Introduction

Purpose

Identify unknown drugs in powder and pill form using presumptive and confirmatory techniques.

Drug Identification

Analysis of controlled and uncontrolled substances is often fairly routine. After a visual assessment and measuring the mass of the exhibit, we will typically perform presumptive testing then use a different technique to confirm the identity of an unknown. While some labs or specialists may need to quantitatively characterize the various components of an unknown drug, we’ll focus on simple identification since that is the most routine task required.

Explanations and examples of presumptive and confirmatory testing are provided below. Regardless of the specific methods used in our analytical scheme, both analyses must yield results that agree with one another in order to confirm the identity of an unknown substance. There are some possible exceptions to this sequence of events, though. One example of an exception is non-controlled pharmaceutical preparations, which is also explained below.

Presumptive Testing

Presumptive testing is a quick, cheap, useful tool that can help inform future analysis and often serves as 1/2 of the concurrent positives required in most drug identifications. A common method to perform this testing is by using one or more reagents that are expected to change color when exposed to certain functional groups. These color test reagents are usually quite simple and vary in reactivity – they may only change one specific color in response to one specific type of drug or they may exhibit a range of colors in response to a wide variety of drugs.

The Marquis reagent is an example of the latter. A simple formulation of sulfuric acid and formaldehyde technically made for detection of alkaloids, the Marquis reagent may turn shades of orange, brown, red, green, or yellow in the presence of a variety of amphetamines, opiates, and other drugs. While lacking in specificity, the results can often be used in conjunction with other tests or with confirmatory results to reinforce identification of these substances.

Controls should always be used with color tests. A known positive should be used to generate the expected reaction and ensure no false negatives (positive control). A known negative, or blank, should be used to ensure a reaction does not occur when the target is not present and ensure no false positives (negative control).

Confirmatory Testing

Confirmatory testing should then typically be performed on presumptive positives. Because confirmatory testing requires expensive instrumentation and personnel, it is helpful to have some direction to save time and money (also – we need two positives most of the time anyways, so confirmatory testing may not be useful without another positive). The gold standard for confirmatory drug testing is usually gas chromatography-mass spectrometry (GC-MS), or, as an alternative, Fourier transform infrared spectroscopy (FTIR).

GC-MS

GC-MS analysis will generate a chromatogram (signal vs. time) and a mass spectrum (signal vs. mass-to-charge ratio, m/z). The chromatogram will show us the retention time (RT) – how long each compound takes to elute, or travel through the column. The mass spectrum will show us the masses of compound fragments found at each retention time. Together, this information makes GC-MS a very powerful discriminatory tool.

Sample Preparation

Sample preparation can vary depending on the physical form of the drug. For normal GC-MS testing, we need our drug to be dissolved in a volatile organic solvent. So, if the drug is an acidic salt, we need to do a basic liquid-liquid extraction (bicarbonate solution + chloroform, e.g.) to analyze the free-base version in the organic layer via GC-MS. Some labs will perform this extraction on most powders that come in – it saves time when we don’t know what it is, and drugs that are already soluble in the organic layer will still be there. Otherwise, presumptive tests or FTIR analysis can help inform the preparation process. Liquids, plants, and other forms of drugs have other sample prep considerations but we’ll only be looking at powders and pills in this class.

Standards

Prior to analysis of unknowns, a control sample must be analyzed using the same instrument method you intend to use on your unknown. This control sample is a mixture containing standards of some or all of the drugs for which that assay is designed and an internal standard (IS). A blank should also be run to ensure there are no false positives resulting from instrumental/method issues or analyte carryover from a previous run.

The internal standard is a compound that can also be added to our unknown mixture and is ideally structurally similar to our analytes. Internal standards can serve a couple of purposes. First, they are used to confirm the instrument and method are working appropriately for that run – this way, if no other compounds appear in our unknown data, we know the drugs we’re testing for are not present. Second, an internal standard can be used to obtain relative retention times (RT of drugs relative to that of IS to account for acceptable variations).

Analysis

For a GC-MS analysis to be confirmatory of a drug identification, there are some parameters that must be met:

• RT of IS must be within a given tolerance of the control RT
  • If this is not the case, the entire run is generally void
• RT of unknown must be within a given tolerance of the control RT
• Major mass fragments for that drug present at that RT
  • Number of peaks required can vary from drug to drug
  • Relative intensity of each m/z should also be within a given tolerance compared to control
• No major unidentifiable m/z peaks

Legally Manufactured Pills

Sometimes, legally manufactured, non-controlled drugs are recovered by authorities. Whether or not they were legally possessed, we still often need to identify them. If there are tablets with a pharmaceutical identifier on them, we can often consult reference materials for a presumptive identification and then confirm with FTIR or GC-MS. If you have access to an attenuated total reflectance (ATR) FTIR, you can confirm by placing a tablet directly on the ATR crystal, though grinding into a powder will usually yield a more intense signal. Capsules that can be broken open always need to be emptied prior to confirmatory testing.

If the FTIR results do not support the ID from the pharmaceutical identifiers, GC-MS analysis should generally be performed to confirm the FTIR results.

Materials and Methods

Supplies (Part 1)

1. internet access
2. camera (phone is acceptable)
3. FTIR or ATR-FTIR

Procedure (Part 1)

Pills – Visual Identification

RxList maintains a free pill lookup resource that allows you to enter markings, shape, and color of pills. If it is a legally manufactured pill, it’s pretty reliable for returning accurate results. The DEA maintains its own Logo Index with over 30000 pills. There is also now software labs can buy to enter photos of pills and automate the identification process.

1. Take a close-up photo of any pill evidence you’ve been given
2. Enter what information you have into the identifier at https://www.rxlist.com/pill-identification-tool/article.htm

Pills – Analytical Confirmation

To confirm the identity of the pills, we can use FTIR and compare to known results for the same pills.

1. Obtain a blank spectrum
2. For tablets – analyze whole pill first if possible
   • Then, ground the tablet and analyze again to compare
3. For capsules, break open and analyze the contents
4. Compare to reference spectra
5. Take note of parameters and obtain CSVs of your data for your report
6. If results do not confirm your ID from the pharmaceutical identifiers, you’ll perform an extraction later to analyze via GC-MS

Supplies (Part 2)

1. Components for Marquis Reagent
2. Storage bottle
3. graduated cylinder
4. Testing tray
5. Disposable pipette
6. camera (phone is acceptable)

Procedure (Part 2)

Make Your Own Marquis Reagent

Marquis Reagent is made with concentrated (95–98%) sulfuric acid and 40% formaldehyde. You’ll use it to perform colorimetric tests on a variety of samples provided.

1. Add 1 mL of formaldehyde to your storage bottle
2. Carefully add 20 mL sulfuric acid to the formaldehyde
3. You may adjust the total volume with the same ratio if needed (5 mL + 100 mL, e.g.)

Use Your Marquis Reagent

1. Place a small amount of each sample into individual wells of your sample tray
2. Label each sample with a marker and note your labels in your notebook
3. Carefully add a few drops of Marquis Reagent to each well
4. Color changes should happen immediately
5. Consult a color reference chart like that provided in class to try to presumptively identify each unknown

Supplies (Part 3)

1. Control sample with standard mix (TA provided)
2. GC-MS
3. GC sample vials
4. Unknown(s)
5. Pills from Part 1, if additional analysis needed
6. Internal standard solution (details indicated on board)
7. Acid/base extraction solvents
   • Suitable acids may include: hydrochloric (concentrated or diluted) and acetic (concentrated or diluted)
   • Suitable bases may include: sodium hydroxide, sodium bicarbonate, and ammonium hydroxide
   • Suitable organic solvents may include: acetone, ethyl ether, chloroform, heptane, hexane, methanol, methylene chloride, isopropanol
   • DI water
8. Test tubes or other vessels suitable for liquid-liquid extraction
9. Pipettes

Procedure (Part 3)

Sample Preparation

In order to analyze your unknown drugs via GC-MS, you’ll first need to get them into a volatile organic solvent. The type of sample (physical form, acid/base properties, etc.) and your instrumental methodology will determine how to best go about doing this. There are a variety of methods including dry solvent extractions, solvent washing, reconstitution, etc., but we will perform only simple acid/base extractions in this exercise.

1. Place ~1 mg of the unknown in the test tube
2. Use presumptive test results and refer to structures of suspected drugs to determine extraction route
   • Note: drugs within a given class (opiates, amphetamines, etc.) usually tend to exhibit similar acid/base properties
   • Alternately – you can do both acid and base extractions in separate vessels and combine the organic layers (if same solvent used), though this could potentially result in over-dilution in some cases
3. Add ~1 mL of the appropriate acid/base solution
4. Add ~1 mL of organic solvent
5. Mix gently and allow layers to separate
6. Transfer the the organic layer to the GC vial
7. Dilute as instructed if needed
8. Determine how much internal standard solution you need to add for the desired final concentration indicated on the board and add it

GC-MS Analysis

Your control mix will be pre-prepared and the contents detailed on the board. Your instrumental method will be pre-prepared as well, but you should make sure to note the details of the GC and MS settings provided by your TAs for your methods section.

1. Run the control mix
2. Ensure IS and all standards in the control mix are accounted for
   • Review RTs and fragments
3. Run a blank
   • may be omitted at TA discretion
4. Run unknown samples
5. Ensure IS is accounted for and that RT is no more than ~0.1 min different than control
   • Unless instructed otherwise
6. Look over RTs and fragments for unknowns
7. Obtain CSVs of chromatograms and spectra to plot for your report

Supplies (Part 4)

1. Unknown samples
2. FTIR

Procedure

FTIR of unknowns

We may also be interested in using FTIR to confirm the identity of unknown drugs.

1. Obtain blank spectrum
2. Analyze unknowns
3. Compare to reference spectra
4. Take note of parameters and obtain CSVs of your data for your report