“Flip-Flopping” Forensic Analysis

You recently published a paper describing the effectiveness of “flip-flop” chromatography using silica hydride stationary phases, combined with tandem photodiode array UV and single quadrupole mass spectrometry (MS) detection, for general drug screening (1). What is flip-flop chromatography, and what specific advantages does it offer in terms of analysis time and ease of use compared to traditional two-column methods for drug screening?

Flip-flop chromatography is a technique where a multi-modal stationary phase can be used on the same sample for different separation mechanisms, such as reversed-phase liquid chromatography (RPLC) and normal-phase liquid chromatography (NPLC), or combinations of both techniques, without changing the contents of the solvent reservoirs. This technique, for which specificity of identification is significantly increased, has several advantages over the use of the conventional two-column approach in terms of analysis time and simplicity for drug analysis.

A two-column approach uses a classical C18 column in the RPLC mode and a classical silica column in the hydrophilic interaction chromatographic (HILIC) mode. For high performance liquid chromatography (HPLC) or ultrahigh-pressure liquid chromatography (UHPLC) systems with a single-column manager, switching chromatographic modes would involve changing columns. For both single-column and multi-column managers, the use of HILIC involves long equilibrium times. Using a conventional RPLC column in the reversed-phase mode for the general analysis screening for drugs of abuse requires gradient analysis, which includes extra time for equilibration to starting conditions.

For the simultaneous separation of various classes of drugs of abuse, HILIC does not allow for the retention of non-charged analytes such as cannabinoids and synthetic cannabinoids . In contrast, the use of a silica hydride stationary phase allows for relatively uncorrelated separations of seized drugs involving both the RPLC and aqueous normal phase (ANP) modes with rapid equilibration times because of a lack of a retained water layer on the stationary phase. Another advantage of using a silica hydride phase, such as cholesterol, is that it allows for relatively rapid uncorrelated separations using two isocratic separations.

Can you elaborate on the limitations or challenges encountered when implementing silica hydride stationary phases in flip-flop chromatography?

Although the silica hydride stationary phases we investigated undergo flip-flop chromatography, the challenge is finding a phase that exhibits good overall retention under both RPLC and ANP conditions. During our work, we investigated various stationary phases to find which silica hydride column was best suited for the simultaneous analysis of seized drugs (1).

Were there any particular classes of drugs or compounds that posed difficulties in separation, and how did the method overcome these challenges?

The separation of positional isomers for novel psychoactive compounds (NPS), such as synthetic cathinones, is difficult using a single chromatographic system. Using flip-flop chromatography with a silica hydride silica C column employing both the RPLC and ANP modes allowed for the separation of eight positional isomers of pentedrone (2).

How adaptable is this flip-flop chromatography method for routine analysis in forensic laboratories, especially considering sample throughput and repeatability?

Any HPLC or UHPLC system is easily adaptable for routine analysis employing flip-flop chromatography. All that is required is a single column manager and a solvent manager with at least two reservoirs. Sample throughput would be increased, especially for general drug screening, as mentioned earlier, and for basic NPS drugs, of which many analogues of similar structure and positional isomers exist, and which are difficult to separate using a single chromatographic system. The remedy using classical liquid chromatographic techniques would be to use a dual-column approach employing both RPLC and HILIC stationary phases. As mentioned earlier, this approach is time-consuming. Alternatively, flip-flop chromatography using a single column and the same solvent reservoirs represents a faster alternative that can improve sample throughput. Flip-flop chromatography using both RPLC and ANP separations is highly repeatable, similar to that which is obtainable using classical techniques, with run-to run repeatability ≤0.6%.

In another research, you applied gas chromatography–vacuum ultraviolet spectroscopy (GC–VUV) and derivative analysis to analyze cannabinoid isomers, diastereomers, and homologs (3). What were your findings?

For 15 JWH-018 synthetic cannabinoid positional isomers and six synthetic cannabinoid diastereomer derivatives, VUV spectroscopy offered significantly greater specificity in identification than native VUV spectroscopy, particularly using principal component analysis (PCA). When employing second derivative VUV spectroscopy, 14 out of 15 JWH-018 positional isomers were found to be distinguishable by library searches, while 13 out of 15 were discriminated using PCA. Principal component analysis of the first derivative of VUV spectra for synthetic cannabinoid diastereomers provided greater specificity for four out of the six diastereomers using PCA. Derivative VUV spectroscopy did not improve specificity for homologs.

How does derivative spectral processing enhance the specificity of GC–VUV in differentiating isomeric compounds?

Derivative spectral processing provides greater specificity in differentiating isomeric compounds by providing further spectral information, such as minima, maxima, and saddles, out of otherwise similarly shaped spectra.

What are the advantages of GC–VUV over the traditional GC–EI-MS method when analyzing synthetic cannabinoids?

One advantage of employing GC–VUV over GC–EI-MS relates to coeluting peaks. For GC–VUV, software exists for de-convoluting VUV spectra using a spectral fitting algorithm based on the Beer-Lambert Law to model absorbance as a linear combination of reference spectra. The de-convolution of EI-MS spectra is more difficult, requiring chemometric tools that may not be available for instrumental software packages. Another advantage pertains to differentiating positional isomers differing in the orientation of the naphthyl group (1’ vs. 2’). These analytes are easily differentiated using VUV spectroscopy, while EI-MS spectrometry discrimination can be difficult.

Why is the differentiation of synthetic cannabinoid isomers important in forensic chemistry?

The differentiation of positional isomers and diastereomers, such as synthetic cannabinoids, is important for legal purposes. A forensic chemist must identify the correct isomer present in a seized drug. Depending on the legal system, certain isomers are controlled substances, while other isomers are not controlled. Misidentification could lead to a defendant being charged erroneously for possession or sale of a controlled substance. In addition, during a drug trial, the forensic chemist could be impeached as an expert witness if the drug is misidentified.

How could the methodologies discussed improve current forensic drug testing protocols?

The “gold standard” for analyzing mixtures containing seized drugs is GC–EI-MS. This technique is problematic for novel psychoactive substances, particularly for positional isomers and diastereomers. Not only is there the possibility of similar retention times, but certain drug classes also lack molecular ions and/or give similar fragmentation patterns. Therefore, complementary techniques that would increase the specificity of analysis, such as “flip-flop chromatography” and GC–VUV, are desirable.

References

(1) Appia-Kusi, V.; Lurie, I. S. Utility of “Flip-flop” Chromatography Employing Silica Hydride Stationary Phases with Simultaneous Photodiode Array Ultraviolet and Single Quadrupole Mass Detection for the Analysis of Seized Drugs. J. Chromatogr. A 2023, 1707, 464294. DOI: 10.1016/j.chroma.2023.464294

(2) Ploumen, C.; Marginean, I.; Lurie, I. S. The Utility of Silica Hydride-based Stationary Phases for Dual-mode Ultra High Performance Liquid Chromatography Separation of Synthetic Cathinone Positional Isomers. J. Sep. Sci., 2020, 43, 3449–3457. DOI: 10.1002/jssc.202000599

(3) Dombrowski, A.; Le, D.; Lurie, I. S. GC-VUV Spectroscopy of Synthetic Cannabinoid Isomers, Diastereomers and Homologs: Increasing Differentiation by Derivative Spectral Processing. Forensic Chem. 2025, 42, 100635. DOI: 10.1016/j.forc.2024.100635

Ira Lurie is a professional lecturer at George Washington University in the USA, where he has previously served as both an adjunct professor and a research professor. His research interests include the investigation of novel separation and detection techniques for drug analysis. Lurie retired from the U.S. Drug Enforcement Administration after 40 years of service, where it pioneered advanced liquid phase separation methods for seized drug analysis. He holds a Ph.D. in chemistry from the University of Amsterdam, under the guidance of Peter Schoenmakers, and has authored over 90 peer-reviewed articles and book chapters and co-edited a book entitled High-Performance Liquid Chromatography in Forensic Chemistry. Lurie is a fellow of the American Academy of Forensic Sciences (AAFS), for whom he was the winner of the 2015 Paul L. Kirk Award, the highest form of recognition from the Criminalistics Section of the AAFS.