The Evolving Role of SFC in Forensic Analysis

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Ira Lurie of The George Washington University, Washington D.C., USA, describes the role of supercritical fluid chromatography (SFC) in forensic analysis and why the technique has evolved to the point it should become a recognized technique for the analysis of seized drugs.

Q. How is ultrahigh-performance supercritical fluid chromatography (UHPSFC) used for the analysis of seized drugs? 

A:

Seized drugs consist of substances that have potential for abuse and are either prohibited or controlled by federal, state, or local statutes. These substances represent different drug classes (that is, narcotic analgesics, stimulants, depressants, hallucinogens, and anabolic steroids). Recently there has been an explosion in the use of new synthetic psychoactive designer drugs created to circumvent the controlled substances laws.   Modification in the existing controlled substances results in structural analogues, structural homologues, positional isomers, and stereoisomers. Emerging drugs such as synthetic cannabinoids and synthetic cathinones (“bath salts”) are easily available over the Internet and at local “head shops”. For the analysis of these solutes the analytical methodology should have the ability to distinguish between the similar solutes present in the various classes of designer drugs.    Ultrahigh-performance supercritical fluid chromatography (UHPSFC), which typically gives normal phase separations with sub‑3-µm stationary phases, is particularly well suited for the separation of similar compounds such as positional isomers and stereoisomers. Compared to normal phase liquid chromatographic techniques, equilibration is extremely fast, and excellent reproducibility is obtained. SFC is also advantageous since it uses mainly nontoxic and inexpensive carbon dioxide as the predominant component in the mobile phase. The use of UHPSFC would greatly assist the criminal justice system in the adjudication of emerging drug cases. In particular, it would aid in determining which - if any - controlled substances are present in the seized drug.  

Q. How has SFC evolved and has the technique tackled some of the problems associated with it in the past? 

A:

Instrumentation and column technology, especially for packed column SFC, has significantly improved since a flurry of activity in the 1980s. Sophisticated commercial instrumentation allowing independent flow control under both pressure and composition gradients came out in 1992, which increased the applicability of the aforementioned technique (1). Subsequent problems with back-pressure regulation, consistent flow rates, modifier addition, sample injection, automation, detector issues, and stationary phases have been addressed (1). SFC instrumentation integrates the design of the pumping system, modifier module, post-column nozzle, separator, detector, and associated universal software for the generation of a more robust, reliable technique for packed column SFC–mass spectrometry (MS) analysis (2). Most recently instrumentation has been introduced that allows significantly improved SFC separations (UHPSFC) by allowing the incorporation of sub-3-µm particle columns and providing significantly increased robustness, reduced sample volume, and lower limits of detection (3,4).  

Q. You recently presented a lecture at SFC 2015 on SFC for the analysis of synthetic cannabinoids. Could you talk a little about this research? 
What can SFC offer for the separation of these solutes that other techniques cannot?

A:

As I discussed at SFC 2015 (5) and in a recently published article (6), four state of the art 1.7-µm particle silica-based achiral stationary phases containing either 2-picolylamine, diethylamine, high density diol, or 1-aminoanthracene, one state of the art 2.5-µm particle amylose‑based chiral stationary phase containing 

tris

(3.5‑dimethylphenylcarbamate), and two state of the art 2.5-µm cellulose‑based chiral stationary phases containing either

tris

(3.5‑dimethylphenylcarbamate) or

tris

(3-chloro-4‑methylphenylcarbamate) were used with both a photodiode array (PDA) UV detector and a mass detector for the separation of 22 controlled synthetic cannabinoids and JWH-018 and nine mostly noncontrolled positional isomers. Of particular interest was the separation of JWH-018 and its positional isomers, which are much more difficult to distinguish between by gas chromatography (GC) and ultrahigh-performance liquid chromatography (UHPLC) retention time and MS spectrum (electrospray ionization [ESI] and electron ionization [EI]) (7). For these solutes the best separation was obtained with a 2.5-µm cellulose‑based chiral stationary phases containing

tris

(3.5- dimethylphenylcarbamate) and an isopropanol modifier. Near baseline separation of all 10 positional isomers related to JWH-018 was obtained.   In comparison, at best three out of 10 and four out of 10 of the above positional isomers were separated by UHPLC and GC, respectively (7). For the controlled substances with the same conditions as employed for optimized separation of JWH-018 and its positional isomers, 11 out of 22 solutes were resolved with a resolution ≥1 in a 10 min gradient run. By comparison UHPLC resolved 15 out of 22 in a 13 min gradient run while GC resolved 18 out of 22 in a 24 min temperature programmed run (7). HU 210 and its diastereoisomer HU 211, which have not been reported to be separated by LC (8) or GC (9), were well resolved using UHPSFC. In addition, without derivatization, the enantiomers of CP47, 497, its diastereomer 3-epi CP47, 497, its homologue CP47, 497 C8, and its diastereomer 3-epi CP47, 497 C8 were all well resolved in under 4.8 min. This separation was not possible without derivatization using the achiral stationary phase routinely used by UHPLC or GC. The orthogonality of UHPSFC, GC, and UHPLC for the analysis of synthetic cannabinoids was demonstrated using principal component analysis. 
    Although there was extensive overlap of the 22 controlled substances by UHPSFC with UV detection, extracted ion chromatograms allowed the deconvolution of all compounds that have different molecular masses. In this vein it was of interest to compare the performance of both the PDA UV and mass detector for the analysis of synthetic cannabinoids. For UV detection at 215 nm linearity was obtained over two orders of magnitude with 0.9996≥R

²

≥1.0000, while for MS detection for extracted ion chromatograms linearity was achieved with well over one order of magnitude range for most solutes with 0.9936≥R

²

≥0.9998. For most analytes MS detection offered lower limits of detection ranging from a half to an order of magnitude. At three concentrations representing low, moderate, and high concentration ranges, comparable peak area precision was obtained by both detectors (peak area UV 0.18≥% RSD≥2.92 and peak area MS 0.17≥% RSD≥2.97).  

Q. How easy is it to implement SFC in your work? 

A:

I believe it would be very easy to implement UHPSFC for forensic drug analysis. Its instrumental footprint is 
similar to a binary HPLC system, with 
one of the solvents being replaced with carbon dioxide arising from a carbon 
dioxide tank. In our laboratory we employ liquefied carbon dioxide assessed with a dip tube, and find this solvent source lasts for over a month. The instrumentation uses larger pump heads to facilitate the pumping of liquid carbon dioxide. The other significant difference is that makeup solvent is added to allow the column effluent to be MS-compatible. This requires a splitter after a PDA UV detector (split between MS and back pressure regulator), and an additional splitter for the addition of MS-compatible effluent by an auxiliary solvent delivery pump. In spite of the additional complexity, for the most part we have experienced minimal instrumental issues. In regard to the technique itself, it can be easily adopted by anyone with familiarity with HPLC. In essence the operating principle of UHPSFC is similar to conventional normal phase 
HPLC with the base solvent hexane or cyclohexane being replaced with carbon dioxide.   

Q. Why is SFC not a recognized technique for the analysis of seized drugs?

  A: SFC has experienced minimal use for the analysis of seized drugs since its advent commercially in the 1970s. This is largely because of the instrumental issues mentioned earlier. Our experience with capillary SFC in the mid-1980s for the analysis of seized drugs was very negative. Although the instrument vendor promised us more GC-like separations in terms of peak efficiency, HPLC-like separations were still obtained. Supercritical extraction as an on-line sample preparation technique was also explored with little, if any, success. After about three years of investigating applications for seized drugs, the project was terminated.   An additional reason for the lack of interest in SFC for the analysis of seized drugs was the poor chromatographic performance obtained using packed column SFC for strong bases, such as amphetamine and methamphetamine, because of secondary interactions with the residual silanols on the stationary phase (10). Although successful chromatography was obtained after derivatization, the time and effort required by this additional step would appear to have defeated any advantages over GC analysis. Dispass (11) demonstrated that good peak shapes could be obtained for power compounds (including primary and secondary amines) using SFC, without the use of derivatization and with a proper choice of stationary phase, modifier, and additive. The stigma attached to SFC still exists today. I experienced this reluctance to adapt SFC first-hand, at a joint National Science Foundation sponsored conference between academia, government, and industry, where one of the participants pointed to the old issues that existed for SFC. It is clear from our research on synthetic cannabinoids and recent work on bath salts that UHPSFC is an easy to operate and rugged technique, as evidenced by near turnkey operation, minimal instrumental downtime, excellent run-to-run retention time precision (synthetic cannabinoids 0.01≥% RSD≥0.32; bath salts 0.01≥% RSD≥0.55), excellent day-to-day (one week period) retention time precision (synthetic cannabinoids 0.12≥% RSD≥0.50), and good day-to-day (one week period) relative retention time precision (bath salts 0.53 ≥ %RSD ≥ 1.52).    At the 2016 Forensic Science R&D Symposium hosted by the National Institute of Justice on 23 February at the Annual American Academy of Forensic Science Meeting in Las Vegas, I presented a lecture entitled “Should Forensic Laboratories Embrace Ultrahigh-performance Supercritical Fluid Chromatography as a Separation Technique for the Analysis of Seized Drugs?” My conclusion based on the techniques relatively high resolving power, excellent repeatability, relatively low limits of detection, and orthogonality to GC and UHPLC is that UHPSFC belongs in the compendium of techniques for the analysis of seized drugs.   

Q. What other projects are you working on using SFC?

A:

As I mentioned earlier, I am also working on the UHPSFC analysis of bath salts. My goal, identical to my aim with synthetic cannabinoids, is to determine the utility of UHPSFC for the analysis of seized drugs. In this vein, UHPSFC separations of a mixture of controlled bath salts, as well as the separation of positional isomers of several of these solutes will be compared with GC and UHPLC.   

Acknowledgements

  This project was supported by Award No. 2014-R2-CX-K009, awarded by the National Institute of Justice, Office of Justice Programs, and U.S. Department of Justice. The opinions, findings, and conclusions or recommendations expressed in the lecture are those of the author(s) and do not necessarily reflect those of the Department of Justice.  The author would like to acknowledge the contribution made by Stephanie Breitenbach, a graduate student in forensic chemistry, who performed most of the hands on work. In addition I would like to acknowledge Professor Walter Rowe from The George Washington University and Professor Bruce McCord from Florida International University for their technical assistance. Finally I am grateful to Waters for the loan of the UHPSFC instrumentation.  
   

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Ira Lurie

received his BA in chemistry from Queens College, his MS in chemistry from Rutgers University, and his Ph.D. in chemistry from University of Amsterdam under the guidance of Professor Peter Schoenmakers. Dr. Lurie served almost 40 years as both a forensic chemist and senior research chemist with the Drug Enforcement Administration, where he last served as the agency’s expert in liquid phase separations. He presently resides at The George Washington University as a Research Professor. He is the author of over 70 publications including several book chapters and a co-edited book entitled HPLC in Forensic Chemistry. Professor Lurie is the winner of the 2015 Paul Kirk award, the highest form of recognition one can receive from the criminalistics section of the American Academy of Forensic Sciences.