Introduction

Purpose

Identify and quantitate substances present in a urine sample.

Urine Toxicology

Urine is the most common matrix used for most toxicology testing. It is easy to collect from living or deceased specimens and tends to have the highest concentration of drugs and their metabolites. Typical analytical schemes consist of some form of sample clean-up, gas or liquid chromatography, and mass spectrometry.

Drug Metabolism

Metabolites are the result of the body’s attempt to make a drug less active or more water soluble through modification (like oxidation or reduction) and/or conjugation (like methylation or glucuronidation). Understanding the pharmacological behavior of these substances is needed in order to know what metabolites to even look for – consulting peer-reviewed literature is usually a good jumping off point.

Sample Preparation

Sample preparation for urine samples can vary pretty extensively from lab to lab. The end goal is usually to reduce matrix effects caused by the many ionic and organic compounds present in urine aside from the analytes we want to measure. This can be accomplished with varying levels of effectiveness depending on the preparation method. Liquid/liquid extractions with organic solvent can be prone to lack of sensitivity, simple dilution tends to reduce but by no means eliminate matrix effects and can quickly result in a dirty sample inlet, and solid phase extraction can be very effective but more expensive and time consuming.

Solid phase extraction uses a stationary phase adhered to a filter in a syringe or well-plate. The stationary phase is designed to retain the drugs and metabolites while the rest of the sample is rinsed through with a weak solvent. Then, a strong solvent can be used to pull the analytes through the filter in a more purified solution. This can either be directly analyzed or dried down and reconstituted.

Liquid Chromatography – Tandem Mass Spectrometry

Liquid Chromatography – Tandem Mass Spectrometry or LC-MSMS is a useful method for aqueous or mixed aqueous solutions like those often encountered in toxicological testing. While traditional GC-MS requires a thermally labile analyte in a volatile organic solvent, LC-MS instrumentation can utilize a variety of mobile phases, stationary phases, and ionization sources that more readily facilitate the analysis of biological matrices.

Materials and Methods

Supplies (Part 1)

1. Urine samples
2. Reference drugs and metabolites
3. DI water
4. Methanol
5. Phosphate Buffered Saline (PBS)
6. Acetonitrile
7. Solid Phase Extraction (SPE) Cartridge
8. Internal standard

Procedure (Part 1)

Prepare Urine Sample(s)

1. Add 3.0 mL of methanol to SPE cartridge
2. Add 3.0 mL of DI water to SPE cartridge
3. Add 1.0 mL of PBS to SPE cartridge
4. Add 1.0 mL of Urine sample to SPE cartridge
5. Wash SPE cartridge with 2.0 mL of DI water
6. Wash SPE cartridge with 2.0 mL of methanol
7. Let SPE cartridge dry for about 20 minutes
8. Elute sample with 2.0 mL of acetonitrile
9. Add internal standard
10. Store for Part 2 analysis

Prepare Calibration Solutions

1. Determine an appropriate range of calibration solutions for the reference materials you’re provided with the help of your TA
2. Create the calibration solutions and store for Part 2
3. Add internal standard (be sure to account for volume in concentration calculations)

Supplies (Part 2)

1. Prepped urine sample
2. Methanol
3. DIwater
4. Acetonitrile
5. Standard solutions
6. LC-MS

Procedure (Part 2)

Analyze Part 1 Solutions

1. Run standard solutions using pre-made method via LC-MS
   • Check for internal standard
   • Run in triplicate
2. Run urine sample solution
   • Check for internal standard
   • Run in triplicate
3. Determine the identity of drugs in the urine sample
4. Obtain CSV of at least one chromatogram of combined standards (highest concentration is probably preferable)
5. Obtain CSV of one mass spectrum for each compound in combined standards
6. Obtain CSV of chromatogram and mass spectra for urine sample
7. Obtain area measurements for all data