Agilent 6100 training course

1. Introduction to LC-MS using the SCU Agilent 6100 Single Quadrupole

2. Introduction Training outline LC-MS introduction Basics of LC-MS as Analytical Technique Introduction to the Agilent 6100 Running a sample Data Analysis : Qualitative and Quantitative analysis Best Practices: Operating/Troubleshooting What does LC-MS stand for?? Liquid Chromatography – Mass Spectroscopy

3. Introduction

4. Introduction Requirements of an Analytical technique: What are the questions we expect an Analytical technique to answer? What is in the sample? Qualitative analysis e.g. Was there really caffeine in that coffee? 2) How much is present? Quantitative analysis e.g. How much caffeine is present? Analytical technique needs to identify the component unambiguously, needs to be sensitive, reproducible and needs to complete analysis within a reasonable time cost and ease of use. LC-MS

5. Introduction Pharmaceutical sciences -Metabolic Profiling -Identification and purity -Formulation -Pharmacokinetic profiling Forensic sciences -Explosives Performance enhancing drugs: cortiosteroids -Adulterants in consumer products: Bisphenol A Clinical Sciences -Metabolic Disorders -Therapeutic Drug monitoring Environmental Chemistry -EPA Appendix VIII -Pollutants : chlorophenoxy herbicides Biochemistry and Biotechnology -Post translational modifications -Non covalent complexes -Peptide mapping Synthetic Chemistry -Combinatorial Libraries -Reaction products Food Chemistry -Drug residues: Bovine hormones -Artificial Sweeteners -Flavor components: wine antioxidants -Fungal metabolites: mycotoxins Petroleum Chemistry -Oil exploration - Fractionation of oil : Polycyclic aromatics

9. LC-MS-UV Basics of LC-MS Ion source:makes ions Data System UV detection HPLC:provides separation Mass Analyzer: Separates ions Detection Device 1:9 split Electrospray APCI Maldi Quadrupole Time of Flight Ion Trap Reverse Phase Ion Chromatography Aqueous Normal Phase Chromatogram Mass spectrum LC-MS Chromatogram Providing separation of components and mass spectrum of each.

11. Stream of solution sprayed out of capillary at high voltage (ca. 3 – 5 kV).

12. Charged droplets formed by spray.

13. Solvent evaporated by stream of warm N2.

14. As droplet shrinks, charge density increases until analyte ions ejected.

15. Pseudomolecular [M+H]+ - positive mode or [M-H]-ions formed or water adducts.

17. Stream of solution sprayed out of capillary at high voltage (ca. 3 – 5 kV).

18. Charged droplets formed by spray.

19. Solvent evaporated by stream of warm N2.

20. As droplet shrinks, charge density increases until analyte ions ejected.

21. Pseudomolecular [M+H]+ - positive mode or [M-H]-ions formed or water adducts.

23. Basics of LC-MS In general TIC data is more favored than SIM data since it contains all the m/z values For the eluting components. TIC’s are often processed to give Extracted Ion Chromatograms -EIC – where the trace for only one m/z value is “extracted” from the TIC highlighting the presence of a component of interest. TIC and EICs of carbamate pesticides spiked in a tomato extract

25. Is concentration dependant- can be used for quantitative analysis.

26. Can be coupled to Reverse phase HPLC separations – amenable to analysis of a wide range of compounds. - It is an “Active” detection method.

27. Basics of LC-MS LC-MS is an “Active” detection mode as opposed to LC-UV. The interface accomplishes the physiochemical process of ionization, evaporation, pressure reduction, and in some cases molecular fragmentation. These can be affected profoundly by Solution phase chemistry –pH, and mobile phase composition.

28. Intro to Agilent 6100 Agilent LC-MS 6100 : where do I start? Degasser UV detector Solvent reservoirs Column compartment Mass Spectrometer Mass Analyzer and detector Data System HPLC Ion Source Pump Autosampler Reference: Agilent 6100 Series Quadrupole LC/MS system, Concepts guide.pdf, Quickstart guide.pdf, Familiarization guide.pdf

29. Intro to Agilent 6100 Introduction to the Chemstation software Method and Run control tab

31. Set up methods to control LC, ion source and MS

33. Intro to Agilent 6100 START-UP gray SHUTDOWN Agilent 6100 STARTUP AND SHUTDOWN 1 Ensure that you are wearing your PPE and Lab glasses. 2. Observe instrument and workspace - Did previous operator complete shutdown? Are all the components present- e.g. HPLC column? Is LC connected to MS through a split? Are solvent reservoirs for HPLC full with appropriate mobile phase? Are the Autosampler wash bottles full? Is workspace clean? Are all components powered up? Any visible red lights on instrument? 3. In the software ensure that you are in the in the Method and Run Control Tab 4. From the Method and Run Control view, click the button on the system status bar to activate the system. 5. Ensure all components are “green” - Autosampler, Pump, UV, MS. 6. Depending on how long the system has been idle, one can warm up/ “equilibrate” the system by turning on the HPLC pump – Instrument/Pump at a low flow- ca. 0.5 ml/min- for 15 min. And/or one can run 1-2 blanks. SHUTDOWN - standby mode Suggested exercises: Startup instrument and observe at rest values for LC pressure and MS vacuum. Equilibrate system for 10 minutes and observe values. Are those values normal? – Refer Familiarization guide.

34. Method and Run Control Running the 6100 Agilent 6100: loading methods and running a single sample Ensure that you have completed LC-MS Startup procedure. Activate the single sample toolset by clicking the button on the toolbar. Load a method by selecting Method/Load method or by clicking the the button. 3. If you need to create new method, load “DEF_LC.M” and save as a new name, e.g. “pc061010_sulfa_analysis_1.M” 4. To edit the method use Method/Edit entire method or icon. A series of dialog boxes will appear allowing you to set and save desired parameters for Autosampler, LC, UV and MS. Make changes and save method. 5. Place the sample vial in position as you specified in the autosampler method parameters and click the Start button Suggested exercises: - Create a method with your initials and date e.g. PC060710_1 using suggested parameters given in Sample Experiment 1

35. Data Analysis Agilent 6100 Data Analysis view

36. Data Analysis Agilent 6100: loading data files

37. Data Analysis Agilent 6100: Qualitative information from your loaded chromatogram [M+H]+ m/z 271 Mass Spectrum of component eluting at 1.885 min Fragment ion m/z 156 m/z 156 1. Use the cursor to expand the chromatogram in the Data Analysis view. 2. Use the Spectrum Icon from the tool bar to click on the peak to obtain a Mass Spectrum of the component eluting at that time point. 3. This provides Qualitative information – molecular ion and characteristic fragment Of the component eluting at 1.85 minutes- Sulfamethiozole.

38. Data Analysis Agilent 6100: Generating EIC (Extracted ion chromatogram) data Use File/Load signal to load your initial TIC Use File/Extract Ions to extract ions Enter the desired m/z values in the dialog box and click OK EIC’s will be displayed within same window as TIC as shown below Qualitative Analyses suggested exercises: 1) Complete exercises 1-3 Pages 30-41 6100 Familiarization guide for analysis of sulfonamide Drugs.

39. Data Analysis Agilent 6100: Quantitative Analysis Quantitation is the process of determining how much compound is present in a sample. In LC-MS this is achieved usually by comparing Peak areas of component in your sample with the area generated by a standard or set of standards of known concentrations. Peak Areas are generated by integration for which the correct parameters must be set Use File/Load signal to load the TIC for your lower level standard. Use File/Extract Ions to generate EIC suitable for quantitation. Click on the icon to activate Integration tool set. Click on the auto integrate tool to complete initial integration. Click on the Icon Edit Integration table which displays below screen and Adjust integration events manually if auto integration is unsuitable. 6. Save this integration method as part of your overall LC-MS method.

40. Data Analysis Agilent 6100: Quantaitative Analysis Quantitation of samples may be done by activating the calibration Icon and tool set. 2. A calibration curve table may then be constructed by loading signals for standards and entering the known concentrations as shown below for caffeine. 3. This generated calibration may then be used to quantitate samples ran within the Sample set for which peak areas have been calculated Qualatative Analyses suggested exercises: 1) Complete exercises 1 & 2 Pages 63-76 Agilent 6100 Familiarization guide quantitation of caffeine.

44. Best practices Trouble shooting No Signal- no peaks? Or very low signal? - Check that LC flow is normal and back pressure is normal and steady. - Check that there is spray from the nebulizer. - Check that you have vial in correct position and that enough sample is present. - Check LC-UV chromatogram, are peaks present? - Check that ion Source parameters – Capillary Voltage, Drying gas flow and temp are set to normal ranges. - Check that you are running a full scan method in the expected range for your analyte. - Check that your sample is fresh- prepare fresh standard if in doubt. - Consider your solution phase chemistry. Are you providing sample to MS in a volatile solvent at a pH which promotes ionization? - Remove source and check orifice entrance. Has it become blocked?

47. M+˙, molecular ion—the ionized molecule; the molecular ion peak is the peak representing the ionized molecule that contains only the isotopes of natural abundance

48. Base Peak—the peak in the spectrum that represents the most abundant ion

49. Daughter Ion—the product produced by some sort of fragmentation of a larger ion

50. Isotopic Peak—a peak in the spectrum that corresponds to the presence of one or more heavier isotopes of an ion

51. “A” Element—an element that is monoisotopic

52. “A + 1” Element—an element with an isotope that is 1 amu above that of the most abundant isotope, but which is not an ‘A + 2’ element

54. General Instructional Objective Students shall gain the basic competency of Agilent 6100 hardware and software to complete an LC-MS Electrospray separation of a multi-component mixture according to an established procedure. Students shall further develop the skills to identify the components and determine the method sensitivity with established parameters. Specific Instructional Objectives Students will learn how to prepare and LC-MS sample. Students will learn how to prepare the 6100 for operation. Students will learn how to create and save a method given the parameters. Students will learn how to inject successive samples as per established method. Students will learn how to process data to produce mass spectra and identify components in a mixture. Students will learn how to process data and plan further injections and dilutions to determine the limits of sensitivity of a method. Prerequisites. Students will have completed initial 30 minute training session and 20 minute laboratory familiarization with the instrument. Future Objectives Depending on time constraints this experiment can be easily extended to investigate the effect of key ion source parameters (e.g. fragmentor voltage) on qualitative data or the effect of solution phase chemistry (different pH) on separation and sensitivity.

55. Preparatory work. 1. Fill mobile Phase A reservoir with deionized water/5 mM Ammonium Formate and mobile phase B with Methanol/5 mM Ammonium Formate . Place the Betasil C8, 50 x 2.1 mm, 3 micron HPLC column in correct position in the column compartment. Start up LC-MS-UV as described in initial training section. Experimental Procedure: Prepare a 1: 10 dilution of Electrospray LC Demo kit sample (which contains the four sulfonamides. Dilute 100 μL of the sample with 900 μL of 90:10 Merhanol:water/5 mM Ammonium Formate. Filter this sample into a 1 ml autosampler vial. Concentration of the samples is then 10 ng/ml. Prepare a solvent blank by filtering 1 ml of 90:10 Methanol:Water /5 mM Ammonium Formate into a 1 ml autosampler vial. Load DEF_LC.M in Method and Run control tab . 4 Save this method as SULFA_MS_SCAN_1.M Go to Set up Instrument method and click on Autosampler tab. Click Injection Volume and enter a value of 1uL into dialog box. Save. Click on the Pump tab in Set up Method and set up the following gradient parameters Flow = 0.4 ml/ml, Stop time = 10.0 min.

56. Experimental Procedure- continued 7. Set up the following gradient table: Time %A %B 0.0 88 12 1.0 88 12 3.0 0 95 6.0 0 95 7.0 88 12 10.0 88 12 8. Leave DAD (UV) settings in Set up instrument method tab at default settings. 9. Right click MSD icon and select Set Up MSD signals. Enter signal 1 in Scan mode, positive polarity. Enter the following parameters Scan range: 100-500; Fragmentor: 100 V; Gain: 1.0; Threshold: 150; Stepsize: 0.1. 10. Set up Ion Source by right clicking MSD icon and Select Spray Chamber. Set Drying gas to 9L/min, Nebulizer to 40 psi, Drying gas to 300°C and Capillary voltage to 3000V. 11. Save method and print for your experimental report. 12. Place blank vial on position 1 of autosampler and run blank in single sample mode as described in your Initial training material. 13. Place sulfonamide sample in position 2 and inject sample.

57. 14. Post run Open data file in Data Analysis tab as described in initial training document. Print out TIC (as shown in Figure 1) and the Mass spectrum associated with each peak. Assign each peak in the TIC to determined sulfonamide. 16. Dilute Sulfonamide sample and determine by subsequent injections the LOD Limit of detection (defined as 3 x Signal/noise) of each sulfonamide by this method. Figure 1: TIC of sulfonamide mixture in Fullscan positive mode at 10 ng/ml Reference: Agilent 6100 series Familiarization guide, Sections 2 and 3.

58. Sample Experiment 2: LC-MS methods to support QbD (Quality by Design) approaches to Active Pharmaceutical Ingredient manufacturing. Introduction Gabapentin is an anticonvulsant medication indicated in the treatment of epilepsy and neuropathic pain. Since October 2004, It has been launched as a generic drug. This compound is an γ-amino acid which under the right circumstances may form a lactam as shown in Figure 1. The study of conditions which favor this process is of importance since the lactam displays a certain toxicity and must therefore be avoided as far as possible in the formulated drug. Recent FDA initiatives have suggested a Quality by Design (QbD) approach to minimizing the occurrence of such impurities in Drug products1. These studies have contrasted conventional approaches to minimizing such impurities (reduced expiration dates for batches, modified packaging, reduced storage temperatures) with a QbD approach (greater understanding of drug substance degradation pathways, and incorporating this understanding into formulating and manufacturing processes). This QbD approach stresses the need for more selective and sensitive Analytical methods to be applied to gain understanding of the conditions which lead to impurity formation. Figure 1. Gabapentin lactamization

59. Introduction continued Pregabalin a related compound has also shown effectiveness as an anticonvulsant and is similarly capable of lactamization as shown in Figure 2. Figure 2. Pregabalin lactamization Previous analytical methods for assaying the kinetics have established that two acid base equilibria are involved as shown in Figures 3 and 4. These methods have relied on Fluorometric detection which required a derivatization step or NMR monitoring which lacked sensitivity Aim of Experiment To develop sensitive direct LC-MS methods capable of detecting and quantitating the lactam of Gabapentin and Pregabalin. To apply these methods to determine the rate of Lactam formation at pH 3.0 and 10.0 and at 37°C and 80°C

60. Introduction continued Figure 3. Equilibrium between different forms of Gabapentin as a function of pH2. Figure 4. pH-rate profile for the lactamization of Gabapentin at 80°C and 0.5 M concentration3. References FDA Advisory committee for Pharmaceutical Science and Clinical pharmacology meeting, April 14, 2010 meeting. <http://www.fda.gov/AdvisoryCommittees/CommitteesMeetingMaterials/Drugs>AdvisoryCommitteeforPharmaceuticalScienceandClinicalPharmacology 2. Zambon et al, Tetrahedron, 2008, 64, 6739-6743 3. Kearney et al, Int. J. of Pharm, 1992, 78, 25-34.

61. General Instructional Objective Student will use previously developed expertise to develop sensitive LC-MS methods capable of detecting and quantitating the lactam of Gabapentin and Pregabalin. Student will apply these methods to determine the rate of Lactam formation at at pH 3.0 and 10.0 and at 37°C and 80°C and concentration of 0.5 M for both compounds. Specific Instructional Objectives Students will develop and optimize an LC-MS method to obtain best separation and sensitivity for the Gabapentin and Pregabalin lactam. Student will set up an experiment at two pH values and two temperatures investigating lactam formation from Gabapentin and Pregabalin. Student will sample the experiment at set time points and run the samples using the developed LC-MS method. Students will process the data and quantitate the amount of lactam formed over time at each condition. Student will use this data to determine First order rate constants. Of the conditions investigated, student will determine those which lead to a greater than 1% lactam formation and which should be avoided in a manufacturing process. Student will determine difference in lacatam formation between both amino acids under given conditions and account for this. Prerequisites. Students will have completed LC-MS basic training and LC-MS experiment set 1 as well as Pre reqs for Chem 111 and Chem 154

62. Experiment Design Gabapentin/Pregabalindissolved in pH buffer at 0.5 M Incubation @ at pH 3.0 and 10.0 and at 37°C and 80°C Reaction quenched at 10, 20, 40 and 60 minutes in ice bath 10 x Dilution with 50:50 = H2O:ACN at neutral pH Analyzed with LC/MS method

63. Preparatory work. 1. Set up LC-MS as in Experiment 1. Obtain pH buffers and set up incubator at 37 and 80°C. Obtain 5M stock of Gabapentin and Pregabalin from Freezer. Obtain 0.5 M stock of Gabapentin lactam and pregabalin lactam from freezer. Install LC-MS divert valve. Set Autosampler to low temperature within sample method. Experimental Procedure: Dilute lactam stocks 10X and develop LC-MS fullscan positive mode separation as in Experiment 1. Determine Retention times to set divert valve switching time. Prepare standard setofGabapentin and Pregabalinlactam for quantitation. Dilute Gabapentin and Pregablin 10X into pH buffers and set on incubator. Sample at 10, 20, 40 and 60 minutes. Quench and dilute 10X as per scheme. 4 Analyze standard set and time point samples by LC-MS method. Determine concentration of lactam present in each sample. Complete plot of ln [lactam] versus time and determine slope, which is equal to –k. Hint: LOD for both lactams on LC-MS should be in the region of 1-10 ug/ml. Notes: Depending on time constraints this experiment may be need to be divided into a Number of lab sessions- method development, experiment completion, analysis. It can also easily be simplified to 1 compound, 1 pH value or expanded to form a more detailed study.

64. Acknowledgements Xenoport Analytical research team: Quincey Wu Senior Director Analytical Research. Bernd Jandeleit, Associate Director, Medicinal Chemistry. Mark Gallop, Senior Vice president, Research. Mark Gao, Staff Scientist, Analytical Research.