Almost 44,000 North Americans die each year from drug overdose, according to overdoseday.com. Determining the drug type as well as amount ingested can be a challenge for forensic investigators. Even linking the drug ingested to the source can be a difficult endeavor. Many forensic labs make use of analytical instrumentation to analyze drug and toxicology evidence. In this study, you will act as the manger for a forensic laboratory who is tasked with linking unknown pill samples to a set of known over the counter (OTC) analgesic drugs.
Police responded to a call of a possible overdose in Worcester Country. They found several different pills on a nearby bathroom countertop and are actively working to identify the drugs. Are the pills OTC, prescribed, or illicit? Many of the markings on the drugs indicate they are likely OTC, but analytical evidence is needed. While another lab is working to identify the drugs and drug metabolites present in the victims stomach and blood, they will need evidence that the drugs present at the crime scene are consistent with those found in the body. That’s where your lab comes in.
As manager of the lab, you asked your technician to develop a liquid chromatography method to separate seven common OTC analgesic compounds using three different column chemistries. While he determined the best mobile phase combination of protic solvents (55% phosphate buffer to 45% methanol), he has not yet determined which column is best for the separation. The LC is equipped with a diode array UV-Vis detector allowing for identification based on multiple wavelengths. As the lead contact with police, you will be the one providing information and so you want to see the data yourself.
The chromatograms he collected are provided here in various packets. The green trace is from detection at the wavelength listed in the caption. The black line is from detection at 245nm. Many of these chromatograms are zoomed in on peaks (they don’t start at 0 min retention time). In this case, you’ll see a smaller chromatogram in the top right of the rectangle that outlines the entire injection run. Supplemental information, including column types and analyte structures, is provided as well. Your goal is to determine if you can conclusively identify the unknown tablets taken from the crime scene using your lab’s method.
- What type of liquid chromatography (reversed or normal phase) is being performed? What would you expect elution order to be based on?
- First, compare all columns under pH 7.0 for the separation of all seven analgesics (page 1). Based on the chromatograms, which column would you choose for these analytes? Why?
- Next, look at the effect of pH on the separation. You are provided with RP-Amide and C18-PFP data (pages 2-7). Which pH would you choose for these analytes on the column you chose in question two? Why? Why are the peak shapes so different from pH to pH? Why do you suspect some of these peaks are tailing? What are some possible ways you could investigate the reason for the tailing?
- Using identification runs for 55% pH=7 phosphate buffer on the C18-PFP column, identify peaks in the “All Analgesics” run at these conditions (page 6). Briefly explain how you arrived at your answer.
- Based on chemical structures, physical properties, chromatographic conditions, and your answer to question one, propose a comprehensive justification of retention order of “All Analgesics” analytes on C18-PFP at 55% pH=7 buffer (page 6).
- Now find the chromatograms of the unknown pills (page 8). Try to identify the peaks in each tablet using the comparable known chromatograms (page 6). Make a list or table for each tablet. Can you conclusively identify the drug in each pill? Can we confidently rule out prescription or illicit substances from the unknown tablets? Explain your findings.
- If there are any unknowns you aren’t confident identifying, which additional testing methods would you recommend? Why? Remember, you’re in a forensic lab, so only traditional equipment is available.
- Since you’re the boss, you would get called in to testify if necessary. How would you explain LC and the different columns to a judge and jury?