LC MS

why LCMS? Advantages and Challenges

1. LIQUID CHROMATOGRAPHY MASS SPECTROMETRY (LC-MS) AN OVERVIEW Presented by: Akshay Prakash Nehe Roll no. 52 Third year B pharmacy (sem-6) Amrutvahini college of pharmacy, Sangamner

2. Content – WHY LCMS? ADVANTAGES & CHALLENGES Limitations of GC and GCMS Advantages of LC & LCMS Challenges of Interfacing LC with MS The Early Developments in the Interfaces and Ionization. The current state of LCMS technology - Atmospheric Pressure Ionization-(Electrospray Ionization-ESI) - Formation of Positive ions and Negative ions in LCMS - Atmospheric Pressure Chemical Ionization (APCI) - Atmospheric Pressure Photo Ionization (APPI) Illustrative Examples Frank.P (2010) CSOI: The fascinating history of development of LCMS UK, University of Greenwich at medway.

3. Applicability of various ionization techniques Frank.P (2010) CSOI: The fascinating history of development of LCMS UK, University of Greenwich at medway.

4. Why LCMSMS ? A few Advantages of Liquid Chromatography - Analysis of labile analytes - Analysis of more polar compounds without derivatization - Analysis of significantly higher masses - Reduction of lengthy clean-up Advantages of MS-MS? - Additional structural elucidation - Further reduction of clean-up - Specificity - Useful MS modes Frank.P (2010) CSOI: The fascinating history of development of LCMS UK, University of Greenwich at medway.

5. Early stages of LCMS Development Liquid Chromatograph Interface/ Ionization Source Mass Analyzer EM Detector Limited Pumps Limited Columns DLI/EL PB/EI Thermospray Dyn. FAB Single Quad Triple Quad 3D- Trap Low Sensitive Under high vacuum Very few Manufacturers/ Less Competition Slow Computers/ Software Frank.P (2010) CSOI: The fascinating history of development of LCMS UK, University of Greenwich at medway.

6. Present Day LCMS&LCMSMS Choice of Pumps - ESI Choice of Columns - APCI Good Literature - APPI Knowledge Base Atmospheric Training Pressure High Vacuum Powerful Computers/SW Very Good Competition for Technology and Quality Liquid Chromatography Ionization Source Mass Analyzer HED-EM Quadrupoles 3D/2D Traps TOF Hybrids Orbitrap Sensitive Detectors Frank.P (2010) CSOI: The fascinating history of development of LCMS UK, University of Greenwich at medway.

7. A Few of Commercially Available LCMSMS Systems Thermo Waters Agilent Sciex

8. Types of Ionization & Sources Electrospray Atmospheric Pressure Chemical Ionization (APCI) Atmospheric Pressure Photo-Ionization (APPI) Eleanor.R. & Steve.B(2017)CSI:MS-API use& applicability University of Southampton.

9. Atmospheric Pressure Ionization (API) Revolutionized LC/MS opening it to a wide array of applications Desolvation and/or ionization of analytes occurs at atmospheric pressures Gas phase ions are sampled by the high vacuum mass spectrometer Eleanor.R. & Steve.B(2017)CSI:MS-API use& applicability University of Southampton.

10. Atmospheric Pressure Ionization

11. ESI Spray Process and Formation of Ions

12. Electrospray: Overview Eleanor.R. & Steve.B(2017)CSI:MS-API use& applicability

13. ESI: Droplet Size Reduction & Fission Droplet size reduction occurs by the continual repetition of two processes: Desolvation (evaporation of neutral solvent and volatile buffers) Droplet fission caused by electric repulsion between like charges Eleanor.R. & Steve.B(2017)CSI:MS-API use& applicability

14. ESI: Production of Charged Droplet A large voltage (up to 6kV) is applied between the end of a capillary carrying the LC mobile phase and the mass spectrometer entrance. Ions (of the polarity) are drawn out toward the counter electrode (curtain plate) pulling the mobile phase along. When the excess charge at the tip of the capillary overcomes surface tension, a droplet is formed. Eleanor.R. & Steve.B(2017)CSI:MS-API use& applicability

15. ESI: Pros and Cons Pros: Soft ionization technique, resulting in little decomposition of labile analytes. Generally produce only molecular ions. Multi charged analytes easily produced, allowing proteins to be analyzed. Wide range of analytes Highly efficient ion production. Cons Cannot ionize Non polar compounds Eleanor.R. & Steve.B(2017)CSI:MS-API use& applicability University of Southampton. 05/04/202

16. APCI Process- Nozzle Detail George.k,Eugenio.A(2012).Mass spectrometry chapter 9

17. APCI Mechanism APCI is useful for less polar compounds. Therefore the mobile phase will have more organic eluents In the mobile phase a solvent such as Methanol is added if not already present, the methanol undergoes protonation. The protonated Methanol then Transfers one proton to the non polar molecule to form the (M+H)+ ion Eleanor.R. & Steve.B(2017)CSI:MS-API use& applicability

18. APPI Ionization Eleanor.R. & Steve.B(2017)CSI:MS-API use& applicability

19. Typical APPI compounds Estrone Hydrocortisone Fluorathene Eleanor.R.(2017)CSI:MS-APPI use& applicability

20. How do we choose the type of Ionization Based on Specific Applications. Increasing polarity and molecular weight and thermal instability favors electrospray. - Most drugs of abuse are highly polar and are easily analyzed using electrospray. - High molecular weight proteins also require electrospray Lower polarity and molecular weight favors APCI or APPI. - Lower background but compounds must be more thermally stable. Denise.W & Jason.W (2014) CSOI: Development of highly selective LCMS Ionization.

21. Case study with examples LC-MS in Food Analysis (Targeted Analysis) 1. VETERINARY DRUGS AND GROWTH PROMOTERS - Multimethods were developed for the analysis of a large number of veterinary drugs in milk, fish, eggs and meat samples. - Veterinary drugs, especially antibiotics, are used for the control of various diseases. - For antibiotic, maximum residue limits (MRLs) are established because of their possible harmful effect on human health. Robust & sensitive technique for detection LC-MS/MS Utilization of APCI in LC-MS/MS increased method’s sensitivity Extract ion chromatograms for a milk sample fortified at 10 ug /L. George.k,Eugenio.A(2012).Mass spectrometry chapter 9

22. Ion chromatograms of milk sample George.k,Eugenio.A(2012).Mass spectrometry chapter 9

23. 2. Mycotoxins Mycotoxins are toxic chemical products which are produced as secondary metabolites by fungal species on agricultural commodities. Their presence in food products can cause serious harm to human and animal health. Sample preparation technique used- Liquid- liquid extraction (LLE) Accelerated solid extraction (SPE) Solid phase extraction (SPE) Analytical technique- LC-QQQ-MS system For determination of mycotoxins in eggs, olive oil, wheat, maize, barley, oats, sweet pepper and moldy food George.k,Eugenio.A(2012).Mass spectrometry chapter 9

24. An overlap total ion chromatograph (TIC) of 17 mycotoxins and zearalenone as internal standard (IS) under +ve & -ve modes, with all peaks eluting in 6 min

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