Drug Testing Methods

Chapter 6 Objectives

  • Explain the general principles of each analyte method

  • Discuss the limitations of each analytic method

  • Compare and contrast the various analytic techniques

  • Discuss existing clinical applications for each analytic technique

  • Describe the operation and component parts of the mass spectrometer and gas chromatograph

  • Outline the quality assurance and preventative maintenance procedures involved with the mass spectrometer and gas chromatograph

Chromatography

  • Principle:

    • Separates a complex mixture into individual components based on the differences in their relative affinities for two different media.

    • Differences are due to physical interactions with those media.

    • The mixture is dissolved in a fluid solvent (gas or liquid) called the mobile phase, which carries it through a system (a column, capillary tube, plate, or sheet) where a material called the stationary phase is fixed.

  • Categories of Chromatography:

    • Several methods are employed to achieve separation, including Adsorption, Partition, Steric Inclusion, and Ion-Exchange.

Components of Chromatography

  • Mobile Phase:

    • A moving solvent that can be either gas or liquid; it carries the complex mixture (sample).

  • Stationary Phase:

    • A solid or liquid through which the mobile phase flows.

  • Separated Components:

    • Referred to as eluate; separation is based on the differential partitioning between the mobile and stationary phases.

Modes of Separation

  • Adsorption (Liquid-Solid):

    • Molecules most soluble in the mobile phase move fastest.

  • Partition (Liquid-Liquid):

    • Relative solubility in non-polar (organic) solvent and polar (aqueous) solvent.

  • Steric Inclusion:

    • Separates solute molecules based on size and shape; packing material for column can be gel or rigid inorganic materials (e.g., silica or glass).

  • Ion-Exchange:

    • Separation occurs via the magnitude and charge of ionic species; resin has charged functional groups immobilized on resin beads.

Thin-Layer Chromatography (TLC)

  • Stationary Phase:

    • A thin layer of adsorbent material, e.g., alumina, cellulose, dextran, or silica gel, coated on a solid support (e.g., plate or sheet).

  • Procedure:

    • Sample is applied as a small spot at the base of the plate.

    • The mobile phase (solvent) is placed in a closed container, saturating the atmosphere with solvent vapor. The plate stands in edgewise in the solvent; solvent migrates up the stationary phase via capillary action.

    • Sample components dissolve and move at different rates based on solubility.

  • Retention Factor (Rf):

    • Rf=distance travelled by the componentdistance travelled by the solventRf = \frac{\text{distance travelled by the component}}{\text{distance travelled by the solvent}}.

  • Developing Stage:

    • The solvent reaches a predetermined height, then remove from solvent and dry the plate.

  • Interpretation Stage:

    • Quality control requires standards to reach specific Rf's and reactivity at various stages as predicted.

    • Identification is done by comparing the Rf of components to standards.

Sample for Drug Testing by TLC

  • Typically uses random urine samples; a minimum of 10 mL is required for both acid and basic drug testing.

  • Other samples may include blood, bile, and gastric specimens.

TLC for Drug Testing Procedure

  1. Extraction Stage:

    • Sample added to extraction tubes.

    • Tube “A” extracts basic and neutral drugs at pH 9.

    • Tube “B” extracts acidic and neutral drugs at pH 4.5.

    • The tubes are centrifuged.

  2. Concentration Stage:

    • Supernatant is removed and concentrated.

  3. Inoculation and Development:

    • Sample is inoculated onto a plate (or sheet) for development.

Identification of Drugs by TLC

  • Drugs are identified post-development through stages involving staining and comparison with standards. Each drug must react as expected in each stage.

  • A compendium with over 200 different drugs is typically used according to standards from H. Wagner and S. Bladt.

Troubleshooting in Identification

  • If known drugs are available but no chromatogram, drugs can be tested using a pharmacy pill for comparison.

Notes on TLC for Drug Testing

  • Advantages:

    • Broad applicability for various substances.

    • Capable of processing several samples simultaneously.

    • Requires a small amount of sample.

  • Disadvantages:

    • Labor-intensive; difficult to automate.

    • Samples usually require pre-treatment and multiple chemicals (OH&S considerations).

    • Subjectivity in observing results by analyst; factors affecting migration can lead to reproducibility issues.

    • Non-specific method—cannot conclusively identify a particular drug or metabolite.

High Performance TLC

  • Semi-automated sample applicators are used.

  • Plates feature uniform sorbent thickness, finer particles, and better solvents than manual TLC.

  • Densitometer can measure each spot and calculate concentration against standards, allowing quantitation of separated compounds.

Gas Chromatography (GC) Methods

  • Separates mixtures of volatile compounds by forcing them through a stationary phase (solid or liquid).

  • Two types of gas chromatography are:

    • Gas-Solid Chromatography (GSC): uses a solid stationary phase.

    • Gas-Liquid Chromatography (GLC): involves a non-volatile liquid stationary phase.

  • Mobile Phase:

    • Inert gas such as nitrogen, helium, or argon, referred to as the carrier gas.

Components of Gas Chromatography

  • Flow Controller: maintains the flow rate of the carrier gas.

  • Sample Injector: introduces the sample into the mobile phase.

  • Column: where the separation occurs; configurations include:

    • Packed columns filled with inert particles coated with non-volatile liquid phase.

    • Capillary wall-coated column.

Gas Chromatography: Sample Injection and Characteristics

  • Injection must occur through a septum as a gas or at above boiling point to allow effective separation.

  • Retention Time:

    • Molecules with low affinity for stationary phase maintain presence as gas and have shorter retention times versus those with higher boiling points that exhibit longer retention times.

Chromatograms in Gas Chromatography

  • The effluent passes through a detector, generating an electrical signal proportional to the concentration, displayed as a series of peaks. The number of peaks indicates mixture complexity.

Identification and Quantitation in Gas Chromatography

  • Qualitative identification is made by comparing peak positions with those from reference solutions.

  • Quantitation is achieved by measuring peak size relative to standards, which corresponds to the amount of analyte present.

Quality Control in Gas Chromatography

  • Assessments compare retention times and peak heights of standards to expected values.

Detectors for Gas Chromatography

  1. Thermal Conductivity (TC):

    • Employs wire filaments that change resistance with variations in temperature due to the presence of different analytes.

  2. Flame Ionization Detector (FID):

    • Sample is combusted in a hydrogen flame, producing current proportional to ion concentration; noted for high sensitivity.

Gas-Liquid Chromatography for Volatile Compounds

  • Applicable for qualitative and quantitative assessments of various compounds such as ethanol, methanol, isopropyl alcohol, and others.

High-Performance Liquid Chromatography (HPLC) Methods

  • Utilizes a liquid mobile phase, typically at ambient temperatures, with detectors commonly including spectrophotometers.

  • A pump forces the mobile phase through the column, with direct sample injection.

  • Detection mechanisms include visible or UV spectrophotometers, fluorescence, amperometric, or mass spectrometry.

Reversed Phase HPLC

  • The stationary phase consists of nonpolar molecules on silica gel particles, separating ionic, non-ionic, and ionizable samples using buffers for ionic characteristics.

Mass Spectrometry (MS)

  • Primarily used for definitive identification of samples, commonly employed as a detector for GC or HPLC.

  • A mass spectrometer determines the ratio of a molecule's mass to its charge by volatilizing and ionizing samples, separating them according to their mass-to-charge ratio.

Components of a Mass Spectrometer

  • Includes:

    • Sample inlet

    • Ionization source

    • Mass analyzer

    • Ion detector

MS Sample Introduction and Ionization Procedures

  1. Electron Ionization (EI):

    • Most frequent in GC/MS; molecules are bombarded with high-energy electrons to produce charged ions.

  2. Atmospheric Pressure Ionization:

    • Liquid introduced into the ionization source directly results in charged droplets.

Components of Mass Analyzers

  • Measurement of mass-to-charge ratio occurs within four types of analyzers: quadrupole, ion trap, tandem mass spectrometry, and high-resolution MS.

  • Quadrupole Mass Analyzer:

    • Allows only ions of specific m/z values to pass to the detector, scanning for a full spectrum of m/z.

High-Resolution Mass Spectrometry

  • Achieves mass of the compound per the peak width, and measures a wide array of analytes simultaneously.

  • Orbitrap:

    • Uses tangential ion injection to create stable orbits correlated to m/z values.

Applications of Mass Spectrometry

  • Utilized in conjunction with GC as a detector for confirmation and quantitation of drugs, providing highly specific results with minimal false positives.

  • Essential in structural information and weight determination for compounds such as Vitamin D, testosterone, and others.

  • The technique also plays a role in advanced studies such as proteomics, where it investigates protein products related to human diseases, and microbial identification.

Common Sources of Error in Chromatography and Mass Spectrometry

  • Factors affecting results can include:

    • Temperature variations

    • Column wear

    • Gas purity

    • Uninterrupted flow rates

    • Buffer charges in HPLC and MS analyses

    • Ion suppression in LC/MS

    • Technical handling errors by the analyst.

References

  • Bishop, M.L., Fody, E.P., Schoeff, L.E., Clinical Chemistry: Principles, Techniques, and Correlations, 8th Edition. Wolters Kluwer, 2018.

  • Burtis, C.A., Ashwood, E.R., Bruns, D.E., Tietz: Fundamentals of Clinical Chemistry, 6th Edition. Saunders Elsevier, 2008.

  • Armbruster, D. et al., Toxicology and Drug Testing in the Clinical Laboratory: Approved Guideline – Second Edition, Clinical and Laboratory Standards Institute, 2007.