC-MS system, after eluting from the column, samples are ionized and enter the mass analyzer for analysis.

GC operates based on similar principles. In GC, a sample is injected and heated to produce a vapor, which is then carried through the column by a gas. The GC column is lined with particles, i.e. the stationary phase, similar to that of the LC column. Due to interaction of the sample with the column, compounds separate within the column and elute from the column at different retention times. In a GC-MS system, after eluting from the column, samples are ionized and enter the mass analyzer for analysis.

Three UHPLC columns were tested for separation of the controlled cannabinoids as well as for the JWH-018 isomers: SPP C18, SPP Phenyl Hexyl, and SPP PFP. A mobile phase of 0.1% formic acid in acetonitrile and water 70:30 was used. Samples were prepared for analysis by dilution in methanol to either 1.0 or 10.0 mg/mL, depending on which concentration for the compound was deemed appropriate by the authors. A mixture of controlled cannabinoids was analyzed by each method, as well as a mixture of the uncontrolled JWH-018 isomers.

Of the 23 controlled substances, 15 substances were resolved by the C18 column with separation of 1 min or greater between each. Similarly, 15 were resolved by the Phenyl Hexyl column, and 14 were resolved by the PFP column. Samples were then analyzed by GC-MS, where 17 compounds were chromatographically resolved. All individual controlled compounds could be identified with a combination of the GC retention times and resulting mass spectra. Significant differences in retention times between LC columns suggest that all 23 controlled cannabinoids could be well resolved with a combination of two UHPLC systems utilizing different columns. However, guidelines set forth by the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) state that uncorrelated techniques are required if two chromatographic separation techniques are to be used. Therefore, if UHPLC is to be used, a combination of UHPLC and GC are required to satisfy SWGDRUG guidelines and provide resolution of all 23 cannabinoids. Principal component analysis (PCA) of retention times was used to evaluate similarities and differences among each chromatography method. PCA is a method that can determine the similarities and differences between samples by clustering similar samples together based on their LC or GC retention times. Little correlation was found between GC and LC data, whereas LC methods were found to be more closely correlated to one another. This is not surprising, due to distinct differences in the chromatographic methods of LC vs GC.

Of the JWH-018 isomers, i.e. uncontrolled cannabinoids, widespread overlap was observed for each LC column. PCA showed the lowest amount of correlation between GC and the Phenyl Hexyl LC results, indicating the possibility of a viable LC/GC method using a Phenyl Hexyl LC column. GC-MS analysis also resulted in overlap among isomers, but the isomers could be identified by their individual mass spectra.
According to SWGDRUG guidelines, a MS method producing only molecular mass information, i.e. no fragmentation, does not satisfy Category A requirements. Fragmentation provides additional information that may provide more discriminatory analysis and aid in identification. UHPLC-MS methods were then modified to induce fragmentation. The authors examined exit voltages between 90V (where compounds are not fragmented) and 400V (where extensive fragmentation was observed for each compounds), to determine the optimal fragmentation voltage. 150V was determined to produce some discriminatory fragmentation for the JWH-018 isomers, which share a common molecular mass (i.e. weight of the unfragmented molecule). Most controlled cannabinoids did not share common molecular masses. However, JWH-019 and JWH-122 are controlled cannabinoids that are isomers of one another and therefore share a common molecular mass. These were chromatographically separated by all methods, but could also be distinguished based on the fragmentation patterns observed. Upon comparison of GC-MS and UHPLC-MS with fragmentation, GC-MS was still found to be more discriminatory in identifying compounds.

The authors conclude that, even with fragmentation during UHPLC-TOFMS analysis, GC-MS produces more discriminatory results for synthetic cannabinoids, based on MS data. However, the authors indicate that a combination of UHPLC and GC techniques may significantly improve the ability to identify synthetic cannabinoids based on retention times. The authors suggest that, due to its more rapid analysis in comparison to GC-MS, UHPLC-TOFMS could be used as a screening method. As a screening method, UHPLC-TOFMS can still provide high accuracy mass data, which can be used to determine a molecular formula. Confirmatory analysis can then be performed using GC-MS, if deemed necessary.

Scientific Highlights

  • The authors suggest that UHPLC and GC may be combined to produce more discriminatory retention time data, although the method has not yet been examined.
  • UHPLC-TOFMS was determined to produce less discriminatory results than GC-MS, even with inducing fragmentation.
  • UHPLC-TOFMS provides more rapid analysis compared to GC-MS, and may serve as a screening method prior to confirmatory analysis by GC-MS.


Synthetic cannabinoids often possess similar structures, and several isomers may exist for a particular molecular formula. Investigators’ goals are to analyze synthetic cannabinoid samples efficiently and to provide accurate determinations of the compounds, since some compounds are scheduled while others are not.

Potential Conclusions

  • UHPLC-TOFMS may provide a screening technique for synthetic cannabinoid analysis, which may be followed by confirmatory analysis by GC-MS.
  • • Further research to enhance chromatographic separation of synthetic cannabinoids is required.