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In the United States, a vast network of forensic labs at the city, county, state, and national levels is taxed with the analysis of record numbers of samples of prohibited substances. Following proper analysis and identification of each substance, conclusive findings must be presented in U.S. courts. For proper criminal proceedings, identification of active ingredient samples and other species, such as adulterants, excipients, and reaction byproducts, must be definite and conclusive. It is imperative that analysis of seized substances be performed and determined accurately.
In order to meet the stringent requirements of the court system, most forensic labs currently follow the recommendations outlined by the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG)1 for the qualitative and quantitative analysis of seized drugs (see www.SWGDRUG.org). For conclusive qualitative analysis, SWGDRUG requires 1) a minimum of two analytical methods to form an acceptable analytical method, and 2) that each method must yield conclusive and definite results (precluding false positive). Today, the new solid phase GC-FTIR hyphenated systems coupling gas chromatography (for separation/detection), and solid phase infrared spectroscopy (for detection), meet both requirements in one reliable and accurate instrument. In fact, the use of highly specific infrared spectroscopy2 reduces the risk of false positive identification by rapidly and accurately providing detailed structural information such as isomeric differences.
Detailed structural information is becoming invaluable for forensic analysts as they are increasingly confronted to new recreational synthetic drugs such as the JWH series with numerous “legal or not” potent analogs. Numerous so-called “research labs” have popped up to manufacture these “soon-to-be banned” analogs and take advantage of the current loopholes in the U.S. legislation on controlled substances. In particular, the interpretation of the term “substantially similar” in section A(i) is not specific enough to give a definite answer as to whether an analog of a controlled substance falls under the federal analog act3 or not, forcing legislators into a potential cat and mouse game with the manufacturers of designer drugs.4,5 In addition, if the manufacturers make sure to indicate that the drug is not for human consumption, then all bets are off.
Increasingly, forensic scientists are forced to deal with this problem by accurately identifying controlled substances and their analogs to support the court system. This application note will provide examples of the use of Direct Deposition GC-FTIR technology in the forensics environment, including demonstration of the Direct Deposition GC-FTIR instrument’s unique ability to deal with large volumes of samples, while highlighting the specificity of the technology by differentiating typical street drug isomers.
Methods of Analysis and Identification of Controlled Substances Categories of Analytical Techniques
The SWGDRUG requires the use of multiple independent identification techniques. They have divided currently-used methods into three categories, with “category A” techniques providing the best discriminating power.
The following are some of the guidelines put forth by SWGDRUG:
Recommendation on Methodology for Controlled Substances Analysis
Analytical identifications that include a “Category A” method require the use of at least one other independent method from category A, B, or C. When a “Category A” technique is not used, then at least three different validated methods must be employed. The combination shall identify the specific drug present and shall preclude a false positive identification. All “Category A” techniques shall have reviewable data. In addition, when hyphenated techniques are considered (e.g. gas chromatography-mass spectrometry, liquid chromatogram-diode array ultraviolet spectroscopy), they will be treated as separate techniques provided that the results from each are used.
The Use of Hyphenated Methods: GC-MS or GC-FTIR
Samples of clandestine lab materials and ethical drugs typically contain multiple ingredients. Examples include residual starting materials, side-reaction products, excipients, and isomeric forms of constituents. In general, highly discriminating analytical techniques require isolation and purification of an analyte to achieve unambiguous identification. Classical methods of sample preparation (and purification) are extremely time consuming steps for small samples, and influence the speed, discrimination, and accuracy of analytical results. Direct linkage of high resolution chromatography and spectroscopy can provide an automated single procedure to fill these requirements. In particular, the combination Mass Spectrometry from “Category A” and Chromatography (gas or liquid) from “Category B” has been widely adopted in the forensic community as a sensitive and rapid automated for analyte components identification. By providing molecular weight of parent ion and fragmentation products, mass spectrometry enables rapid identification of clandestine lab materials. Infrared Spectroscopy can now be successfully substituted for Mass Spectrometry for the analysis of controlled substances (Light Pipe solution, Direct Deposition using cryogenic technology). The substitution of Mass Spectrometry by Infrared Spectroscopy enables an identification based on the absorption of specific infrared frequencies that are characteristic of the structure exposed to the infrared region of the electromagnetic spectrum ( specifically between 400cm-1 to 4000cm-1). Because Infrared Spectroscopy provides a different basis for identification than Mass Spectrometry, these two spectrometric methods are said to be complementary. For example, Infrared absorption band positions and relative intensity provide a unique fingerprint enabling the discrimination of isomeric forms of a molecule (position and stereo) by Infrared Spectroscopy. This discrimination by MS is very limited or requires extensive analysis of fragmentation products (for example by Tandem MS-MS).
Quality Assurance/ Uncertainty
In October 2008, the SWGDRUG made recommendations regarding quality assurance and uncertainty. Uncertainty encompasses limitations of the qualitative methods used to determine the identity of the samples at hand. SWGDRUG considers an understanding of uncertainty to be fundamental to the interpretation and reporting of results and recommends the concept of uncertainty be considered for all analytical results. SWGDRUG Guidelines for qualitative results:
- Relevant limitations of an analytical scheme (e.g. inability to distinguish isomers) should be documented and may need to be included in the report.
- It is expected that an appropriate analytical scheme will result in, effectively, no uncertainty in reported identifications (absence of false positive identification).
The specificity of Infrared Spectroscopy reduces uncertainty in the identification of isomers, which reduces uncertainty in the identification of controlled substances.
Gas Chromatography-Infrared Spectroscopy
Progress in detector and rapid scanning interferometers in the mid-seventies made the hyphenation of gas chromatography to infrared spectroscopy possible. Improvement on the first reduction to practice led to the development of Light Pipe (LP) GC-FTIR that is commonly used in some forensic labs. The typical arrangement for a LP GC-FTIR drives the effluent from a GC column to a heated measurement gas cell called light pipe. At the same time, a modulated infrared beam is passed through this gas cell and captures information of absorbing material through multi-bounce internal reflection. After exiting the LP flow cell, the infrared light is collected by an MCT detector and analyzed by Fourier Transform. The on-the-fly nature of the technology negatively impacts the detection sensitivity of this hyphenation.
Figure 1: Light Pipe System6
Detection limits for this technique can be as low as 1µg for poor infrared absorbers and less volatile compounds. The desire to improve detection limit led to Direct Deposition System GC-FTIR where sample absorbance and/or observation time could be increased to obtain GC-MS like detection. For a typical arrangement of a direct deposition system GC-FTIR, the effluent stream (carrier gas + eluant) exiting the GC column via a heated transfer line, flows through a deposition tip and eluants are deposited as a collimated jet onto a cryogenically cooled sample collection disk. The Infrared transparent disk rotates/translates under the deposit tip to result in a spiral deposit track of samples. As analytes elute from the capillary and onto the cold disk they form a solid phase deposit on the disk. A beam of infrared light from an interferometer passes through the disk along the deposition track, and infrared spectra are collected onto an MCT detector. By laying down a solid phase (i.e. frozen) track of eluant mimicking classical transmission instruments, the Direct Deposition system dramatically improves detection to nanogram level.7 In addition, transitioning from gas phase spectra to solid phase brings a better resolution for the infrared spectra. For example, the absence of rotational motion in the frozen tracks improves infrared spectrum resolution by narrowing absorption bands. This is particularly an advantage where weak characteristics bands are at play and identification of isomers is necessary.7
Figure 2: Direct Deposition using a cryogenically cooled disk GC-FTIR.
Example 1: Clan Lab SX
This example8 illustrates the use of Direct Deposition GC-FTIR in the analysis of materials seized from a clandestine laboratory. It demonstrates the infrared chromatogram generated by the Direct Deposition GC-FTIR and individual spectra selected from various portions of that chromatogram. Components were identified by comparing to a library of Solid Phase Infrared spectra which were prepared from known standards.
Experimental Conditions Summary
Figure 3 shows the infrared chromatogram of peaks generated during the sample elution. The elution times of the four peaks were 2.79 min, 2.97 min, 3.57 min, and 5.9 min.
Figure 3: Infrared chromatogram of a clan lab sample.
The Direct Deposition GC-FTIR collects spectra at 0.3 sec intervals during the run. Figure 4 displays spectra taken from the data set at the peak maxima of the first three elution peaks. The spectra are strong, and possess the fine structural detail typical of solid phase spectra.
Figure 4: IR spectra from three of the clan lab elution peaks.
A library of drug spectra was prepared, using the Direct Deposition GC-FTIR and known standards. The spectra shown in Figure 4. were matched against the prepared library. The peak eluting at 2.79 minutes was identified as methamphetamine (Figure 5). The match was derived from a PLS algorithm common for spectral matching.
Figure 5: Identification of the 2.79 min peak as methamphetamine.
In a similar fashion, the other three peaks were identified by matching their spectra against the drug library. The 2.97 peak was determined to be CMP [1-(1’,4’-cyclohexadienyl)-2-methylaminopropane], a byproduct formed in the synthesis of methamphetamine. Peak 3 was identified as pseudoephedrine and the fourth peak is a hydrocarbon residue. Components are thus identified by two independent methods; their infrared spectra and their chromatographic elution times.
ISOMER Identification: GC-FTIR and GC-MS
While Mass Spectroscopy identification is made based on the molecular weight of the parent ion and its fragmentation pattern, Infrared Spectroscopic identification is based on the unique constellation of absorption bands that the analyte molecule generates. Because of intramolecular resonances within the molecule, seemingly minor chirality differences can affect profound differences in the resultant spectra. The following examples compare the analytical power of infrared spectrometry when dealing with similar compounds which possess the same molecular weight.
The following examples show three cases of similar molecular structures commonly encountered in the forensic laboratory. Samples of such drugs are readily separated into discrete components. Each component can be subjected to spectroscopic examination. Identification of components is made on the basis of chromatographic elution time and spectra of the components. In the three cases shown, pairs of differing analytes have similar mass. GC-mass spectrometry is compared to Direct Deposition GC-FTIR infrared spectroscopy. As in Example 1, identification was based on comparison to condensed-phase library spectra prepared on the DiscovIR using known standards.
The following examples illustrate the ability of Direct Deposition GC-FTIR to readily differentiate between compounds possessing similar or identical molecular weights. The examples are presented in order of increasing molecular similarity.
Example 2: Analysis of Oxymorphone and Dihydrocodeine
These compounds are opioid analgesics of similar structure. Although they have differing molecular composition, they possess almost identical molecular weights. These ethical drugs are produced in various administration forms and both are subject to illicit usage.
Figure 6: Mass spectra of Oxymorphone and Dihydrocodeine.
With respect to Mass Spectrometry, Oxymorphone and Dihydrocodeine both generate large base ions and small secondary ions. Identification based on the base ions is not possible, and the limited secondary ion fragmentation patterns make identification quite uncertain. The infrared spectra of these two compounds, however, show distinctive differences, and permit ready identification, as shown in Figure 7.
Figure 7: Infrared spectra from a chromatograph of a sample of Oxymorphone and of Dihydrocodeine.
The carbonyl function present in the oxymorphone presents a strong absorbance at 1728 cm-1, and the broad hydroxyl peaks in the 3500-3000 cm-1 region are much stronger for the Oxymorphone. Strong functional group bands for shared molecular structure such as the aromatic group are evident. In the 2000-800 cm -1 region, the intramolecular vibrational differences of the two molecules result in different sets of bands. These two structures are readily differentiated and identified by IR spectra.
Example 3: Constitutional Isomers
Methamphetamine is one of the principal products of clandestine labs in various parts of the world. The positional isomer, Phentermine, is produced for use as an appetite suppressant. It is similar to the amphetamines and is classified as a Schedule IV controlled substance under the U.S. controlled substances act. A chromatography separation and deposition provides the following spectra.
Figure 8: Infrared spectra from a chromatogram of a mixture of Methamphetamine and Phentermine.
These two substances have identical molecular composition. Mass Spectroscopy reveals identical parent ions and very minor differences in the fragmentation ions. The structural differences of the isomers yield distinctly different patterns in the IR fingerprint region, as well as the methyl stretches of the methyl groups adjacent to the primary and secondary amines.
Example 4: Diastereomers Ephedrine and Pseudoephedrine
Samples of diastereisomers typically produce similar mass spectra and chromatographic retention times. Ephedrine and pseudoephedrine are both employed in various OTC and prescription therapeutic agents. Either of these materials is also used by clandestine laboratories as a precursor to methamphetamine. As such, the identification of these substances is useful in forensic identification of residuals in methamphetamine preparations and in the analysis of processing equipment and materials.
These diastereisomers possess two chiral centers. The enantiomers with opposite stereochemistry around the chiral centers (1R,2S), (1S,2R) are designated ephedrine. Pseudoephedrine, by contrast, has the same stereochemistry around the chiral carbons (1R,2R), (1S,2S). The molecular diagrams show that these isomers differ only in the R/S configuration at the carbon holding the OH group.
Figure 9: Molecular structure of ephedrine and pseudoephedrine.
It would be problematic to use gas-phase spectra to distinguish these two compounds. Solid-phase infrared spectroscopy, however, produces high resolution spectra that readily distinguish the two steroisomers. Shown in Figure 10 are the spectra of ephedrine and pseudoephedrine. The isomers give rise to intra-molecular resonances that yield distinctly different sets of spectral peaks. In the fingerprint region, we see that three major bands in the 1100-1200 cm-1 are shifted in frequency from one spectrum to the other, as well as the band near 750 cm-1. These features provide ready differentiation of the stereoisomers.
Figure 10: Spectra from a separation of ephedrine and pseudoephedrine.
GC-FTIR coupling infrared spectroscopy to gas chromatography can answer the needs of modern forensic drug analysis laboratories. With the modern sampling capabilities and data management software sold by the major scientific instrument vendors, a GC-FTIR can provide the capability of unattended automated operation, data archiving, and software for identification of samples of seized products. The technology is particularly useful in dealing with isomers and chiral molecules which are often found in recreational drugs. The examples presented here also illustrate the complementary aspects of GC-FTIR and GC-MS for the confirmatory needs of the forensic labs.
- SWDDRUG. http://www.swgdrug.org/Documents/SWGDRUG%20Recommendations.pdf
- United States Pharmacopeia 34. NF 29 Chapter <1097>
- Federal Analog Act 21 U.S.C. § 813
- Case of USA v. Damon S. Forbes et al. (1992) 806 F.Supp. 232
- Case of USA v. Washam (2002) 312 F.3d 926, 930
- C.P. Sherman Hsu, Handbook of Instrumental Techniques for Analytical Chemistry, Prentice Hall, Inc. Frank Settle Editor Chapter 15 page 264
- Le Quéré, J.-L. 2006. Gas Chromatography/Infrared Spectroscopy. Encyclopedia of Analytical Chemistry.
- Spectra Analysis wishes to thank Alabama Department of Forensic Sciences, courtesy of Stephanie M. Fisher and J. Gary Wallace, Mobile Lab in providing the analytical data for the Clan Lab samples and the comparative data for Oxymorphone/Dihydrocodeine.
Frederic Prulliere received a BS and MS in organic chemistry and materials science from the Ecole Supérieure de Chimie et Electronique de Lyon and the University of Houston. He also received a MBA from the Darden School of Business at the University of Virginia in 2007. He worked for Encysive pharmaceuticals as a medicinal chemist from 2003 to 2006. He went on to work for Mettler-Toledo and Thermo Fisher Scientific as a product manager developing particle sizing probes (FBRM and PVM) and Infrared/Raman Spectroscopy handheld Instrumentation (TruScan). In 2011, Frederic Prulliere joined Spectra Analysis Instruments, Inc. where he is the Vice President of Sales and Marketing. He is responsible for the development of LC-IR and GC-IR instrumentation markets.