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Presumptive and Confirmatory Forensic Testing on Controlled Substances

Objectives: Differentiate Preliminary and Certified Weight; Define Presumptive Colorimetric Analysis; Define Screening Procedures for Biological Specimens for Controlled Substances; Discuss Confirmatory Testing Procedures for Controlled Substances; Explain and Define Areas of Concern for Presumptive and Confirmatory Testing Procedures as they Relate to Controlled Substances; Recognize Necessary Documents for Argument on Controlled Substance Analysis.

I. Controlled Substance Certified Weight Analysis

a. A certified weight of any controlled substance can be performed by any analyst on an analytical balance in a location that serves as an appropriate location for handling and storing controlled substances.
b. Preliminary weights are sometimes done from patrol car at the time of seizure and/or by an evidence custodian gaining insight for an approximate weight for charge/arrest purposes. These weights should be considered preliminary and approximate only.
c. Accurate reporting of any weight is accomplished by using internal an internal calibration and/or external calibration weights issued by an approved manufacturer providing proof and certification for those weights. NVLAP (National Voluntary Laboratory Accreditation Program) is an approved organization for certifying weights. Troemmer is an approved manufacturer of certified weights. ASTM (American Society for Testing Materials) and NIST (National Institute of Standards and Technology) are other organizations that develop standards for testing and measurement.

II. Certified Weight Analysis Process

a. Balance is allowed to warm-up and stabilize. A balance that stays on at all times will remain warmed-up and stabilized.
b. An internal calibration is completed if the balance is equipped for an internal calibration. An internal calibration should, in good laboratory practice, be checked with and external certified weight to ensure proper function of the balance.
c. An external calibration may be done in place of or in addition to an internal calibration. A series of certified weights issued by an approved manufacturer and with supporting certificates.
d. Weight of controlled substance(s) is obtained one container/bag/box/vessel at a time. When multiple containers are involved in the seizure each container should be weighed separately and contained separately to eliminate cross contamination in the event a re-examination is desired. Each value obtained should be then added together to obtain the final certified weight of the seizure.
e. A final confirmation of an external certified weight should be done at the end of the analysis to ensure balance was in proper working order throughout the analysis. If an extended period of time elapses during analysis, a confirmation weight may be done at intervals (generally defined by a laboratory Standard Operating Procedure) throughout the weight examination. This is done to ensure continued proper performance of the balance.
f. Certified weight analysis can be confirmed by an independent analyst for confirmation.

III. Law Enforcement and Corrections Standards and Testing Program

a. Sponsored by the Office of Science and Technology of the National Institute of Justice. The program is an applied research effort that determines technological needs and sets minimum performance standards for specific instrumentation or devices.
i. Testing of commercially available equipment.
ii. Reports results to criminal justice agencies nationally and internationally.
b. Law Enforcement and Corrections Technology Advisory Council (LECTAC) is comprised of recognized parties from the federal, state, and local agencies.
i. Assess technological needs and sets priorities for research programs.
ii. Sets priorities for items to be tested and evaluated.
c. Office of Law Enforcement Standards (OLES) develops national performance standards for compliance testing.
i. Ensure items and equipment are suitable for use in the criminal justice agencies.
d. National Law Enforcement and Corrections Technology Center (NLECTC) is operated by grants
i. Supervises national compliance testing program operated by independent laboratories.
ii. Standards developed by the OLES are the performance benchmarks by which commercial equipment is measured.

IV. National Institute of Justice Standard for Color Test Reagents/Kits for Presumptive Testing

a. Color Charts:
i. Centroid – No longer considered to be the international standard of color. The numbers are listed and available for historical purposes only.
ii. Munsell – The Master Atlas of Color
1. Colors are made according to specifications listed in the final report of the subcommittee of the Optical
Society of America on spacing of Munsell colors.
2. Colors measured by spectrophotometry and are visually inspected.
3. There are nearly 1600 color chips.
4. There are 40 pages of colors, each page presents one hue, hues 2.5 steps apart.
5. Hue=value/chroma
iii. Final color = color that remains after any intermediate colors have disappeared (generally after 1-2 minutes). Intermediate colors may appear by the addition of reagents.
b. Detection of Controlled Substance – Presumptive Substance Specific Kits
i. A List of specific substances combined with reagents assembled in a kit may be used to make tentative identifications.
ii. Kits will contain a statement of usage for presumptive identification purposes only and more definitive examination for confirmatory testing should be performed by a qualified analyst or scientist in a properly equipped crime laboratory.
iii. Kits will contain a statement that users of the kit are appropriately trained and that the reagents of the kit may give false positive or false negative results.
iv. Kits will contain statement that reagent or sample contamination could lead to misleading results.
v. Kits will contain a statement regarding proper storage conditions for buildings and vehicles.
c. Kit Labeling
i. Reagent Identification
ii. Identity of drug/drugs of detection per container
iii. “Danger” when appropriate
iv. Marked with an expiration date when appropriate
d. Kits should be visually inspected for broken or inoperative catches, hinges, or containers and reagent leakage.
e. Color References for Kits
i. The kit will include samples or reproductions of colors produced by each reagent when reacted with each drug listed on the label.
ii. There will be sufficient reagent per kit to permit differentiation between each drug listed.
f. Kit Test Methods
i. General Conditions
1. Ambient temperatures at the time of test should be between 10oC and 40oC (50oF -104oF).
2. Safety recommendations specific to each kit should be followed.
3. Storage shall comply with conditions of storage specific to reagents in the kit.
a. Oxidizers
b. Flammables
c. Acids
d. Bases
ii. Place 500ug of drug dissolved in chloroform or in powder form into 3 wells of a plate or 3glass tubes.
iii. Use disposable pipettes to transfer reagent into tubes or wells. A separate pipette should be used for each reagent required.
iv. Transfer one drop of reagent being tested in the sequence specified by the kit manufacturer to each of the 3 wells or tubes.
v. Compare colors within the specified time limits provided by the kit manufacturer.
vi. Typical Reagents:
1. Cobalt Thiocyanate Reagent
a. Cocaine HCL
b. Methadone HCL
2. Marquis Reagent
a. D-Amphetamine HCL
b. Codeine
c. Diacetylmorphine HCL
d. LSD
e. Mescaline HCL
f. Methadone HCL
g. D-Methamphetamine HCL
h. Morphine Monohydrate

V. Limitations of Colormetric Kits

a. Cobalt Thiocyanate Test for Cocaine:
i. Ambient temperature affects equilibrium of the test results
1. Cooler temperatures yield a higher sensitivity, increasing the potential for a false positive.
2. Warmer temperatures yield lower sensitivity, increasing the potential for a false negative.
ii. Subject to false positive and false negative results
1. Positive test result does NOT confirm Cocaine
2. Negative test result does NOT indicate lack of Cocaine
3. Chemical interferences such as cutting agents can interfere with testing.
4. Adulterants and dilutants will interfere with the test and also decrease the amount of cocaine placed in the well, tube, or kit.
iii. Quantity of material used for testing
1. Insufficient and excessive quantities of cocaine have been documented to yield false negative results./
b. Ambient Temperature Limitations for Kits
i. NIJ sets a generic standard for the color kits whereby recommending ambient temperatures between 10oC and 40oC or 50oF and 104oF.
1. Studies showed kit temperatures at 4oC or 39oF yielded greater sensitivity and accuracy.
2. Studies show kit temperatures at 45oC or 113oF and 60oC or 140oF had a higher occurrence of false positive and inconclusive results. Note: Temperatures of the inside of vehicles and the inside of trunks of vehicles in a parked lot have been known to reach more than 127oF.
3. Storage of 10oC or 50oF may be too high and sacrificing sensitivity of the test.

VI. Biological Sample for Use in Drug Testing

i. Blood
ii. Urine
iii. Hair

VII. EMIT (Enzyme Multiplied Immunoassay Technique)

a. Immunoassay testing is quick, highly sensitive, and relatively inexpensive but lacks specificity

VIII. ELISA (Enzyme Linked Immunosorbent Assay)

a. Detects the presence of antibodies and enzymes linked to antibodies.
b. Utilizes flourogenic, electrochemiluminescent, and real-time PCR reporters to create quantifiable signals.

IX. Limitations of Immunoassay Testing

a. Medications and Substances Causing False Positives.
i. 161 prescription and over the counter (OTC) medications have been studied.
ii. 65 medications produced false positive results in commonly administered urine tests for drugs.
iii. Cross reactions or false positives from medications:
1. Marijuana – Pain relievers such as Advil, Motrin, and Midol, all drugs containing Ibuprofen, and passive marijuana smoking (passive inhalation in an area where others are smoking and the ventilation is low or less than adequate).
2. Amphetamines – Nasal sprays, Sudafed, any substance or drug containing ephedrine or pennypropanolamine.
3. Opiates – Dextromethorphan (cough medications), Primatene-M (containing perylamine), Demerol, Elavil, Quinine Water, Poppy Seeds.
a. According to the Journal of Chemistry Vol. 33 #6, 1987, one only needs to ingest 25-40 grams (one or two poppy rolls, bagels, or cake) to produce a false positive test for opiate abuse. “Not only is it difficult to distinguish heroine or morphine abuse from codeine, but dietary poppy seeds can give strong positive results for urinary opiates for several days duration that is confirmed by GC/MS analysis.”
4. Methadone – Nyquil
5. Cocaine – Antibiotics (Ampicillin, Amoxicillin)
6. PCP – Diazapam, some ingredients in cough medications, Dextromethorphan.
iv. Endogenous Excretion of Enzymes
1. The list of agents which can cause false positive results in the urine have also been described for endogenous (produced inside the body) excretion on enzymes in the urine.
2. Study conducted by Dr. James Woodford of Emory University showed a percentage of persons of African origin, Orientals, and Pacific Islanders may be testing positive for marijuana secondary to a mechanism which involves the pigment melanin, which approximates the molecular structure of the THC metabolite causing laboratory cross reaction for marijuana.

X. Confirmation Testing

a. GC/MS (Gas Chromatography/Mass Spectroscopy)
i. Excellent methodology if done properly
1. Clean equipment
2. Standard Operating Procedures of the laboratory implemented and followed.
ii. Considered the “gold standard” in confirmatory testing
iii. GC is effective at separating compounds into its various components yielding results in retention times proportional to the concentration
iv. MS provides specific results for mass but produces uncertain qualitative results.
v. GC/MS Analysis
1. GC instrument feed to the MS instrument and used for conclusive proof of substance identity.

XI. Limitations of GC/MS Analysis

a. Although erroneous results are not expected, false positive and false negative results are possible.
b. Lack of complete separation of the compound leads to an impure MS feed.
i. This is usually seen as “background noise” (a raised or unstable baseline) in MS.
c. Carrier gas from the GC not correctly deflected into the MS may also yield “background noise”.
d. MS suffers from an inexact method or practice of interpreting mass spectra
i. Analyst and computer discretions of calculations and system conditions.
e. Memory bank of most MS is about 5000 spectra for a particular group of compounds.
i. There are over 200,000known existing chemicals

XII. Case Studies of GC/MS False Positive or False Negative

a. Journal of American Toxicology published an article describing a false negative THC metabolite confirmed by GC/MS. Urine tested positive by EMIT Cannaboid 100ng assay and positive via TLC (Thin Layer Chromatography). Subsequent work up of the sample yielded a high level of ibuprofen. The sample was analyzed again by GC/MS with an increased amount of methylating agent and finally yielded a positive result.
b. Controlled study of 60 subjects, producing 510 specimens, which had taken ibuprofen/Naproxen. Approximately 20 specimens (4% of the specimens) tested false positive for cannabinoid.
c. Several controlled studies have shown as little as one poppy seed muffin (about 15g of seeds) can produce detectable amounts of morphine and codeine by both immunoassay and GC/MS.
i. Federal government thresholds have been increased defining a positive screen in urine for morphine or codeine from 300ng to 2000ng in an effort to reduce food consumption false positives.

XIII. Conclusive Results

a. Can be corroborated with proper system conditions documented, spectra and data reviewed as a part of the case file, verifying all procedures have been performed to the defined laboratory standards and operating procedures.
b. No analytical procedure for obtaining results should be Without doubt.

1. 2008. NFSTC Forensic Technology Testing and Evaluation Report Form
2. 2011,
3. 2011,
4. 2003, DEA Resources, Microgram Journal, Voume 1
5. 2011,
6. 2011,
7. Vincent, E. MD, Oct 2006, vol.55 no.10 pp.893-897, Clinical Inquires
8. 2011,
9. Bunk, S.D., JAT vol. 12 issue 5 p.290-291, Sept/Oct. 1988

Disclaimer: While every effort has been made to ensure the accuracy of this publication, it is not intended to provide legal advice as individual situations will differ and should be discussed with an expert and/or lawyer.For specific technical or legal advice on the information provided and related topics, please contact the author.

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