Toxicology

• Link to the State Crime Lab’s procedure
• Characteristics:
  • ELISA can be performed on blood and urine.
  • Immunoassays measure the presence or concentration of a substance in urine or other fluid by using antibodies that specifically bind to drugs and their metabolites (the compounds that form after the body has metabolized a drug).
• How it works:
  • An antigen is a foreign substance that induces an immune response by triggering the production of an antibody.
  • In immunoassays, antibodies that contain enzymes react and bind to a specific drug or drug metabolite. This reaction generally produces a signal, such as a color change, or can be measured using other methods of measuring enzyme activity.
• Substances it tests for:
  • in blood:
    • Barbiturate
    • Benzodiazepines
    • Carisoprodol
    • Cocaine Metabolite (Benzoylecgonine-BE)
    • Cannabinoids (THCA/CTHC)
    • Methadone
    • Methamphetamine
    • Opiates
    • Tramadol
    • Zolpidem
    • 3,4-methylenedioxymethamphetamine (MDMA)
  • in urine:
    • Barbiturate
    • Benzodiazepines
    • Cocaine metabolite (Benzoylecgonine-BE)
    • Methadone
    • Methamphetamine
    • Opiates
• Results:
  • Understanding how the laboratory reports results is imperative. The State Crime Laboratory reports results stating that a specific drug or classes of drugs are positive or negative. These results must be confirmed by a confirmatory test.
  • ELISA immunoassays measure the color change that occurs when a sample tests positive for a specific drug. The State Crime Laboratory uses the Tecan/Immunalysis Freedom EVO ELISA Analyzer to measure the color change. This machine measures the color change by determining how much light is absorbed by the sample, also known as the absorbance.
  • Absorbance and concentration are inversely related. If a sample’s absorbance is low, the concentration of the drug present in the sample is higher. If a sample’s absorbance is high, the concentration of the drug present in the sample is lower. (See Section 5.8). This chart demonstrates how results are reported:

• All results that test positive or elevated will continue to confirmation testing, including extraction and either GC/MS or LC-MS/MS analysis.
• Limitations:
  • Because immunoassays are only screening tests, positive results must be confirmed using a confirmatory test.
  • False positives with are possible. This website lists the cross reactivities of some drugs. Another list of cross-reactivities is available here.
  • ELISA testing for cannabinoids cannot be performed on urine samples and will only be performed on blood samples.
• Links:
  • This video demonstrates how an ELISA test is performed and how the science works.
  • This website provides and in-depth explanation of the science of the ELISA test.

Blood and urine contain a wide variety of compounds. Confirmatory tests (GC/MS, LC-MS/MS) cannot be performed on whole blood or urine samples since there are too many compounds. Some compounds may interfere with the machine’s ability to interpret the presence of a substance of interest. To solve this problem, prior to confirmatory analysis, the analyst must perform an extraction to isolate the testable portion of the sample. An extraction is scientific technique that separates immiscible compounds (ones that do not mix easily) based on their solubilities. The State Crime Laboratory uses both liquid-liquid extraction and solid phase extraction in their procedures. Those procedures are described below:

• Acidic/Neutral Drug Solid Phase Extraction (ANSPE)
  • This procedure is to be used for extraction of acidic, neutral, and basic drugs from blood, serum, and urine.
  • It should not be used for cannabinoids or phenethylamines.
  • The sample will be separated into acidic/neutral and basic fractions via an extraction column.
  • Typically, the acidic/neutral fraction will contain barbiturates, carisoprodol, meprobamate, and some benzodiazepines. Alkaloids, amphetamines, opiates, zolpidem, tramadol, and most benzodiazepines elute in the basic fraction.
  • Some compounds may need to be further separated by derivatization. The technique of derivatization does not change the drug being tested, it only removes the insignificant ions that interfere with GC testing.
  • Morphine and benzoylecgonine are examples of drugs that need to be derivatized for detection. Some benzodiazepines will need to be derivatized if they already screened as positive or elevated in a sample and no corresponding signal was detected in subsequent GC/MS testing.
• Phenethylamine Liquid-Liquid Extraction (PHEALLE) for Analysis by GC-MS – This procedure is to be used to extract phenethylamine drugs (such as methamphetamine, MDMA, amphetamine, ephedrine, or pseudoephedrine) from blood or urine.
• Blood Cannabinoid Liquid-Liquid Extraction (BCLLE) for Analysis by LC-MS/MS – This procedure is to be used for extraction of cannabinoids from a blood sample. This procedure is not to be performed on urine samples. This test is used to test for presence of THC, THCA, and 11-OH-THC.

Once the desired portion of the sample is isolated it is then conclusively identified and if appropriate, quantitated. The State Crime Laboratory tests blood, urine, and serum using two methods: Gas Chromatography/Mass Spectrometry (GC/MS) and Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS).

• Gas Chromatography/Mass Spectrometry (GC/MS)
  • This technique is used after an extraction performed using the PHEALLE (liquid-liquid extraction) method or solid phase extraction method.
  • For more information about the science behind GC/MS, how a GC/MS works, and the limitations/considerations of GC/MS testing, click here to be redirected to the NCIDS Drug Analysis page.
  • Toxicology-specific limitations/considerations of GC/MS testing:
    • Subtraction: The State Crime Laboratory’s procedure for Toxicology GC/MS testing allows for the analyst to subtract interfering signals from the chromatogram. (See Section 5.6.12.2.4.) The procedure proscribes that when additional major ions are present, the source of the extraneous, background ions may be subtracted prior to formal analysis (peaks may be removed from the chromatogram to better visualize the remaining peaks, which are then compared to known reference standards). Only a trained expert will be able recognize if and when an essential peak has been erroneously subtracted and should be consulted as needed.
    • The State Crime Laboratory Procedure lists its criteria for a positive match for GC/MS data in Section 5.6.12.2 and 6.6.12.3. These criteria include:
      • The mass spectrum shall be compared to reference mass spectra.
      • Probability Based Matching (PBM) is a computer-calculated figure that represents the probability that the peak of interest matches the reference peak. Ex. Unknown peak has 98% probability of matching cocaine. PBM shall be used to aid in identification but should not be used as the sole means of identification.
      • The mass spectrum must contain all of the major and diagnostic ions unique to the analyte.
      • The signal to noise ratio (the response at the baseline or valley immediately before the internal standard signal) of the internal standard must be greater than 5:1.
      • To be considered a match, the ion’s retention time must be within 2% of the reference standard’s retention time.
      • This method is not to be used to distinguish between optical isomers.
• Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)
  • This technique is used only in conjunction with the BCLLE extraction method, for blood cannabinoid testing. The State Crime Lab’s procedure is available here.
  • How it works:
    • Liquid Chromatography (LC) is similar to Gas Chromatography (GC), except that the mobile phase used to separate molecules is a liquid instead of a gas. Molecules are separated by a liquid mobile phase so that they can be analyzed after separation by a mass spectrometer.
    • Tandem Mass Spectrometry/Mass Spectrometry (MS/MS) is the technique used here to further separate the molecules into measurable fragments. When a molecule travels through the mass spectrometer, and breaks apart, the resulting pieces are called fragments. The fragments are measured by the detector at the end of the mass spectrometer which creates peaks on a mass spectrum. A higher volume (number) of a specific fragment will generate a higher peak on a mass spectrum. The size and position of the peaks on a mass spectrum will exclusively identify unknown compounds. Tandem mass spectrometers have more than one analyzer that operate simultaneously and further separate complex molecules into more defined fragments. See the Drug Analysis page for additional explanation of the science of mass spectrometry. Tandem MS/MS analysis is specifically used for determining the structure of a molecule, while MS alone is used to determine the molecular weight of the molecule. Tandem mass spectrometry can be used to distinguish between molecules that are very similar.
  • Limitations:
    • The State Crime Laboratory procedure warns that this procedure shall not be used to distinguish between optical isomers. See Section 6.1.
    • A calibration curve must be generated for quantitation of THC present in the sample. The procedure lists when to accept or reject the calibration curve. See Section 5.6.3.2.
    • The positive control must be within 25% of the expected concentration. See Section 5.6.4.1.
    • The State Crime Laboratory’s procedure permits manual integration of a some peaks. See Section 5.6.5. The analyst must attach the paperwork accompanying the manual integration in the laboratory packet. Manual integration should only be performed under appropriate circumstances. Only a trained expert can recognize when a manual integration should or should not have been performed.
    • All body fluids must be homogeneous (same throughout). The blood/urine/serum must be well-mixed prior to sampling (taking an aliquot) so that what is taken from the sample is representative of the entire sample. If a homogeneous sample cannot be obtained, the procedure requires that this be noted in the report. See Section 5.4.
    • The internal standard must be within 50% to 200% of the average internal standard range. See Section 5.7.2.
    • Typically, if a batch fails, the analyst will repeat testing on a new aliquot. If not enough sample remains, the failed batch data may still be reported if certain criteria are met. See Section 5.7.3.

• Link to the State Crime Lab’s procedure
• Characteristics:
  • GC Headspace is not an altogether different method of toxicology analysis, but simply just a different way of introducing the sample into the machine.
  • Volatile liquids (such as alcohol) are favorable types of samples to be tested with GC Headspace.
• How it works:
  • The analyst will fill a GC vial with a proscribed amount of the Internal Standard Solution and the blood sample and seal the vial with a cap.
  • The Internal Standard Solution contains ethanol, methanol, isopropanol, and acetone. It is added so that the analyst can later compare the peaks produced by the known amounts of these standards with the peaks produced by the evidence sample.
  • The volatile analytes (ethanol, methanol, isopropanol, and acetone) will begin to vaporize inside the vial (similar to a carbonated drink building up pressure in a sealed bottle.)
  • The vial will contain a layer of blood at the bottom and a layer of gas on the top called the headspace. After time, headspace will have equilibrated (the contents of the gaseous layer will be equally distributed throughout that layer) and the headspace can be tested by the machine.
  • The machine will draw a sample for testing from the headspace. The headspace gas sample will continue through the gas chromatograph as described in the gas chromatography section of the NCIDS Drug Analysis webpage. See this video for additional explanation of headspace sampling.
  • After the headspace sample is separated into its component parts by the gas chromatograph, the components will enter the Flame Ionization Detector (FID) which measures the amount of analyte present. A hydrogen flame will cause the organic compounds in each component to combust. The strength of the combustion is proportional to the amount of the organic compound present and is measured by the height of the flame. The height of the flame will be represented as a peak on the resulting graph.
  • This animation demonstrates how a flame ionization detector works. Note that the animation involves a liquid sample rather than a headspace sample.
  • Identification of ethanol is determined by comparison of its retention time with the retention time of a known standard in the Internal Standard Solution.
• Limitations:
  • GC Headspace primarily tests for the presence of alcohols (ethanol, methanol, and isopropanol) and acetone. For quantitation purposes, a calibration curve must be made for each analyte.
  • The blood is sampled and tested four times (in replicate). Each run will have a resulting concentration of all four analytes. Therefore, there will be a total of 4 resulting concentrations for each analyte. The four concentrations are averaged and reported as a mean. See Section 5.6.3. If any of the requirements below are not met, then the sample must be reanalyzed:
    • Each of the four concentrations for ethanol must be within 6% of the mean.
    • Each of the four concentrations for methanol must be within 9% of the mean.
    • Each of the four concentrations for isopropanol must be within 8% of the mean.
    • Each of the four concentrations for acetone must be within 20% of the mean.
  • No further analysis is performed on DWI submissions where the BAC was found to be 0.08 g/100 mL of whole blood unless:
    • The case involves the death or personal injury of someone other than the driver of the vehicle.
    • Upon approval of a request from the District Attorney’s office. See Section 6.2.
  • Homogeneous samples are required for analysis. If a homogeneous sample cannot be obtained due to clots, blood cells becoming separated from the liquid, or other reason, the concentration must be converted to whole blood alcohol concentration by dividing the alcohol concentration by 1.18 to compensate for the water present in the sample. See Section 6.1.