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 quatrapole 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).

Reagents and Standards:

1.      Milli-Q water

2.      C8 RP column

3.      HPLC-MS acetonitrile (Fisher gold) formic acid

4.      Syringe filters

5.      Analytical balance

6.      Pipettes and disposable tips

7.      Volumetric flasks

8.      Standards: Acetaminophen, acetylsalicylic acid, caffeine, and ibuprofen

9.      Analgesic medication of unknown concentration.

Laboratory week 1:  optimization.

Standards of acetaminophen, acetylsalicylic acid, caffeine, and ibuprofen are supplied to the students.  Prepare a  10 ug/ml solution of each standard using the stocks and diluting solution provided in the 1.5 mL centrifuge tubes.  1.5 mL will be sufficient.

Filter all solutions through a 0.45 um syringe filter.  Fill the MS syringe pump apparatus with the filtered solution, connect to ESI unit with syringe connector and peek tubing and infuse the solution into the MS by turning on syringe pump. Purge for a few seconds (injects 200 ul/min) and leave on run (which injects 20 ul/min of sample). 

The ion source, detector and RF should be on.  Click on them if they are off. They will be green and show larger print when on.

The MS should be set to full scan mode which is in the active segment window.  Set the range from 105 to 500 so the instrument is not producing a lot of excess data.  Monitor the ions in the left hand bottom screen and identify the major ion(s) of interest.  Your ion will be the M+H ion (MW plus 1 H ion). If the major ion, that correspond to an ingredient in your sample, is not present it may be present as the M + Na (+23) ion or as a breakdown product ion.

For each of acetaminophen, acetylsalicylic acid, caffeine, and ibuprofen optimize the needle voltage, capillary voltage and % RF loading in the MS on the major ions independently.  This can be done under the optimization plots screen, inputting the molar mass of the ion to optimize in plot these masses window, and selecting the parameter to optimize in dependant parameter window. The MS will optimize each of needle voltage, capillary voltage and %RF as you select them and pressing start icon.

Set the instrument in MS-MS mode (under active segment), set the optimization parameters (just determined) for each of the major ions of interest, one at a time and set the CID voltage to 0 and you will see on the screen the parent ion of interest. 

To see the product ions produced from each precursor ion; fragment the ions using the MS-MS mode. To do this you must determine the optimum CID (excitation voltage) voltages to produce product ions.  Choose method development (AMD) and adjust the CID voltage (the table allows you to use increasing values in different channels) and monitor product ion production in the different channels.

Once the optimum CID voltages have been determined (usually you want to see a little precursor ion) these can be set in the MS-MS mode window and product ion production monitored.  This can be done for each ion of interest.  Collect the spectral data of the precursor and product ions after optimization.  

Repeat the MS-MS ³ and follow all product ions.  Make a diagram of product ions breakdown (formation) and demonstrate in lab. write up how they are connected (fractionated) to precursor ion.  Try and identify as many of the major product ions (molecular formula) as you are able.

Collect all your data (your data should go into a folder as a descriptive name of run under 3590, 2015, Group). Your chromatograms can be copied into word and from word copied into your write up.

HPLC Seperation:

Check to see how the elution solvent(s), HPLC pumps, solvent mixer, auto-sample and injector, column (C8 reverse phase), and ESI unit are connected.

Start up HPLC pumps making sure they are primed and are pumping solvent.  Using the windows screen open the pump control screen (212.24 ) set the pumps to pump a combined (if using both pumps) 300 ul/min.  Set pump “B’ to 5% ACN and the ramp to 1 min.  Starting ramp will start pumps.

Pump the first five min. into waste to prevent salts and contaminates entering MS.

Note:  Always increase pressure on column and pumps slowly by ramping over time to prevent pressure surges on columns which can create dead space at top of column which will decrease quality of separation.

Note:  All material going through column ends up in MS ion detector.  It is important not to add contaminates or too much material to the MS or the only ions that will be seen will be the contamination.  It is always good to start with very small amount of sample and increase if needed.

Run solvent through the column to equilibrate it for at least 6 to 10 volumes of solvent (5-10 min.).  Switch from waste to load and monitor the ions.  The ions of interest can be monitored individually by typing their mass numbers (M +1) in the middle screen, one at a time.

Set up a full scan program.  Open a method file and save as your group and date under 3590.  Set pump to 5% B at start, increase to 15% B over 5 min., then increase to 30% B over 2 min., return to 5% B over 1 min and keep at 5% for 4 min. Set acquisition time to 12 min.

To inject sample (filter samples before injection) from auto-sampler you can use quick inject if you are only injecting one sample or you can select the sample table screen and set the parameters in this screen, including sample name, type,  volume injected, and auto-sampler  number.

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Inject 10 ul of one of standards (20 ug/ml) to determine the retention times of acetaminophen, caffeine, acetylsalicylic acid and ibuprofen.  

Start the run by clicking the begin icon at the very bottom of the sample window. 

After completion of the first run, the retention times of the various constituents in your sample are known.

Make a combined standard of the four standards, acetaminophen, acetylsalicylic acid, caffeine, and ibuprofen at concentrations ranging from 5 to 100ug/ml (use diluting solution).

Week 2:

Using the retention times from week 1, retention time segments (in method window) can be chosen to correspond with the retention time of each ion.  Each time segment can have its own set of conditions to optimize collection of the ion of interest, and/or to fragment the ion to product ions.  Set the CID voltage(s) to produce the 1^st (most abundant) product ions for each precursor ion. 

The conditions are set by making a new method and selecting MS-MS mode and setting the various parameters under MS/MS parameters and the sub windows.  You will need to set the capillary voltage, CID voltage, % RF, drying gas pressure (35 p.s.i.)) and temperature (keep at 350^oC) and nebulizer gas pressure (50 p.s.i.) values for each time segment.

Rerun your standards and samples, tylenol, ASA and the mixture using your new MS/MS method with the time segment information added to your method.  In this run you will get the major product ions in each time segment.

Integrate a product ion of standards and sample (hold I key down and use cursor to draw baseline) to get area related to concentration.  You can also integrate using the integration icon in the tool bar.  

Identify compounds by product ions.

Compare the one standard run at full scale, to it’s product ion, to determine if sensitivity and/or selectivity is better with product ion (transition) or full scale precursor ion  

Draw standard curve in “Excel” and determine concentration of medication.

See if label agrees with your analysis.

Note:  Make sure the material going into the needle of the ion spray is always washed out with solvent before the pumps are shut off or syringe pump is stopped or the needle will plug and have to be replaced.

Data:

Identify each medication by retention time and product ions.

Identify the fragmentation patterns of the original and product ions and compare to literature patterns.

Determine concentration of medication and compare to claimed concentration.

Discussion:

Discuss your results in detail. Discuss the methodology, improvements that are possible.  Discuss the fragmentation patterns and what is occurring using literature sources.  Discuss concentration results, how it compares with label and reasons which might affect results, etc., etc.

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?