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High-Throughput Sample Preparation on a 96-Well Plate and LC-MS Analysis of Thimerosal in Biological Products

Khakinejad, Mahdiar | 2010

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 41913 (03)
  4. University: Sharif University of Technology
  5. Department: Chemistry
  6. Advisor(s): Bagheri, Habib; Eshaghi, Ali
  7. Abstract:
  8. Thiomersal is an organomercury compound. This compound is a well established antiseptic and antifungal agent. It has been used as a preservative in vaccines, immuno-globulin preparations, skin test antigens, antivenins, ophthalmic and nasal products, and tattoo inks. Thiomersal is very toxic by inhalation, ingestion, and in contact with skin, with a danger of cumulative effects. It is also very toxic to aquatic organisms and may cause long-term adverse effects in aquatic environments.
    Thiomersal's main use is as an antiseptic and antifungal agent in vaccines. In multidose injectable drug delivery systems, it prevents serious adverse effects such as the Staphylococcus infection that, in one 1928 incident, killed 12 of 21 children inoculated with a diphtheria vaccine that lacked a preservative. After this incident addition of preservative to mulitdose vaccines becomes an obligation.
    In vaccine industry it is essential to determine the concentration of thiomersal. High concentration of thiomersal is hazardous due to its mercury contained, in the other hand low concentration of thiomersal leads to low perseverant vaccine. Because of too many samples in the vaccine industry, the mentioned analysis must be fast and reliable, so in this study, the objective is to develop sensitive, reliable and high-throughput method to analyze the thiomersal in vaccines.
    For the first time, a high-throughput ultrasonic-assisted micro-liquid-liquid extraction on 96 well-plates analysis followed by ultra high performance liquid chromatography-tandem mass spectrometry was developed to analyze thiomersal in biological samples. The previously used methods are rather time consuming and tedious using ditiazon complexing agent and UV-Vis spectrophotometry. Very fast chromatography runs couple by ion trap Ms2 determination, makes this analysis fast, selective and sensitive. Many parameters optimized as Ion trap parameter, liquid chromatography and extraction parameters. Optimum extraction parameters obtained, 0% NaCl for salt effect, PH=4, 1-decanole as extraction solvent and extraction time of 5 min. The dynamic range was obtained 10-200μg/L and detection limit reach to 3μg/L. Relative standard deviation percentage was 5.3% at 20μg/L. The developed method was successfully applied to six different types of vaccines providing fast, reliable, low solvent consumption and sensitive analysis
  9. Keywords:
  10. Liquid-Liquid Extraction ; High Performance Liquid Chromatography ; Mass Spectrometery ; Thiomersal ; Biological Products ; 96 Well-Plate

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