Toxicology is the study of the effects of drugs on biological systems. Specifically, forensic toxicology focuses on the detection of alcohol, drugs, and other toxic substances in the human body. The State Crime Laboratory currently uses screening and confirmatory tests to indicate the presence of a drug in a defendant’s blood, urine, or other bodily fluids. For information about the identification of bulk samples of suspected controlled substances, click here. This page was created in August 2014. Attorneys should check that the lab procedures referenced are the most up-to-date versions.
Procedures for Testing Bodily Fluids for Drugs
Toxicology samples that are being tested for drugs are screened using a presumptive test, such as the ELISA test. If the screening yields a positive result, the sample must undergo an extraction and be tested using a confirmatory test to conclusively identify the substance that is present and potentially quantify the amount of the substance that is present.
- Presumptive Test
- ELISA Immunoassay
- Identification and Quantification
- Identification and Quantitation
- Link to the State Crime Lab’s procedure
- 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:
- Cocaine Metabolite (Benzoylecgonine-BE)
- Cannabinoids (THCA/CTHC)
- 3,4-methylenedioxymethamphetamine (MDMA)
- in urine:
- Cocaine metabolite (Benzoylecgonine-BE)
- in blood:
- 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.
- 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.
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 188.8.131.52.4.) 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 184.108.40.206 and 220.127.116.11. 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.
- 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 18.104.22.168.
- The positive control must be within 25% of the expected concentration. See Section 22.214.171.124.
- 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.
Procedures for Testing Blood for Alcohol
The Toxicology Section at the State Crime Laboratory performs Headspace Gas Chromatography to both confirm the presence of alcohol and quantitate the amount of alcohol present in a blood sample.
- Link to the State Crime Lab’s procedure
- 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.
- 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.
Reports and Publications
Department of Justice article regarding issues raised by drug court programs regarding drug testing.
Available from the NC Office of the Chief Medical Examiner
This National District Attorney Association has these and other guides for prosecutors available on their website:
- Cross-Examination for Prosecutors
- The Drug Evaluation and Classification (DEC) Program
- Drug Toxicology for Prosecutors
- Hardcore Drunk Driving Prosecutorial Guide: A Resource Outlining Prosecutorial Challenges, Effective Strategies and Model Programs
- Horizontal Gaze Nystagmus—The Science and the Law: A Resource Guide for Judges, Prosecutors and Law Enforcement
- Overcoming Impaired Driving Defenses
The National Highway Traffic Safety Administration offers information about the effects of impairing substances.
The Washington State Patrol has posted the following manuals online: DWI Detection and Standardized Field Sobriety Testing, DRE, and Advanced Roadside Impaired Driving Enforcement (ARIDE). These manuals are available for download.
This 2014 article lists the therapeutic, toxic, and fatal blood drug concentration levels for many controlled and non-controlled substances.
Reference cited by the NC State Crime Laboratory. Provides the normal, therapeutic, toxic, and lethal blood level for various drugs and their metabolites.
From the Blog
- An announcement from Becton Dickinson and Company (BD) may raise doubts about the validity of some blood alcohol tests conducted across the country since August 31, 2018. On May 30, 2019, with a correction made June 12, 2019, BD announced a recall for its “BD Vacutainer® Fluoride Tubes for Blood Alcohol Determinations” product. Vacutainers are …
- Forensic Toxicology Online Symposium, 4/9/2019RTI International is offering their 2nd Annual Online Symposium: Current Trends in Forensics & Forensic Toxicology program May 13-17, 2019. The program is free of charge. The program will feature speakers on various topics of toxicology and seized drug analysis. The program is intended for laboratory scientists who work in these fields so that they can receive …
- ANSI/ASB Best Practice Recommendation 037, Guidelines for Opinions and Testimony in Forensic Toxicology, 1/29/2019The American Academy of Forensic Science Standards Board (ASB) has published ANSI/ASB Best Practice Recommendation 037, Guidelines for Opinions and Testimony in Forensic Toxicology, First Edition. This document delineates guidelines for best practices in forensic toxicology opinions and testimony, including human performance toxicology (e.g., driving under the influence of alcohol or drugs), postmortem forensic toxicology, …
- OCME Toxicology Lab Procedures available, 9/18/2018The IDS Forensic website has posted the toxicology lab procedures from the NC Office of the Chief Medical Examiner here. Attorneys who would like to learn more about the procedures that the OCME toxicology lab uses to test evidence can read through the procedures. For casework, attorneys should obtain the lab procedures that were in …
- Houston in the Blind, 5/8/2018Blind studies and procedures are the gold standard of evaluating the quality and reliability of scientific results. Unfortunately, this has long been lacking in forensic science. Fortunately, strides are being made to introduce blind testing to forensics, most notably in the Houston Forensic Science Center (HFSC). Currently, forensic scientists are tested periodically on their knowledge …
- Any practitioner that handles driving while impaired charges knows the significance of field-sobriety tests (“FSTs”) to the investigation and prosecution of drunk driving. Scientific research supports the basic idea behind the tests — that test performance on FSTs is an indicator of alcohol impairment. What about when the impairing substance isn’t alcohol at all? Do …
- Toxicology Testing by NMS Labs – FAQ, 7/22/2016Many attorneys have asked us about blood-drug (and some blood-drug and alcohol) testing performed by NMS Labs. We have compiled the questions and our answers here. If you have additional questions, please post them in the comments and we will try to answer them as well. Let’s make this a living document! The toxicology evidence …
- The NC Department of Public Safety (DPS) has begun relying on less accurate presumptive testing for urine screens for drugs for probation, prison, and DSS cases. The focus of this post will be probation cases, though the testing is the same for prison and DSS cases. Testing Prior to 2014 Until February 2014, the Department …
- Defense attorneys may decide to make a motion for independent testing of forensic evidence in cases where either the State has chosen not to complete forensic testing of an item of evidence or where the State has tested the item and the defense would like it to be re-tested. This issue has come up more …
- Bullcoming and Blood Alcohol Testing, 10/10/2011The National Association of Criminal Defense Lawyers’ amicus brief in Bullcoming v. New Mexico is an excellent resource for attorneys who want to understand gas chromatography measurements of blood alcohol concentration (BAC) and possible challenges to that technique. Gas Chromatography or Headspace Gas Chromatography is the typical method used in North Carolina when the State …
- Gas Chromatography-Mass Spectrometer (GC/MS): In Scientific Evidence, Even ‘Gold Standard’ Techniques Have Limitations
by Joanna Gin and Edward Imwinkelreid. UC Davis Legal Studies Research Paper, available for free download. Like nuclear DNA testing, GC/MS analysis has important limitations. Courts should not assume it is a nearly infallible technique. When GC/MS is used in drug testing, the court must inquire as to the mode of analysis: full scan, selective ion reliance, or selective ion monitoring. When GC/MS is employed to identify ignitable liquids in arson investigations, the court should inquire as to the condition of the sample tested: Has it been subjected to weathering, microbial degradation, or pyrolysis?
Article from US Pharmacist that addresses potential false-positives and false-negatives in urine screens.
2nd Annual Online Symposium hosted by RTI and ForensicEd on May 13-17, 2019. This program offers information on best practices in forensic toxicology, drug analysis, and trace analysis such as sample preparation, method development, and forensic method validation. Presentations are geared toward forensic practitioners, but several of the sessions should be of interest to attorneys.
The National Forensic Science Technology Center created this website to explain in simplified terms the principles of each type of forensic analysis and how the analysis is performed. Topics include DNA, digital evidence, fingerprints, firearms, trace evidence, blood stains, and more.
The ABFT is a non-profit organization certifies forensic toxicologists and accredits forensic toxicology labs that comply with standards of the ABFT.
The CLIA program works to ensure quality laboratory testing in all clinical laboratories regulated by the Centers for Medicare and Medicaid Services.
A collaboration between the Orlando Public Defender and the National Center for Forensic Science at UC Florida. The site has links to many helpful training videos that help attorneys understand forensic science evidence.
Lists numerous websites, webinars, and other helpful online resources, prepared by Peter Stout, Ph.D., D-ABFT
SOFT is an organization of practicing forensic toxicologists. SOFT’s annual meetings provide a forum for the exchange of information and ideas among toxicology professionals. SOFT sponsors workshops, newsletters, proficiency testing, and technical publications with the goal of improving toxicologists’ skills and knowledge. These Forensic Toxicology Laboratory Guidelines by SOFT and the American Academy of Forensic Sciences provide basic requirements for forensic toxicology labs, including guidelines on the use of screening versus confirmatory tests.
Motions and Briefs
Sample discovery motion regarding drug chemistry or toxicology evidence.
Motion to exclude expert testimony based on failure to satisfy requirements of the new Rule 702 of the North Carolina Rules of Evidence.
Order granted in attorney James Davis’s case in 2013 under the new Rule of Evidence 702.
2013 motion to be used for independent testing or re-testing of toxicology evidence.
Toxicology in the News
- Forensic experts question reliability of breathalyzer used by Minnesota law enforcement agencies, KSTP, 7/14/2019
- Thousands of DWI cases in Harris County thrown into question after blood vial recall, by Samantha Ketterer and St. John Barned-Smith, Chron, 6/21/2019
- Thousands of Harris County drunk driving cases affected by blood vial recall, by KHOU Staff, KHOU, 6/21/2019
- NC lawmakers see hemp as the state’s next big cash crop. But police are opposed., by Will Doran, Herald Sun, 6/12/2019
- North Carolina Advances Delayed Ban on Smokable Hemp, by Amanda Morris, US News, 6/5/2019
- Hemp production, slowly gaining support, could hit road block, by Taylor Knopf, NC Health News, 6/5/2019
- N.J. wants more cops trained to spot stoned drivers; critics question science behind methods, by Joe Hernandez, WHYY, 5/16/2019
- Philly DA’s Office won’t prosecute cannabis users for DUI unless they are actually stoned (PA), by Sam Wood, Philadelphia Inquirer, 4/29/2019
- Hugh J. Burford, Waynesville, NC
- David Lewis Burrows, Ph.D., Holly Springs, NC
- Dr. Yale H. Caplan, Ph.D., D-ABFT, Baltimore, MD
- Andrew D. Ewens, Ph.D., DABT, Cary, NC
- Barry Funck, Tallahassee, FL
- Jay M. Gehlhausen, Ph.D, DABFT-FD, Apex, NC
- Fran M. Gengo, Pharm.D FCP, Amherst, NY
- Lyle Liechty, Indianapolis, IN
- Andrew P. Mason, Ph.D., DABFT, DABCC-TC, Boone, NC
- Brian A. McMillen, Ph.D., Greenville, NC
- William Alexander (Alex) Morton, Jr., PharmD, Brevard, NC
- Dr. E. Don Nelson, Cincinnati, OH
- Guy Oldaker, Ph.D., Lewisville, NC
- Bethany P. Pridgen, MFS, Wilmington, NC
- Mustafa Selim, Greenville, NC
- Cynthia Slagle, RN, Greenville, NC
- Douglas L. Smith, MS, FTS-ABFT, Durham, NC
- Alfred E. Staubus, Pharm.D., Ph.D., Columbus, OH
- Darrell Sumner, Ph.D., Winston-Salem, NC
- John Vasiliades, Ph.D., DABCC, DABFT, Omaha, NE
- Patra Watson, Columbia, SC
- Carl E. Wolf II, Ph.D., M.S., F-ABFT, Richmond, VA
- James Woodford, Ph.D., Nashville, TN
- Errol Zeiger, Ph.D., JD, ATS, Chapel Hill, NC