Drug Analysis

• Instructional Videos for Chemical Color Tests:
  • The Home Scientist – Forensic Presumptive Drug Testing
  • Ohio University Forensic Chemistry Lab – Chemical Color Tests
• Concerns for color tests:  (1) False positives. Some over the counter cold medication or other substances may test positive for illegal substances using presumptive color tests. Because some drugs behave similarly, a confirmatory test is needed. (2) Color tests may only give an indication of a class of drug present. (3) A color test cannot conclusively identify the presence of a substance.
• Case Law: In State v. Carter (2014), the NC Court of Appeals found the trial court abused its discretion by allowing an officer to testify that a narcotics indicator field test kit indicated the presence of cocaine on items of evidence where the State failed to demonstrate the reliability of the kit.
• To reduce the variability in results due to subjective analysis, good laboratory practices include running a positive control for visual comparison with the evidence sample.
• A lab report from the State Crime Lab will only include the name of the test and the resulting color. The Lab does not use a color standard for comparison or a photo or video to record the result.
• Law Enforcement and Corrections Standards and Testing Program – National Institute of Justice provides standards for color test reagents and kits for the preliminary identification of controlled substances. Attorneys can use this publication to look up a color test and see what substances produce which color changes.
• Helpful questions to consider: Was the required quality control check performed? The reagent lasts for only X days – do you know the age of the reagent? How was the resulting color measured – was it compared to a color standard or simply eye-balled? Was the resulting color recorded (photographed)? What precautions were taken to avoid cross-contamination? If performed by a law enforcement officer, what training does he/she have to perform this test?
• Law enforcement officers may use color tests to check for controlled substances while in the field. They typically use a kit that is produced commercially, for example, by NC company Sirchie. Below are the names of some of the color tests that might be used in the kit or in a lab:
  1. Marquis Reagent
  2. Cobalt Thiocyanate Reagent (Scott’s Test)
  3. Duquenois-Levine
  4. Ferric Chloride
  5. Koppanyi
  6. Potassium Permanganate
  7. Ehrlich’s, Van Urk’s, or p-dimethylaminobenzaledhyde (PDMAB)
  8. Froehde’s
  9. Mecke’s
  10. Other tests

Includes techniques (visual examination and microscopic examination) to understand the physical properties and characteristics of substances.

• Visual Examination – for pills, the State Crime Lab identifies a pharmaceutical preparation by physical characteristics and markings and through instrumental analysis of one pill. This means the pill is compared to pictures and descriptions in reference materials such as Micromedex, The Physician’s Desk Reference, The Logo for Tablets and Capsules or websites. Visual examination is to be used for preliminary examination only. In State v. Ward the NC Supreme Court held that visual identification of controlled substances is not reliable enough to be admitted in criminal trials, and that a chemical analysis is required. For further discussion see the various posts on the subject on the NC Criminal Law blog. SWGDRUG allows pharmaceutical indicators as a class B identification of pills. This means that 2 other identification methods are necessary in order to positively identify a pill as a controlled substance.
  • Concerns: Pills produced in clandestine labs rather than in a pharmaceutical lab are common. It may be difficult to distinguish a pharmaceutical preparation from a clandestine lab pill.
• Microscopic examination – using a microscope to examine the characteristics and properties of a substance too small for the naked eye.
  • Polarized light microscopy – contrast-enhancing technique that improves the quality of the image obtained by equipping these microscopes with a polarizer and an analyzer (second polarizer).
  • Microcrystalline test – a substance reacts with a reagent forming an insoluble crystal. The shape of the crystal suggests the type of drug. These tests are rapid and do not require the isolation of the drug prior to testing.
  • Polarized light microscopy with microcrystalline tests can be used to presumptively identify drugs. The following list indicates various reagents that can be exposed to the drug for analysis using the microcrystalline test with microscopy.
    • Heroin and caffeine: mercuric chloride, hydrochloric acid
    • Barbiturates: Wagenaar reagent (cupric sulfate), sodium hydroxide, sulfuric acid
    • Cocaine and phencyclidine (PCP): gold chloride in acetic acid, hydrochloric acid
    • Plant particles from marijuana, hashish: concentrated sodium hydroxide, petroleum ether, chloroform, chloral hydrate
    • Amphetamines and methamphetamines: gold chloride in water, gold chloride in phosphoric acid, sodium hydroxide
  • Concerns: (1) Impurities may cause unusual crystal formation. (2) Check reagents with known standard of drug before performing an actual procedure. (3) Proper storage of reagents.
• Macroscopic examination – examining the properties and characteristics of the substance that are observable by the naked eye.
  • For descriptions and images of illegal drugs, see the following sources:
    • DEA Resource for Parents – provides images of drugs of abuse and information about the effects and legal status of drugs.
• For Marijuana – the State Crime Lab cannot distinguish between hemp and marijuana at this time. Quantitative determination of %THC levels are required by statute. The lab does not have the procedures, instrumentation, and resources to determine the %THC levels in a substance. Private labs do provide this service.

• Characteristics
  • Specific
  • Considered the “gold standard” of drug identification
  • Most definitive and reliable of the confirmatory tests
• How it works
  • GC
    • A sample is injected via a port into the gas chromatograph and converted to gas form (vaporized). The vaporized sample is then transported by helium gas through a long coiling column. The column is then subjected to varying temperatures which results in the separation of compounds based on volatility (how easily a substance can be vaporized). Eventually, all molecules will reach the end of the column where they enter the detector. After detection, a computer program will generate a chromatogram, which will show peaks for each of the molecules separated by the GC. The higher the quantity of molecules of the same kind that reach the detector, the higher the peak for that molecule will be.
      • Video demonstration of how a GC column works.
      • Video of a scientist performing the GC procedure.
    • The amount of time it takes for a molecule to move through the entire machine and be detected by the detector is called the retention time. Analysts may use retention times to differentiate between different compounds. However, the retention time alone are not conclusive indicators of what compound is present. A specific compound will have a known retention time, but multiple compounds may have the same retention time in the same chromatogram. In order to distinguish and identify which compounds made the peaks, a mass spectrometer is used.
  • MS
    • A mass spectrometer will determine the exact molecular weight of a molecule or compound. A previously separated sample (flowing directly from GC after detection) must be ionized for mass spectrometry. The sample, now broken down into molecules, is subjected to an ionization source, a beam of steadily flowing electrons, which blasts the molecules and causes them to break apart into positively charged particles, or ions. Only positively charged ions will continue through the machine. Next, the ions travel through the mass analyzer, or quadrupole filter. The mass analyzer uses an electromagnetic field to separate the ions based on molecular weight. In a routine drug analysis performed by the laboratory, the analyst will already know the molecular weights of the drugs that are frequently detected. To isolate these specific drugs at their known molecular weights, the scientist will manipulate the experimental environment (such as flow rate, type of gas used, temperature, etc.) in previously tested and scientifically accepted ways to produce the range of molecular weights required for analysis. This manipulation will allow only ions between the desired molecular weights to pass through the machine. Ions that flow through the mass analyzer without being filtered will reach the mass detector. The mass detector will count the number of ions with a specific mass. These detections will be translated by a computer program into a mass spectrum.
  • Together
    • A chromatogram (from the GC) and a spectrum (from the MS) can be used together to determine a molecule’s identity. Solely using GC results to identify a compound may lead to inaccurate conclusions. As mentioned earlier, a molecule will have a specific retention time, but a specific retention time does not identify a specific molecule. The analyst will use the spectra to determine the molecular weight of the molecule that generated the peak at a specific retention time. The molecular weight at that retention time will eliminate incorrect identities and narrow down the potential identities to one. The retention time, molecular weight, and patterns of both the chromatogram and spectrum must all match a known standard to be conclusively identified. Differences should not be disregarded unless specifically permitted in the lab procedures.
• Limitations
  • A mass spectrometer cannot distinguish between compounds in mixed samples. A sample must be separated prior to MS testing. A gas chromatograph is used to separate the impurities out of a sample in preparation for mass spectrometry. The mass spectrometer will measure the atomic weight of the compounds that were separated by the gas chromatograph.
  • MS cannot differentiate between molecules that have the same molecular weight but have chemical structures that are mirror images (also known as enantiomers). This is the case specifically with pseudoephedrine and ephedrine. Subsequent FTIR testing may be necessary to distinguish enantiomers.
  • GC/MS will detect the presence of methamphetamine, however, it will not differentiate between methamphetamine stereoisomers. L-Methamphetamine is the active ingredient in the over-the-counter cold medication Vick’s VapoInhaler and other generic preparations. This ingredient is completely legal. It is commonly labeled as levmetamfetamine to signify that is the legal form of methamphetamine. The other isomer, D-Methamphetamine, is not legal, but will create the exact same peaks on a GC/MS as the L-form does. It is important for the lab to quantitate how much of each stereoisomer are present, which can be done by means of percentages. Some laboratories will allow up to 20% of the D isomer to be present before reporting positive test results for illegal methamphetamine. This blog post explains the concepts of chirality and stereochemistry as they apply to GC/MS testing of suspected methamphetamine.
  • If a sample contains a high concentration of pseudoephedrine or ephedrine, it is possible for an “artifact peak” to appear on a GC/MS chromatogram. If not correctly identified as an artifact peak, the sample may be misidentified as methamphetamine. This peak is created by a reaction at the injection point under certain experimental conditions. At higher temperatures (see Hornbeck et al, “Detection of GC/MS Artifact Peak as Methamphetamine,” Journal of Analytical Toxicology (1993) which determined that at 300 degrees an artifact peak existed, while at 185 degrees it did not) pseudoephedrine and ephedrine will react with 4-carboxyhexafluorobutyrl, pentafluoropropionyl, heptafluorobutyryl, and a few other substances to create an artifact peak that is similar to that of methamphetamine. This artifact peak can be eliminated by adding sodium periodate to the urine sample before GC extraction.
  • Malfunctions in the equipment can occur. The injection point septum is a part of the machine that has the potential to wear. This port only lasts 100-200 injections. The injection port temperature, which is selected by the analyst, may be high or low causing a shortened septum life span, decreased sensitivity, poor separation of liquid material, or decomposition of the sample.
• Considerations
  • Is the machine is properly maintained and calibrated? Were the samples prepared properly by the analyst? Are any reagents out-of-date? Were negative controls, positive controls, and blanks properly used? Was the analyst following the protocol properly?
  • Did the analyst perform an individualized interpretation or did s/he rely on the computer generated comparison to the library of standards?
  • Interpretation of only the chromatogram (chart generated by the gas chromatograph) or a spectrum (chart generated from the mass spectrometer) is not sufficient. Both the chromatogram and spectrum must be interpreted together and a final identification must be supported by both.

• Characteristics
  • Highly specific.
  • Many substances will create an unequivocal IR spectrum that is easily identifiable by analysts.
  • An IR spectrum by itself does not provide an exact chemical structure of a compound, but will provide information about functional groups that are present in the molecule.
  • Presence or absence of certain functional groups will guide an analyst as to the possible identity of a compound.
• How it works
  • Infrared light is light that is not visible to the human eye. FTIR measures the amount of infrared light that is absorbed by a sample. Molecules (multiple atoms held together by bonds) are constantly moving. The bonds that hold the atoms together will bend, rock, stretch, compress, or twist. These actions collectively are called vibrations. Different functional groups, mentioned above, will absorb infrared light differently and will cause the atoms to vibrate more or less frequently. How quickly the molecule vibrates is called the frequency. More frequent vibrations will create a higher frequency, and fewer vibrations will generate a lower frequency.
  • An infrared light beam, or source, is aimed directly at an instrument called an interferometer. The interferometer uses a combination of a beam splitter, a mobile mirror, and a stationary mirror to separate the beam of light into individual wavelengths. First, the beam splitter splits the beam of infrared light at right angles into two smaller beams. One beam will hit the stationary mirror and the other will hit the mobile mirror. The angles of the mirrors allow the two separated beams to reflect, meet again, and reconstitute as one beam. The mirror mechanism separates the light into different wavelengths that are measured at the point of reconstitution. The interferometer records this information and creates a graph called an interferogram. An interferogram records information about every frequency of light from the infrared source. Most importantly, every frequency is measured at once instead of individually as earlier scientific devices required. Once the frequencies of infrared light have been measured by the interferometer, the reconstituted beam continues through the machine to the sample. The beam is applied to the sample where it will either transmit (go through) or reflect (come back towards the source). At this point, specific frequencies of energy will be absorbed by the sample and some will transmit through. The light that does transmit through the source will reach a detector on the other side. Not only does the light that reaches the detector generate information, but the absence of some wavelengths of light, the light absorbed by the sample, also generates information for analysis. The information gathered from the detector is finally sent to a computer where a chart is created specifically for that sample. This computer generated chart is called an IR spectrum.
  • Analysts use the IR spectrum and compare the sample’s spectrum to a known reference sample. This can be done either by computer comparison, or by an individual analyst manually comparing the two spectra. Each “peak,” explained below, is representative of a bond between two atoms. Specific well-known bonds that also have definable physical/chemical characteristics are called functional groups. Common functional groups include alcohols (- OH), ketones (=O), esters (- COOCH3), ethers (-COC-), and carboxylic acids (-COOH), to name a few. Each of these functional groups, if present, will create a distinct peak that typically is easily identifiable by a trained analyst. These peaks will be a specific intensity and be found within a specific wavelength range. When analyzing a spectra, it is important to know that the presence of a peak in a certain place does not necessarily indicate that a specific functional group or bond exists in a sample’s chemical structure. However, absence of a peak in the area where a known functional group or bond would present itself does indicate that that functional group or bond does not exist in the sample’s chemical structure. Some peaks will be more or less intense due to the bond’s nearness to other bonds (the molecule’s stereochemistry), the number of the exact same type of bonds present in a molecule (aromatic C-H bonds), and many other more complex reasons. Identification of compounds by IR spectra is an art which takes a lot of practice. Analysts must not only remember the locations of where peaks should be, but must also remember the patterns common to certain types of molecules that could be present in a larger macromolecule.
• Limitations – limitations of the analysis are based on closely related materials, mixtures, and improper preparation of the sample.
  • IR cannot differentiate between isomers of the same drug.
  • For an accurate spectrum, the substance must be reasonably pure (generally >90%). Testing of pharmaceuticals using IR may be more successful than the testing of street drugs which are often impure.
  • Diamond cell IR, if used, allows smaller amounts of samples to be tested. If diamond cell IR is not used, then the amount of sample required for testing is much higher.
• Considerations
  • Peaks from an IR spectrum are read “upside-down.” A peak, obviously poorly named, is actually observed at the lowest point on the graph, or the valley of the observed area. IR measures absorption, the opposite of transmission. Absorption and transmission are inversely related: the more a sample absorbs light, less light is actually transmitted through the sample. The more light that transmits through a sample, less light is absorbed by that sample. The y-axis of a spectra measures the percent transmission of a sample. Samples with the highest percent transmission will generate higher (deeper) peaks, and those with the lowest percent transmission will show a lower (shallower) peak or possibly no peak.
  • Relative intensities of the bands are important, any significant mismatch with reference spectrum negates identification. To make an identification, an analyst considers intensity (weak, medium or strong), shape (broad or sharp), and position (cm-1) of the peaks in the spectrum. Many compounds look similar, but an exact match is necessary to confirm the identity of an unknown.
• Links
  • FTIR Identification – this blog post by Dr. Fred Whitehurst discusses the limitations of the FTIR for use with forensic samples.
  • Organic Chemistry Lecture – by David Van Vranken, Ph.D. of UC Irvine explaining the science behind IR Spectroscopy.
  • Video on the science of FTIR
  • Video of an FTIR run from start to finish.