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Chemistry LibreTexts

Instructor's Notes

Determination of Drugs in Blood Plasma by GC/MS

References

Cody, John T.; FoItz, Rodger L. GC/MS Analysis of Body Fluids for Drugs of Abuse. In Forensic Applications of Mass Spectrometry; Yinon, Jehuda Ed.; CRC Press: Boca Raton, FL, 1995; pp 13-19.

Jatlaw, Peter I. Drug of Abuse Profile: Cocaine. ln Analytical Aspects of Drug Testing; Deutsch, Dale G., Ed.; Wiley: New York, 1989; pp 229-245.

Thurman, E.M.; Mills, M.S. Solid Phase Extraction: Principles and Practice; Wiley: New York, 1998; chapter 8.

McLafferty, Fred W.; Stauffer, Douglas B. The Wiley/NBS Registry of Mass Spectral Data; New York,1989; 7 vols.

Shuster, Louis. Pharmacokinetics, Metabolism, and Disposition of  Cocaine. Cocaine: Pharmacology, physiology, and clinical strategies; Lakoski, Joan M., Galloway, Mathew P., White, Francis J., Eds.; CRC Press: Boca Raton, FL, 1992; pp 1-6.

Goal

To determine the identity and quantity of any illicit drugs present in a blood plasma sample or urine sample.

Suggested Method of Analysis

Solid phase extraction to isolate the drugs; gas chromatography – mass spectrometry for identification and quantitation

Preparation of standards, samples, and reagents

Choose an illicit drug to use in the experiment.  Stock solutions are available from chemical supply houses (e.g. 1000 µg/mL stock from Sigma-Aldrich) without requirement of a DEA license.  Drug metabolites, e.g. benzoylecgonine from cocaine, are more realistic, but often the standards are more expensive.  Most drugs are amines and thereby basic.  These procedures have been written for use of such a basic drug.  If an acidic drug is used instead, the procedures may need to be modified.

Prepare a working standard solution containing about 10 µg/mL of the drug in methanol. Use a syringe or pipet to place a few µL of drug stock solution in a 5-mL volumetric flask.  Dilute with methanol to the mark, mix the solution, and store the sample in a glass vial.  Store some of the standard in a glass chromatography vial and refrigerate it.  The working standard solution may only be good several days.  Use this solution to confirm the chromatographic determination and mass spectrometry of the drug.

Prepare an aqueous standard solution containing about 0.50 µg/mL of an illicit drug. Use a syringe or pipet to place a few µL of drug stock solution in a 50-mL volumetric flask.  Dilute with deionized water to the mark, mix the solution, and store the sample in a glass vial.  Refrigerate.  The standard solution may only be good several days.  Students had difficulty detecting cocaine when the sample had been prepared more than a week prior to testing.  Use this solution to check the collection efficiency of the SPE process.

Prepare a simulated blood plasma or urine sample solution containing about 0.30 μg/mL of an illicit drug. Use a syringe or pipet to place a few µL of drug stock solution in a 50-mL volumetric flask. Dilute to within a few mL of the mark with deionized water. Adjust the color of the solution, as described elsewhere, to simulate blood plasma or urine. Add water to the mark, mix the solution, and store the sample in a glass vial. Refrigerate. The sample solution may only be good for several days.

Solid phase extraction of a drug from a blood plasma or urine sample

Condition a mixed-mode (C8 and strong cation exchanger) cartridge (3-mL volume; 100 mg resin), load the sample, wash the cartridge, and elute the drug in the following manner:

  flow rate:

condition

3.0 mL methanol

3.0 mL 0.050 M, pH 6.0 phosphate buffer

 

5 mL/min

5 mL/min

load

5.0 mL sample plus 3 mL pH 6.0 buffer

 

2 mL/min

wash

3.0 mL water

3.0 mL 0.1 M HCl

3.0 mL methanol

 

2 mL/min

2 mL/min

2 mL/min

elute

5.0 mL 80:18:2 ethyl acetate: methanol : NH4OH

 

2 mL/min

Set the flow rate by adjusting how much vacuum is applied. Do not let the resin dry out at any time during the procedure. Elute the drug with 5 mL of mixed solvent, at a flow rate of 2 mL/min, into a disposable test tube. Aspirate about 1 minute at higher vacuum to collect all of the eluate.

Reduce the volume of the eluate to just about zero by solvent evaporation (warm the solution to 50 °C while flowing nitrogen gas over top of it). This takes about 30 min. Add 0.50 mL of methanol to redissolve the drug, and then transfer the processed sample to a 2-mL chromatography vial. Store the solution in the refrigerator. Perform the GC-MS analysis as soon as possible.

Gas chromatography - mass spectrometry

The oven temperature program for the gas chromatograph is:

Initial value: 175 °C    Initial time: 2.0 min
Rate: 15 °C /min
Final value: 225 °C    Final time: 5.0 min

Use a split injection of 5 μL with a 20:1 split ratio. The flow rate of helium is 1 mL/min. The mass spectrometer is set to scan from 50 to 350 m/z with a start time of 2.0 min. Record the total ion chromatogram, the peak areas, and the mass spectrum of the drug(s).

Compare the mass spectrum generated by the drug in the simulated plasma sample to known spectra of the possible drugs (prepare a list of possibilities for the students). Find known mass spectra in an electronic library that is part of the mass spectrometer software or in printed compendia from the library*. In the former case, one can also perform a library search and obtain the best matches. Once the drug is identified, the peak areas of the standard and sample can be compared and calculations made to estimate the concentration of the drug in the sample.

*Some mass spectra of drugs can be found in The Wiley/NBS Registry of Mass Spectral Data. For example:

heroin (vol. 4, p. 4165, fig. 11)
phenobarbital (vol. 2, p. 1645, col. 1, fig. 3)
morphine (vol. 3, p. 2484, col. 2, fig. 11)
cocaine (vol. 3, p. 2901, fig. 6).