Bioanalytical Chem: 4590: LC-MSMS of analgesics LC-MS Experiment Liquid Chromatography Mass Spectrometry (LC/MS)

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1 Liquid Chromatography Mass Spectrometry (LC/MS) Prelab Questions: Questions to be answered before doing the experiment. The answers are due at the beginning of each experiment without exception (the questions are for credit and some may appear on your final exam). 1. Compare MS ion trap and electrospray ionization (ESI) technology to the GC- MS quadrapole detection system. 2. Why does the LC-MS tend to be the instrument of choice for many biological samples? 3. Describe the components of the Varian (Agilent) LC-MS (MCAL lab) and how it is used in sample identification. You can follow the sample through the MS and describe what is happening at each step. 4. What is meant by MS-MS, and what are the advantages of an instrument that has this capability? Objectives: 1. Learn the principles and operation of LC-MS. 2. Develop methodology using MS and MS-MS in sample identification. 3. Become familiar with the concept of product ion fragmentation pathways. 4. Apply methodology to sample analysis. Introduction: The MS in MCAL uses electro-spray ionization (ESI) and Ion Trap technology to detect sample ions. The analyte ions are nebulized and dried (and become charged ions). The ions can be scanned in full scale mode, MS-MS mode or can be further fragmented and the product ions scanned. The MS is connected to a binary LC system. Depending on the column used the LC can separate components in a mixture based on hydrophobicity, charge, and molecular size. A major difference between traditional HPLC and the chromatography used in LC-MS is that in the latter case the scale is usually much smaller, both with respect to the internal diameter of the column and even more so with respect to flow rate. The MS will give good sensitivity at flow rates of 200 μl/min or less. There are a lot of other mass analyzers besides Ion Trap that can be used in LC-MS; Single Quadrupole, Triple Quadrupole, TOF (Time of Flight) and Quadrupole-Time of Flight (Q-TOF). Due to different properties of chemicals, the optimal conditions for analysis of one molecule is specific.

2 Reagents and Standards: 1. Milli-Q water 2. Reverse Phase column 3. HPLC-MS grade Acetonitrile 4. Pipettes and disposable tips 5. Eppendorf tubes 6. Standards: Acetaminophen, acetylsalicylic acid, caffiene and ibuprofen 7. Analgesic medications (unknowns) Laboratory week 1: optimization. Standards of acetaminophen, acetylsalicylic acid, caffeine, and ibuprofen are supplied A) Solution Preparation 1. Prepare solution of each standard to a concentration of 40 ug/ml from the stocks provided using the diluting solution. (10% Acetonitrile, 0.1% formic acid). Use the 1.5 ml Eppendorf tubes and micropipettes provided. Note: The stock solution could different concentrations. Make sure you are aware. B) Prepare the instrument - Make sure you are in the 500 MS window. If not; open system control and go to windows (from the toolbar at the top) and select 500 MS. - Make sure the syringe pump is connected to the needle intake. If unsure, ask instructor or TA 1. There is a schematic of the instrument displaying components in the upper left hand side. The ion source, detector and RF should be in green print indicating they are ready. If not; click on them and they should turn from small red to large green type. 2. On the right upper side; you will see tabs named method; active segment; checks and adjustments; module attributes and optimization plots. We will use only active segment and optization plots. 3. Under the active segment tab; select full from the Scan Type drop box and standard from the Scan mode drop box. 4. Under the Electrospray Ionization tab set the nebulizing pressure to 25 and the drying gas pressure to 18. These are specific to direct infusion by the syringe pump as it has a very low flow rate. 5. Under the Full Scan Parameters tab set the Capillary voltage to 80, the RF value to 80 and the mass range to which will cover your range of mass interest. The bottom half of the screen displays data. On the left is the RIC; which provides a measure of each ion s mass and counts on a relative scale (normalized to highest

3 peak). On the right is the TIC; which provides a sum of the RIC over time (this is your background level.) C) Optimization of Instrument Parameters: 6. Load the syringe pump with one of your solutions prepared in step A. Press purge for a few seconds; the release and the syringe pump will run to untill it is empty. 7. Verify that you can see the parent ion of your compound. It should display in the RIC as M+H or M + 23 (sodium adduct). If not; you may need a more concentrated solution or the compound may have already broken. Consult the instructor. Note: you can filter the TIC for your specific compound. Type the mass in the box at the bottom and the plot will split into two showing your compound only. 8. Click on the Optimization Plots tab. a. Select needle voltage in the dropbox under dependant parameters b. Change the starting value to 2000 and leave the end value at 6144 c. Enter your mass of interest on the right hand side. d. Press start. (make sure your syringe pump is still running) e. The instrument will plot parameter value vs. counts and at the end; display the maximum value at the top in the TIC window. Record this value and if you choose; take a screen shot for your report or left click and export to chromatogram pane then copy and paste into powerpoint/word. f. Chang the needle voltage to your optimized value under the Electrospray Ionization tab. g. Repeat a-e for capillary voltage and RF value parameters. Default settings will be fine. h. Under the Active Segment Tab change the scan mode to MS/MS. i. Input your optimized values for RF and Capillary voltage a well as the parent ion mass and set the Excitation Amplitude to O. This means the trap will isolate the parent ion mass; but not provide any collision energy. j. Verify that you only see your parent mass in the TIC with collision energy 0. Then manually change the CID gas in increments of 0.5 and observe the ions. k. Record the fragment ion masses (the product ions). l. Return to the Optimization Plots tab and select CID voltage under the dependant parameters dropbox. Change the plot these masses to the daughter ion mass. Press start and record the optimum voltage when finished. m. Repeat a-l for other compounds. Note: In the lab write up; include a screen shot of the RIC showing fragment ions. Try to demonstrate what fragmentation is occurring according the chemical structure.

4 Laboratory Week 2: HPLC-MS Prior to using the HPLC, there are a few important points to consider: Ensure there is significant level of mobile phase: 10% Acetonitrile. Both pump aspiratiors will be in the same bottle in this case. Ensure the source on the Mass spec is connected to the LC via the red tubing rather than the syringe pump Equilibrate the column for at least 15 min using the solvent and flow rate in your method: Make sure you wash the autosampler before injecting your first sample instructions are in the following steps. Part A: Start up LC-MS 1. At the top of the system control software; select window in the tool bar and select 212 LC to open the pump control window. There will be a graph display plotting each pump s pressure as well as solvent. 2. Click the manual control button and a new window will pop up. Enter 5 under % b; flow rate of 250 ul and ramp time 1 min. Click start and the pumps should start and begin recording in the graph. 3. Monitor the pressure. It should be fairly stable. Slight fluctuations are normal; pay attention to the scale. If it is fluctuating more than 50 p.s.i. there may be a problem such as an air bubble. Consult the instructor if this is the case. Part B: Determine retention time of Analgesic compounds and constituents of unknown: 1) Prepare 2.0 mls of the following using 2.0 ml Eppendorf tubes, then transfer to LC vials: A. Mixture of: 300 ug/ml Acetylsalicylic Acid 50 ug/ml Caffeine 50 ug /ml Acetominophen. Use the stock solutions provided from last week. B. Unknown: Choose 1 of A, B, or C. Dilute A 1 in 20, Dilute B and C 1 in 50.

5 2) Set up an LC-MS method (FULL SCAN) Due to the condition of the instrument, we will be running isocratic using only one pump. The mobile phase is 10% Acetonitrile with 0.1% formic acid. Both pump aspirators will be placed into this bottle. C. Open the method builder. There is a tool bar located at the right hand side of the screen; it is the second from the top. Click File then New Method and save it in the folder. Click next three times and then finish and you will be able to save your method. D. On the left is a navigation pane of all components of the method. Click on pump program and set the following pump parameters in the chart on the right: 0-25 min 100% A DO NOT USE PUMP B The flow rate should be set to 250 ul/min. E. Double Click the Mass Spec Control. A window much the same as in manual operation (when you were doing the optimization) appears 1. Set the first segment of the ms run between 0 and 1 min. 2. Click special applications at the on the right hand side and select Divertor. Under that segment, click the box Divert so that the valve will divert that 1rst minute to waste. (Load position) 3. Make the second segment from 1-25 min. Unclick the Divert box (the machine will now switch to inject position on the valve) at 1 min time. 4. Change the nebulizing pressure to 50 and the drying gas pressure to 30 under electrospray ionization. Drying gas temp should remain at Save your method and upload. To upload, go to the System Control window and there is a button at the top displaying the active method. Left click and select Activate Method. Find yours and open it and it will activate. Set up a sample list and Run the 2 Samples:

6 6. Under the 500 MS window; select File then New Sample list and save it in the folder. (varian ws > data> students>3590> ). 7. Enter name; the correct vial # and an injection volume of 10 ul. 8. Click Data Files in the bottom right hand corner and make sure the data is allocated to your folder. 9. Find the manual control window and end manual control. It s usually hiding behind the main MS window. 10. Click Begin. Observe the tool bar. After a minute or so; you should see the NOT READY change to WAITING and you will hear the autosampler start up. 11. Identify the retention time of each compound present in the standard mixture and unknowns by observing the mass: a. Open the MS data review software (found on the same toolbar as the method editor) b. Locate your chromatogram from your folder on the left hand navigation window and click on it. c. Use the curser and click on the peak of interest in the TIC; this will open the RIC in a window below and display the masses associated with that chromatographic peak. Identify each peak present. Part C: Standard Curve and Quantitation of Unknown Components using MS-MS: 1. Update your method to reflect your optimized parameters. You need to create MS acquisition segments, and program the specific optimized parameters for each compound that you have found present in your unknown: a. Save as your full scan method to create a new one. b. Change the scan type to MS/MS. c. For the earliest eluting compound, create a time segment after the initial waste segment to include the entire peak based on your chromatogram. (ie create a window around it) with 30 seconds buffer on each side. d. Under Electrospray Ionization input the optimized parameters for needle voltage. e. Change the nebulizing pressure 50 and the Drying gas pressure to 30.

7 f. Under MS/MS parameters, input the optimized capillary voltage, Precursor Ion, CID voltage and RF. g. Repeat B-F for second compound if necessary. h. If possible, shorten your total run time on the pump and mass spec control. 2 min past latest eluting compound is sufficient. MAKE SURE YOUR END TIME IN THE MASS SPEC CONTROL MATCHES YOUR PUMP END TIME. 2. Prepare a Standard Curve of the Mixture with minimum 4 standards between the range of 50 to 1 ug/ml. You can use your previous mixture from part B. 3. Integrate the area of each of your peaks in your standard and unknowns to create a curve in excel and determine the concentration of your unknown: a. Open MS Data review and locate your chromatogram b. Locate the set single click action button on the tool bar above the TIC and set it to integrate. c. Click the mouse cursor where you want to start the integration and drag it to where you want to end. The peak will fill in color and provide an area and RT at the top. If you can t see it; you will need to zoom in or out by resetting the single click action back to zoom. d. Repeat for all peaks in all samples. 4. Use your areas to create a curve in excel and determine the concentration of your unknown to report. Ask the Instructor to view the packaging for your specific unknown. Once finished; you need to turn off the pumps from the 212 pump window by clicking Stop Pumps. Also turn of the trap clicking on it in the schematic; then turn the ONLY the ion source back on. Discussion/ Lab report Discuss your results in detail. Discuss the methodology, improvements that are possible. Discuss the fragmentation patterns observed in the MS or full scan mode and what is occurring using literature sources. Discuss concentration results, how it compares with label and reasons which might affect results, etc., etc.

8 Final Questions: 1. What are the reasons to use LC-MS versus GC-MS for the analysis of pharmaceutical and biological samples. 2. What are some of the problems associated with the use of this instrument for quantitative analysis? Compare advantages (disadvantages) of using full scan ions verses product ion from transitions for quantitation, and identification. 3. What parameters would you have to control to produce a library capable of identifying compounds based on their fragmentation pattern? 4. The instrument for this experiment is set to positive mode (can only see positively charged ions). What is the other routine mode the instrument can be set in and when would this mode be used and why?