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4: Drug Analysis of Plasma Samples

  • Page ID
    364569
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    Drug Analysis of Plasma Samples

    You are supplied with extracts* of the woman’s blood for analysis and comparison with samples of powder found in tablet bottles at her home. Analysis is by thin layer chromatography and/or high performance liquid chromatography, depending on time and availability of equipment. You are also supplied with GC-MS data for the extracts.

    *The isolation and identification of an unknown drug in a body fluid is often required as part of forensic and clinical chemistry studies. The first task is to isolate the drug compound from the polar, protein-containing aqueous matrix. Solid-phase extraction was used as follows:

    1. Dilute 1 ml of the serum sample with water (3 ml). Acidify the diluted serum with orthophosphoric acid (87%, 10 µl). Check pH with pH paper. The solution should be about pH 3
    2. Place a C18 SPE cartridge on the vacuum chamber and start the suction.
    3. When you are ready to start wash the cartridge with 1ml of 2% methanol in water to activate the stationary phase. Without letting the cartridge dry out, immediately load the acidified serum sample onto the cartridge and run though. Then allow the cartridge to run dry under vacuum for 1 min. Collect the solution that has run through (which should contain any protonated amines that will not have been retained on the neutral column) and treat as step f.
    4. Elute the cartridge with acidified ethyl acetate containing 1% acetic acid (1 ml). Collect the eluent – this is the acid and neutral fraction.
    5. Dry the cartridge with vacuum (1 min).
    6. Take the collected solutions from part c and basify with 10-M potassium hydroxide (10 µl) and check the pH with pH paper (should be approx pH 9). If it is still acidic add more potassium hydroxide.
    7. Activate the cartridge again with 1ml of 2% methanol in water to activate the stationary phase and immediately load this basified sample (approx 5 ml) onto the cartridge, wash with 2% methanol in water (1ml).
    8. Allow the cartridge to dry under high vacuum (1 min)
    9. Elute with basic ethyl acetate containing 2% 2M NH4OH (1 ml) and collect the eluate, which is the basic fraction.
    10. Acid/neutral and basic fractions can now be analysed.

    As the time available for this experiment is limited, you are supplied with acid/neutral and basic extracts already prepared.

    Samples

    Two extracts from the woman’s blood plasma: an acid/neutral extract and basic extract (both in methanol). Samples of powder found in tablet bottles at the woman’s home. These should be dissolved in 50:50 methanol: water for analysis by HPLC. As you are not carrying out a quantitative analysis, you do not need to prepare accurate dilutions: about 10 mg of powder per 25 ml of solvent should give a suitable concentration for analysis.


    4: Drug Analysis of Plasma Samples is shared under a not declared license and was authored, remixed, and/or curated by LibreTexts.

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