Drug Analysis - Forensic Resources (2024)

• 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:InState 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.
• Typically a lab report from the State Crime Lab will only include the name of the test and the resulting color (or may simply state that the expected color change occurred).
• Link to the State Crime Lab’sprocedure– Note the quality control check that is required on p. 2.
• Law Enforcement and Corrections Standards and Testing Program– National Institute of Justice standards for color test reagents and kits for the preliminary identification of controlled substances.
• 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 companySirchie. An attorney may have to determine which of the tests below is in the kit.
  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,NCSCL’s procedureis for analysts to identify a pharmaceutical preparation by physical characteristics and markings. 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.” (p. 7) InState v. Wardthe 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 Lawblog. 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.
• 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 testscan 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. Proper reagent preparation and procedures are provided by the NC State Crime Lab Guidelines.
    • 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
    • Excipients and diluents: hydrochloric acid, methanol, distilled water
    • Propoxyphene:acetic acid, gold chloride in acetic acid
  • 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.
    • DEA Resource Guide– provides images of drugs of abuse as well as a list of federally scheduled drugs and descriptive information about the major classes of drugs.
    • The University of Texas at Arlington –Pictures of Commonly Abused Drugs
• For Marijuana– The United Nations Office on Drugs and Crimerecommendsa combination of color test, thin-layer chromatography and physical (macroscopic and microscopic) examination as a minimum analytical approach for the identification of cannabis products.
  • NCSCL Technical Procedure for the Identification of Marijuana
  • Polarized Light Microscopy– for viewing suspected hashish/THC samples with the polarized microscope.

• Link to the State Crime Lab’sprocedure
• 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 coilingcolumn. 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 achromatogram, 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. The same is true for the inverse as well.
      • Videodemonstration of how a GC column works.
      • Videoof 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 these retention times to differentiate between different compounds. However, the retention times alone are not reliable indicators of what compound is present. A specific compound will have a known retention time, but multiplecompoundsmay 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) or a pure sample (injected) must be ionized for mass spectrometry. The sample, now broken down into molecules, is subjected to anionization 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, orquadrupolefilter. 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. It is unnecessary and impractical to know the molecular weight of every compound in a substance. Therefore, 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 massspectrum.
  • 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.
  • MScannot differentiatebetween 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 methamphetaminestereoisomers.L-Methamphetamineis the active ingredient in the over-the-counter cold medication Vick’s VapoInhaler. This ingredient has different effects on the human body and 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 laboratorieswill allow up to 20% of the D isomer to be present before reporting positive test results for illegal methamphetamine. Thisblog postexplains the concepts of chirality and sterochemistry 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
  • Non-specific
  • Used for molecules with high molecular weights.
  • Similar to GC, but where the GC is filled with gas, the HPLC column is filled with liquid solvent or solvent mixture.
  • The State Crime Laboratory uses this technique for quantitation of methamphetamine in drug analysis cases.
• How it works
  • HPLC can be used for qualitative identification: figuring out what is in a sample. HPLC is a separation technique that uses a stationary and mobile phase, a pump, and a detector to separate the compounds in a mixture. The result of the process is a computer generated graph known as a liquid chromatogram.
  • First, a small portion of the sample (approximately 5 to 20 µL) is injected into the stationary phase or column. The stationary phase is a small tube, also known as a column that is 3-5 microns in diameter and packed with microscopic particles. Next, the liquid mobile phase is pumped by the exterior pump through the machine. The mobile phase is similar to the gas flowing through a GC. The liquid mobile phase flows through the stationary phase similarly to how water flows through a water filter. The stationary phase separates the molecules in the sample, carrying them down the column at different rates. Eventually, the molecules will travel through the column and reach the detector at the end. The detector will record which molecules are present, and also will record the number of molecules of the exact same chemical makeup are detected. As with a GC, the time it takes the molecule to travel through the column and be measured by the detector is called the molecule’s retention time. Similarly, the greater the number of molecules present in the sample, the higher the intensity of the peaks.
  • HPLC can also be used for quantitative measurement: figuring out how much of a compound is in a sample. A calibration curve is used to calculate the concentration of the sample.
• Limitations
  • The standards used in both GC and HPLC are measured using certain conditions. The analyst chooses the speed of the gas or liquid that travels through the column. They also choose the temperature of the column for each experiment. The temperature of the column, the gas/liquid flow rates, and other factors affect the results. To recreate the experiment and replicate results, the exact conditions chosen by the analyst must be known. The same flow rate and temperature must be used if a second analyst wants to receive the same results on the earlier tested sample. If an analyst wants to compare their results to a known standard sample, they must have performed the experiment under the same conditions that the standard was performed under. As a whole, HPLC results are not as reproducible as GC results.
  • Thisanimationshows how these conditions can change the results in an HPLC chromatogram.
• Considerations
  • A single HPLC chromatogram cannot be used to calculate the concentration of the compound of interest. A calibration curve must be used to calculate the concentration of the sample. The calibration curve must also be within the accepted ranges allowed by the experiment (QC requirements). Always check that the QC and calibration curve were in range. A calibration curve with a correlation coefficient or R2 value equal to 1.000 is the most desirable. An R2 value of greater than 0.995 is required by the State Crime Laboratory for accurate reporting.
  • An HPLC machine cannot test all ranges of concentrations. For a highly concentrated drug, a sample (here referring to a small portion of the overall evidence) will be taken from the whole and diluted to measure the diluted sample’s concentration. A concentration for the diluted sample will be calculated using the concentration curve mentioned above. The diluted sample’s concentration will be multiplied by the dilution factor to find the calculation of the entire evidentiary sample. The concentrations used in the calibration curve must encompass the concentration of the diluted sample. For example, if the analyst is reporting the concentration of the diluted sample of methamphetamine to be 55 mg/mL, and the standards used to create the calibration curve range from 0.5 mg/mL to 50 mg/mL, then the concentration is out of range and cannot be used. The analyst must either further dilute a second sample to be within the range of the calibration curve or create another calibration curve. The curve’s limits may not be extrapolated to determine a sample’s concentration. Only the concentrations within the range of the concentration curve are valid.

• Link to the State Crime Lab’sprocedure
• Characteristics
  • Highly specific.
  • Many substances will create an unequivocal IR spectrum that is easily identifiable by analysts.
  • An IRspectrumby 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
  • Infraredlight 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 aninterferometer. 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. Themirror mechanismseparates the light into different wavelengths that are measured at the point of reconstitution. Theinterferometerrecords 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 anIR 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. The comparison of the sample’s spectra to the known standards is mandatory, either manually or by a computer, and the match must be exact, and forensically, must be supported by another confirmatory test.
• 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 mismatch with reference spectrum negates identification. Significant for the identification of the source of an absorption band are intensity (weak, medium or strong), shape (broad or sharp), and position (cm-1) in the spectrum. Many compounds look similar, but an exact match is necessary to confirm the identity of an unknown.
• Links
  • Overviewof the use of FTIR for analysis of controlled substances.
  • 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.
  • Videoon the science of FTIR
  • Videoof an FTIR run from start to finish.
  • For a fun and rudimentary explanation of light, see Bill Nye The Science Guy’s December 24, 1993Episode on Light and Optics.