Identification of an Unknown Drug CompoundCH 440 Forensic Chemistry

Tim Akers: Department of Chemistry, Northern Michigan University

Background:

In forensic science, analyzing drugs or “suspected” drugs to determine their contents, structure, and potency is an important area in forensics. By knowing this information, it serves as physical proof that with confidence a specific compound is in fact that compound. It is important be able to explain the results from a variety of techniques used. Your findings will be useful in a court of law, and ultimately need to be proved. In this part of forensic chemistry, a vial of 1 mg white powder was received. The goal of this is to perform a variety of presumptive tests, Fourier transform infrared spectroscopy (FTIR), and gas chromatography mass spectroscopy (GC-MS) to determine the molecular formula and structure of the unknown drug compound. In using the results in all of the tests, the certain functional groups and fragments of the molecule should be pieced together to get the final complete structure of the molecule.

Instrumentation and Techniques:

Presumptive Tests:

The first initial tests on the unknown drug upon receiving was to look for what it visually looked like, what the composition of the analyte was, and the color. The next step in determining what kind of drug it is, is to perform presumptive field tests. These tests are similar if not the same as the ones that police officers use in actual drug seizures to initially identify what class of drug it belongs to. By doing this the officer can charge the offender based on the results of the presumptive tests. In this experiment four reagents were used to try to initially identify the drug or class of drugs it belongs to. The Marquis, Mecke, Mandelin, and Simon reagents were tested with controls along with our unknown drug compound. The results were compared to a chart that listed the outcomes for each test for a handful of drugs including amphetamines, psychedelics, and opiates.

The Marquis reagents is a spot test to identify alkaloids and other compounds. The reagent is a mixture of formaldehyde (100 mL) and concentrates sulfuric acid (5 mL). When the drug molecule is exposed to this reagent, it undergoes an electrophilic substitution by protonated formaldehyde. The carbocation reacts with another drug molecule to make a dimer. The dimer is then oxidized, producing the color change. Methanol is used to slow down the reaction so that color change can be viewed more effectively. This reagent is primarily used in ecstasy testing kits and turns a dark purple. The reagent drug mixtures turns are variety of colors based on the components of

the drug.1

The Mecke reagent is a spot test to identify alkaloids. The reagent is a mixture of selenous acid and concentrated sulfuric acid. Morphine for example, when reacted with the reagent goes through a Hussemann-reaction that rearranges morphine into apomorphine, which is then oxidized in the presence of selenous acid to o-quinone. The resulting product has a blue or green color.2 This reagent

also reacts differently to many other drug compounds. This test is popular because it tests for derivatives of MDMA and other hallucinogenic drugs.

The Mandelin reagent is similar to the reagents mention above in that is also is a spot test to identify alkaloids. This reagent is a mixture of ammonium metavanadate and concentrated sulfuric acid.1

Information about the reaction and chemistry it involves could not be found. The Mandelin reagent is used for the detection of ketamine, PMA, MDMA, and other drugs yielding different results.

The last reagent used in the presumptive tests was the Simon reagent. This reagent is also a spot test to identify alkaloids. This reagent is different from the other in that it reacts with secondary amines like MDMA and amphetamines. It contains sodium nitroprusside, sodium carbonate, and acetaldehyde. When it is reacted with the drug, reaction produces an enamine that reacts with sodium nitroprusside to get an imine. The immonium salt is hydrolyzed forming the Simon-Awe complex. When hydrolyzed, the test yields different shades of blue and is selective to certain drugs.2

Testing the unknown drug with all four of these reagents can narrow down the options of what the name could be. If the drug is in fact not on the reference sheet, it may be helpful in determining what class or category it belongs to. It is also beneficial to use all the reagents to see if there are noticeable changes with a control and the unknown compound and to see how well some reagents are and are not working.

FTIR/GC-MS:

The next part of the testing after the presumptive tests, was to perform Fourier transform infrared spectroscopy. In IR spectroscopy, IR radiation is passed through a sample. The sample absorbs some of the infrared radiation while some is transmitted through the sample. This projects a spectrum that represents molecular fingerprint of the sample. FTIR determines the frequencies of the absorbed radiated of the constantly vibrating bonds in order to identify bonds and certain functional groups contained in the molecule. The goal of FTIR is to be able to gather results to identify an unknown

compound, to test the quality of that sample, or determine how many components are in a sample.3 IR spec devices contain a device called a interferometer that produces signals that have infrared frequencies. First, a source of infrared energy is emitted as a beam that enters the interferometer where the beam gets encoded. The beam then

enters a compartment where the sample is held. The beam is either passes through the sample or gets reflected off of it. The frequencies that pass through the sample get absorbed and enter the detector. The detector measures the signal given off. The signal is sent to the computer that makes it possible to visualize and interpret the spectrum.3 The Fourier transform makes it possible for the frequencies of the waves to be determined from the repeating cycle. The sample absorbrs frequencies that are characteristic to their structure.

Gas chromatography mass spectrometry was the another instramental test done to further analyze the different parts of the drug compound to correctly identify it. Gas chromatography is used to separate samples into individual components using a capillary column controlled by temperature.

Smaller volatile compounds that have a lower boiling point travel down the column faster than larger molecules with higher boiing points.4 Mass spectrometry is used to identify the various component by their mass on a spectrum. Every compound measured results in a different spectrum that is unique to that molecule. GCMS analysis can measure solid, liquid, and gases. For this experiement, a liquid was converted to its gas phase to be used for this analysis. The ions are separated by their mass-to-charge

ratios. The compounds are bombarded with electrons that breaks apart the samples into small and large fragments that can be visualized on the sprectum. The sample is injected into a stationary gas that gets carried down into the column where it reacts with a stationary phase. The components in the sample that react with the phases the fastest will elute first followed by the other components. A quadrapole

is used to focuss the fragments through a slit in the detector for certain fragment sizes. The quadrapole cycles different mass to charge fragments until a wide range is covered.. The retention time of a component is the time when the sample gets injected until it is eluded4. This time is where your peaks should show up on the spectrum.

Electron impact ionization (EI) is one of the methods that was used in this experiment. The sample is broked apart from the interaction of electrons. Electrostatic repulsion fuels the loss of electrons, forming positive stable ions. Chemical impact inonization (CI) was also used in this experiement. This method is similar to EI but uses a higher pressure and allows for a more accurate evaluation of molecular mass and certain isotopes. On the spectrum both methods produce a molecular ion peak which is representive of the entire analyte. Daughter fragment peaks are also formed which are the charged fragments of sample that was broken. The base peak of the spectrum is the peak that represents the most stable daughter fragment. Isotope peaks are less abundant surround daughter peaking representing isotopes of certain atoms.

Lab Log:

The first lab period a 1 mg white powdering sample was received. The goal in the first lab period was perform Marquis, Mecke, Mandelin, and Simon reagent presumptive tests to identify what

substances the sample might be similar to, and also to test the reliabilty on these tests. For each reagent, the reagent collected by itself in one pipet, a sugar sample with the reagent in the next, and the unknown sample with the reagent in the last. In order to perfrom these tests, the end of the pasteur pipet was dipped into the different control and unknown samples to force sample into the end of the pipet. The pipet was then placed on a drop of the reagent that naturally drawed up the reagent. The pipet was left to sit and the the results were analyzed and photographed. A downside to these presumptive tests is that the results are not permanent, so the results have to be analyzed right away. Photographs were taken

of the results but doesn’t show a fully accurate depiction of the results as would in person. Some of the tests appeared to to work better than others, yielding positive results for compounds, while some didn’t at all. Overall, the reagent tests showed results but lack reliability.

The next tests done in the following lab period was the Fourier transform infrared spectroscopy. The first method that was used to obtain a spectrum was cracking the end of a pasteur pipet to get a sharp pointy end. This made it possible to scoop some of the analyte sample onto the small zinc crystal. A control of methanol was first used to test the accuracy of the machine. This was compared to another methanol spectra from a different source to make sure there were know frequencies on the spectrum that should’t have been there. The spectrum was obtained by just putting the pure sample on the crystal. The first trial did not produce decent results. Later in the day, another trial was dne by scooping some of the analyte onto the crystal then dropping methanol over the sample. After the methanol was dissolved, the IR was taken with a lot cleaner more accurate results. A control was also done of just methanol prior to obtaining these results.

The next two lab periods were dedicated to performing the GC-MS analysis using both electron and chemical impact ionization. A sample was prepared by dissolving analyte packed in a pasteur pipet dissolved in ~400 microliters of methanol. This liquid in a vial was violatolized to use for GC-MS. EI was performed first. For the gas chromatography the starting temp was changed to 100°C and the final temp to 300°C. Other settings like the column hold time, column flow rate, and injector temp where all adjusted. The MS settings such as the interface temp, solvent cut time, start and ending times, and atomic mass unit levels were all adjusted appropriatly to result in the best spectrum. The class batch of nine samples plus a methanol control and a phenethylamine sample were tested. The resulting EI spectrum showed a molecular ion peak along with a base peak and many daughter peaks. The same setting were kept for the chemical impact ionization except the methane levels were adjusted for a proper pressure. The chemical impact spectrum resulted in a molecular ion peak, with a base peak and some daughter peaks.

Results:

Below are the results from all of the tests perfromed on the unknown drug compound. By analyzing each test, a formula and structure was generated based on the evidence pulled from all data. Some of the data lead to a conclusion that ultimately got ruled out due to structural components.

a) Marquis reagent b) Mecke reagent

c) Madelin reagent d) Simon reagent

Figure 1 Presumptive tests performed with a control, on sugar, and with the unknown drug. a) represents the Marquis reagent. There was no apparent change with X sample (by itself), the salt yieleded a dark yellow color that did not match the chart, and the unknown drug sample turned a bright orange color that matched drugs like amphetamines or 2C-T compounds. b) The Mecke reagent again resulted in a different hue than what was listed on the chart for sugar. The unknown sample turned a dark purple/black color which matched up with 2C-T-7 on the chart. c) Madelin reagent reacting with sugar produced a misleading result while the unknown sample produced a yellow/purple color that did not match any compounds on the refrence list. d) Simon reagent was perfromed without controls because there was a lack of time. The unknown sample did not produce any change resulting in a negative result for this reagent.

Figure 2 The FTIR spectrum from the methanol control. Perfromed by dropping methanol on the crystal, letting it evaporate, then taking the IR readings. Sp3 C-H stretches are shown in ~3,200 wavenumber range. The O-H stretch is the long and narrow signal ~1,000.

Figure 3 The FTIR spectrum from the unknown drug compound. Sp2 C-H stretches on the benzene ring are visible in the ~3,000 range. Secondary amine N-H stretch is visible in the ~1,500 range. The weak bands around ~1,200 represent the S-H bond.The two strong signals from ~1,000-1,200 represent the C-O bonds of the methoxy group. This trial sample was placed on crystal, dropped with methanol, and results taken once methanol dissolved.

Figure 4 The methanol control from The electron and chemical impact ionization of GC-MS

Figure 5 The EI stpectrum from GC-MS of unknown drug sample. A molecular ion peak is visible at 255 m/z, a base peak at 212 m/z, and other daughter fragments at 197, 183, 181, and 153 m/z.

Figure 6 Refrence amphetamine EI spectrum to compare with EI of unknow drug sample.

Figure 7 The spectrum from CI. A m+1 peak is visible at 256 m/z, with strong base peak at 239 m/z, and daughter peaks at 212 and 151 m/z.

Discussion:

First off, the results from the presumptive tests yielded some helpful hints in what the unknown compound might be. Each reagent was ran with two controls. The reagent reacting with itself (air), and mixing the reagent with sugar to see if it showed the results that can be matched to those on the chart. According to figure 1a the first reagent that was tested was the Marquis. In drawing the up the reagent in the pipet and waiting the proper time there was no reaction by itself. Mixing it with the sugar produced a yellow color that did not match the sugar refrence. The unknown compound with the

reagent resulted in a bright oragne color in relation to ampetamine and 2C compounds. This fits with the conclusion that my sample is a derivative of 2C-T-7. It has the same substituent that an amphetamine has with a methyl and amine group. It acts like 2C molecules because they both have sulfur and methoxy substituents. The Mecke reagent in figure 2b produced no change for the unreacted control. The sugar control did not match with the refrence picture of what it should have looked like. The unknown drug sample resulted in a deep purple/black color that closely resembles the refrence from 2C-T-7. This reagent futher supports the identity of unknown compound. The Mandelin reagent in figure 2c did not show any consistent results. The sugar and the unknown sample both revealed colors that did not match up with any compound on the refrence chart. The last reagent used was the Simon in figure 2d. There were no controls performed with this test due to lack of time. After reacting this reagant with my unknown sample, it resulted in no visible changes. This could be due to a failure of performance of the reagent. The Simon reagent is especially specific for testing 2° amines. The unknown drug has a 2° amine that failed to react in this test. With all the results from the presumtive tests, the most useful reagents were the Marquis and Mecke reagents. These two tests resulted in positive matching results that represent the structural similarities between the drug compounds. Although these tests gave positive results, all of the reagents gave unaccurate results for the control, and two of the reagents (Mandelin and Simon) yielded no conclusion in their tests. Overall presumtive tests can show some results, but lack in reliability and accuracy.

The FTIR analysis is a test that further gave evidence to back up the claim that the molecule is a 2C-T-7 derivative. A methanol control (figure 2) was tested first to make sure that the instrament was working properly. The signals on the control sprectrum corresponded to literature signals of methanol. The first trial of the unknown sample on the IR was taken by placing solid sample on the crystal and running the test. The resulting spectrum (not pictured) gave confusing and inconclusive results. A second trial was done by placing solid sample on the crystal, then dropping methanol over so the sample gets evenly distributed over the crystal. The results were taken after the methanol had dissolved. According to figure 3, cleaner and accurate results depicted functional groups that correspond to the drug compound. The first weak signals appeared around ~3,000 wavenumbers that represented the sp2 C-H stretches on the benzene. The next medium signals represented a secondary amine N-H stretch visible in the ~1,500 range. The weak bands around ~1,200 represent the S-H bond.The two strong signals from ~1,000-1,200 represent the C-O bonds of the methoxy group. All of the signals that are shown on the IR spectrum can be linked to structural componetnts found in the drug molecule. The FTIR spectrum evidence further supports that the drug compound is a derivative of 2C-T-7, sharing the same molecular formula.

The GC-MS tests is where the bulk of the evidence is to support the molecular formula and structure of the drug sample. A methanol control was first tested (figure 4) to see if the settings in the EI and CI would result in readable tests. The first GC-MS test that was done was the electrical impact ionization. Figure 5 shows that spectra obtained from this trial. A molecular ion peak is visible at 255 m/z, a base peak at 212 m/z, and other daughter fragments at 197, 183, 181, and 153 m/z. The first visible fragmentation on this spectra is from 255 m/z to 212 m/z. This represents a sigma bond cleavage resulting in a CH3-CH-NH4 fragment. The difference of 255 and 212 is 43 m/z. The fragment mass is 44 amu. An explanation for this proton difference is that before this fragment was cleaved from the molecule, the central carbon was deprotonated before the fragmentation leaving the amu at 43. It is certain that this molecule cleaves in this pattern because a fragmentation of 44 amu was visible on an EI spectrum of amphetamine. The drug compound and amphetamine have the exact same substituent containing and amine and methyl group. This supports the method of fragmentation of this sigma bond. Another fragment pattern visible is a CH2-CH3 group. A difference of 212 m/z and 183 m/z is 29 amu.

This fragmentation represents the CH2-CH3 sigma bond cleavage from the sulfur. The spectrum difference of 212 to 181 m/z is 31 m/z. This signal on the spectrum is weak but believed to be the

cleavage of one of the methoxy groups. Prior to the finding of this structure, there was evidence to support the leaving of a isoproply group that was connected to the amine. This would also show a difference of 43 amu. After adding subsituents on the ring to fufill the evidence and molecular weight, it was realized that the isopropyl group would not fit with the molecule, relieving that structure. The fragmentations on the spectrum make the most sense in puzzling together the structure of the compound. All

of the fragments and the molecule as a whole adds up to 255 amu which is consistent with the molecular ion peak. That molecular mass matches the believed molecular formula of C13H21NO2S from the drug compound. The results from the CI spectra only showed a few, but useful results. The main peak of the spectrum showed a m+1 peak at 256 which corresponds to the molecular mass of the compound. A difference from 255 m/z to 239 m/z is 16 m/z. this fragmentation corresponds with the sigma bond cleavage of the NH2. An alternative fragmentation to the one before is a cleavage of the CH3-CH-NH2. That would leave a difference of 43 amu from the signals 255 m/z and 212 m/z. There was no evidence in the CI that there was cleavage of the methoxy groups, or the CH2-CH3 group. Although the CI yielded only a few results, they can be correlated to possible fragment patterns in the structure of the drug.

Focussing the results from each test performed on the unknown drug compound, it can be said with evidence that the final molecular formula of this compound is C13H21NO2S. Future tests on this compound should strengthen the evidence to support this formula and structure.

Refrences:

Color Test Reagents/Kits for Preliminary Identification of Drugs of Abuse. (2000). National Institute of Standards and Technology. Retrieved February 2, 2016, from https://www.ncjrs.gov/pdffiles1/nij/183258.pdf

George Mason University. (2010). GC-MS Background. Retrieved February 02, 2016, from https://www.gmu.edu/depts/SRIF/tutorial/gcd/gc-ms2.htm

Griffiths, P. R., & A., D. H. (2007). Fourier transform infrared spectrometry. Hoboken, NJ: Wiley-Interscience.

Kovar, K., & Laudszun, M. (1989). Chemistry and Reaction Mechanisms of Rapid Tests for Drugs of Abuse and Precursors Chemicals. Retrieved February 2, 2016, from https://www.unodc.org/pdf/scientific/SCITEC6.pdf