TITLE: Analysis of paracetamol and AT7519 in serum using LC-MS/MS
AUTHORS: Natalie ZM Homer, Joanna Simpson

[DESCRIPTION]
This protocol describes the extraction and targeted mass spectrometry analysis of two drugs - paracetamol and AT7519, a CKDI inhibitor, following enrichment with isotopically labelled internal standards of deuterated versions of the two drugs.  Extraction of the drugs was carried out using automated protein precipitation on a liquid handling robot alongside a calibration curve. Analysis of the extract was carried out by liquid chromatography mass spectrometry (LC-MS/MS) in multiple reaction mode. The amount of each drug (APAP and AT7519) in each sample was calculated using linear regression of the peak area ratio of the analytes to the isotopically labelled internal standards. 

This protocol was published as part of: J Cartwright, J P Simpson, NZM Homer, Adriano Rossi ‘Analysis of AT7519 as a pro-resolution compound in an acetaminophen-induced mouse model of acute inflammation by UPLC-MS/MS’, J. Inflammation (2023)

[BEFORE_START]
Ensure you have all Materials as detailed in Table 1 (Analytical standards), Table 2 (Equipment), Table 3 (Chemicals and Consumables). 
Prepare solutions for extraction according to Table 4
Prepare solutions for liquid chromatography according to Table 5


[GUIDELINES]
Use analytical grade, LC or LC-MS grade solutions and solvents, as detailed in Materials section. Use certified reference materials where detailed. Use the dilution scheme as described to ensure linear calibration curves.

[STEPS]
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
10. Complete a plate map for standards, quality controls and samples (MAKE SURE TO PLACE THEM COLUMN-WISE!) using the design as shown below. The attached excel can be downloaded and filled in with sample specific detail for a real plate map and real samples - change the detail in the second tab (Sample List) and it will populate the plate map view, which you can print out and use at the bench.  

 
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
11.  Defrost biological   and quality control samples (lower limite of quantitation (LLOQ_, low, med and high)

SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
12. Set up Extrahera liquid handling robot for PPT+ extraction:
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
12.1. Turn on Air Compressor. Make sure a pressure of ~9 bar is achieved and that the air compressor goes into Standby (indicated by green flashing light).
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
12.2. Turn on fume cupboard and make sure duct hose is in place in fume cupboard to ensure proper ventilation.
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
12.3. Turn on Extrahera at plug and use twisting switch on right hand side  (turn it over 90 degrees to start it), and wait for it to boot up.
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
12.4. Once the touch screen is active, from the Maintenance menu select Flush Solvent Inlets and Purge line S3 with Acetonitrile
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
12.5. Ensure that a sufficient number of solvent tips (deck position 1) and sample tips (deck position 2) are on the deck. For a full plate you will need 96 tips.
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
12.6. Place a PPT+ plate (Biotage) in deck position 3. Make sure that it is in the correct orientation and is properly clicked in place.
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
12.7. Place a Waters 2mL 96 well collection plate in carousel position A (MAKE SURE WELL A1 IS ON THE OUTSIDE OF THE CAROUSEL NEXT TO THE A1 LABEL!).
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
13. Take a Biotage 2mL deep well 96 well collection plate and add 100 uL 5% BSA solution to each of the standard wells.
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
14. Add required amount of standards to the BSA according to the PREPARATION OF CALIBRATION STANDARDS table section. Due to the small volumes of standards being pipetted, ensure that the standard is pipetted INTO the BSA.
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
15.  For sample, according to plate map, add 50 uL serum plus 50 uL of water to the appropriate wells.
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
17. To samples and standards add 20 uL of internal standard solution (Prepared according to Table - Internal Standard Preparation) using the repeater pipette (Choose 2.5 mL tip, set to 20 uL as above) to all relevant wells
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
18.  Seal the plate using a VWR 96 well plate sealing film and shake the plate on a plate shaker for 2 mins to ensure that the standards and internal standards are sufficiently mixed (shake speed - 200 rpm).
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
19. Remove the plate seal and place the sample plate on the deck of the Extrahera in position 4.
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
20. Select Run Single Method from the Extrahera menu and select the DCA, PPT+ method then press Prepare Run. Select the columns of the PPT+ plate for processing, update the tip numbers/locations if necessary and top up the solvent reservoirs if necessary (when orange). Here you can also adjust the sample volume to fit with the amount you have used. For 100 uL of sample set to 150 uL
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
21. Press Run. The Extrahera loads 400 uL of Acetonitrile into each well. It then transfers the sample plate contents onto the acetonitrile in the PPT+ plate. Following a 10 min wait the Extrahera applies positive pressure to pass the samples through the PPT+ plate and collects the eluent in the Waters 2 mL deep well 96 well plate.
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
22. Once complete, check the volumes of elution solvent in the collection plate are approximately equal - indicating good performance of the positive pressure head. Check that the samples and standards were correctly aspirated from the sample plate. IF the volumes are not equal you may need to push through on the standalone positive pressure unit.
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
23. Place the extracted samples (in the collection plate) on the SPE Dry 96 Dual Sample Concentrator with the gas temperature set to 40°C and the highest flow it will achieve. This step may take up to 30 mins. Keep an eye on the drying phase and make sure needles do not go into the solvent if you alter the height of the plate.
SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
24. Once dry, resuspend all the dry standards and samples in a solvent suitable for LC-MS/MS.
24.4. Shake the plate for 10 minutes on the ThermoShaker (600 rpm) to ensure the samples are resolubilised.

SECTION: Extract calibration standard &amp; Quality Control and samples by PPT
16. Set up a Repeater pipette for adding internal standard solution to the plate. Choose a 2.5 mL tip and set to 20 uL. Make sure you have enough internal standard solution for the full number of samples and standards (Calculate this by counting the number of standards and samples adding 5 and then multiplying by 20 uL)
24.2. Prepare a repeater pipette with 2.5 mL tip and set volume to 100 µL. Pull up full volume into repeater pipette.

24.1. Prepare mobile phase solution for resuspension of samples (90:10 Water/Acetonitrile, v/v). Take 90 mL     and add to 100 mL glass bottle, then add 10 mL   Mix well. Label bottle.

24.3. Add 100 µL of 90:10 Water:Acetonitrile to each well on the plate that is part of the experiment and seal the plate with a Zone-free 96 well plate sealing film.

SECTION: Set up LC-MS/MS for analysis of extracts
25. Prepare Mobile Phase A - H2O + 0.1% Formic Acid
Add 1 L of LC-MS grade H2O to a 1L glass bottle.
Add 1 mL of LC-MS grade Formic Acid to the H2O.
Mix thoroughly.
SECTION: Prepare calibration standard and Quality Control stock solutions
4. Take the 1 mg/mL stock solution of d4-APAP in LC grade methanol 
SECTION: Prepare calibration standard and Quality Control stock solutions
5. Prepare a 1 mg/mL stock solution of d8-AT7519 in methanol using a microbalance and using LC grade Methanol (e.g. 2.5 mg AT7519 into 2500 µL methanol). 
SECTION: Prepare calibration standard and Quality Control stock solutions
6. Prepare a combined 50 ng/mL Internal standard solution, d4-APAP and d8-AT7519, by adding 40 µL x 10 µg/mL d4-APAP and 40 µL x 10 µg/mL d8-AT7519 into 8 mL methanol to give a 50 ng/mL stock solution.
SECTION: Prepare calibration standard and Quality Control stock solutions
3. Prepare a 1 mg/mL stock solution of AT7519 in LC grade methanol using a microbalance (e.g. 2.5 mg AT7519 into 2500 µL methanol). 
SECTION: Prepare calibration standard and Quality Control stock solutions
7. Prepare a combined 50 µg/mL APAP and AT7519 standard solution, by adding 50 µL x 1 mg/mL APAP and 50 µL x 1 mg/mL AT7519 into 1 mL methanol to give a 50 µg/mL APAP + AT7519 stock solution.

Use this  50 µg/mL APAP + AT7519 stock solution to prepare four further stock dilution solutions 
 
 

SECTION: Prepare calibration standard and Quality Control stock solutions
1. The following table details the analytical standards needed for this protocol
 
     Compound      Abbreviation      Supplier/  Manufacturer      Catalogue   Number      Concentration      Storage   Conditions        AT7519      AT7519      Astex Pharmaceuticals      ///      1 mg/mL in MeOH      -20C        [2H]8-AT16043M      d8-AT7519      Astex Pharmaceuticals      ///      1 mg/mL in MeOH      -20C        Paracetamol      APAP      Cerilliant/Merck      A-064-1ML      1 mg/mL   in MeOH      -20C        [2H]4-paracetamol      D4-APAP      Cerilliant/Merck      P-909-1ML      1 mg/mL   in MeOH      -20C       
    

SECTION: Prepare calibration standard and Quality Control stock solutions
5.1.  Prepare a 10 µg/mL stock solution of d8-AT7519 in LC grade methanol by taking 10 µL x 1 mg/mL and adding 990 µL x LC grade methanol.
SECTION: Prepare calibration standard and Quality Control stock solutions
4.1.  Prepare a 10 µg/mL stock solution of d4-APAP in LC grade methanol by taking 10 µL x 1 mg/mL and adding 990 µL x LC grade methanol.
SECTION: Prepare calibration standard and Quality Control stock solutions
3.1. Prepare a 10 µg/mL stock solution of AT7519 in LC grade methanol by taking 10 µL x 1 mg/mL AT7519 and adding 990 µL x LC grade methanol.
SECTION: Prepare calibration standard and Quality Control stock solutions
2.1. Prepare a 10 µg stock solution of APAP in LC grade methanol by taking 10 µL x 1 mg/mL APAP and adding 990 µL x LC grade methanol.

SECTION: Prepare calibration standard and Quality Control stock solutions
2. Take the1 mg stock solution of APAP in methanol

SECTION: Prepare calibration standard and Quality Control stock solutions
8. Prepare quality control solutions as below using the combined standard solutions above and 2% BSA solution. Prepare in bulk and sub-aliquot into 110 μL volumes into eppendorfs (0.5 mL) and freeze (-20oC). Defrost one from each level before each extraction. 
 
 

SECTION: Set up LC-MS/MS for analysis of extracts
26. Prepare Mobile Phase B - CH3CN + 0.1% Formic Acid
Add 1 L of LC-MS grade CH3CN to a 1L glass bottle.
Add 1 mL of LC-MS grade Formic Acid to the H2O.
Mix thoroughly.
SECTION: Set up LC-MS/MS for analysis of extracts
27. Put freshly prepared mobile phases onto the Acquity UPLC system. Purge lines with mobile phase A and mobile phase B on Acquity UPLC system
SECTION: Set up LC-MS/MS for analysis of extracts
28. Install a Waters Acquity BEH C18 (100 x 2.1 mm; 1.7 μm) column into correct position for the method, set column temperature to 45 °Cand equilibrate at 95% A, 0.5 mL/min for at least 15 minutes. Ensure pressure is stable and no leaks on the column. 
SECTION: Set up LC-MS/MS for analysis of extracts
33. In Analyst software, click on 'Acquire' and create a batch using the text from the electronic plate map excel file (From above) to populate all rows needed in a batch file.  Select the APAP and AT7519 method for LC and MS. Name the data file using the following file name convention - yyyymmdd_Eyyxxxxx_Name_APAP-AT7519_initials. Use the same data file name to name the batch.

SECTION: Set up LC-MS/MS and analyse extracts
32. Set a 'system suitability test' solution of APAP, AT7519, d4APAP and d8AT719 to run and check chromatography and peak area response. Retention time should be as shown:  
SECTION: Set up LC-MS/MS and analyse extracts
34. Assess chromatography and peak response while batch is underway.
SECTION: Set up LC-MS/MS and analyse extracts
33. Put plate of extracted samples into the correct position and once SST has shown good results, set the batch to run. Inject 10 μL for each sample. Observe pressure on the column and check that it progresses to inject all samples and collect data.
SECTION: Set up LC-MS/MS for analysis of extracts
29. Create an LC-MS/MS method by building the chromatographic method to be as follows: 

 
     Step      Time (min)      Flow  (mL/min)      A (%)      B (%)      Curve        1      Initial      0.5      95      5      Initial        2      1.50      0.5      95      5      6        3      5.00      0.5      5      95      6        4      7.00      0.5      5      95      6        5      7.10      0.5      95      5      6        6      9.00      0.5      95      5      6   

31. In Method creation use existing method (or create a method) with the following multiple reaction monitoring (MRM) parameters as mass spectrometry settings.

 

SECTION: Evaluation of LC-MS/MS data
35. Use Analyst Explore to view the total ion chromatogram. Extract APAP and d4-APAP and check retention times. Extract AT7519 and d8-AT5519 and check retention times. 
SECTION: Evaluation of LC-MS/MS data
36. Open MultiQuant 3.0.3 and create a new result file. Select the data file collected. Save the result file with the file naming convention (yyymmdd_Exxxx_Analyte_initials). Use a method to evaluate the data that integrates APAP at 2.07 mins; AT7519 at 2.74 mins; d4-APAP at 2.04 mins and d8-AT7519 at 2.7 mins. 
34. In MultiQuant 3.0.3 create a new result file, using the recently collected data file and name it using the file name convention above (yyyymmdd_Eyyxxxxx_Name_APAP-AT7519_initials). 
SECTION: Set up LC-MS/MS for analysis of extracts
30. Start with divert valve going to waste, divert to mass spectrometer at 1 minute, then back to waste at 8 minutes. 
35. Assess the calibration curve for APAP and exclude any points that are outwith 20% accuracy. Aim for a linear regression coefficient of R>0.99. Ensure at least 6 points on the curve

36. Assess the calibration curve for AT7519 and exclude any points that are outwith 20% accuracy. Aim for a linear regression coefficient of R>0.99. Ensure at least 6 points on the curve
34.1. Assign correct sample type - Standards, Blanks, QCs and unknowns. Define standard and QC concentrations. 
34.2. Ensure all peaks have been integrated for APAP, d4APAP, AT7519 and d8AT7519. Check correct peaks have been picked by using the Metric plot function to compare 'Peak Area' of analyte and retention time over the course of the batch.
37. Define the upper and lower limit of the method according to accuracy of the calibration curve and record this to assess the unknowns and how they sit within the method.
32. 
SECTION: Evaluation of LC-MS/MS data
36.1. Define sample type - Blanks, Double Blanks, Standards, Unknowns (samples) and Solvents. 
SECTION: Evaluation of LC-MS/MS data
36.3. Check all peaks have integrated for each analyte - use Metric plots to assess peak area and retention time to ensure correct peaks have been integrated
SECTION: Evaluation of LC-MS/MS data
36.4. Check calibration curves for accuracy (<20%) and exclude those points that are outwith. Ensure there are over 6 points in each calibration curve and a regression coefficient R>0.99
SECTION: Evaluation of LC-MS/MS data
36.5. Copy the data of all peaks and calculated amounts into Microsoft Excel and name the excel file using the  file naming convention (yyymmdd_Exxxx_Analyte_initials) and name the first tab 'RawData'
SECTION: Evaluation of LC-MS/MS data
37. Create a new tab. Go back to 'Raw Data' select 'filter' and select the first analyte in the list (alphabetical). Copy this analyte excel data into the new tab and rename the tab with the analyte name. Repeat with all analytes until you have an excel spreadsheet with the following tabs - Raw Data; APAP 1; AT7519 1
SECTION: Evaluation of LC-MS/MS data
38. In the two tabs for the analytes - rename 'calculated amount' on the APAP tab to '[APAP] ng' and rename in the AT7519 tab to '[AT7519] ng'
SECTION: Evaluation of LC-MS/MS data
36.2. Insert standard curve values and QC valules in 'actual amount' according to the calibration table and QC table. 
SECTION: Prepare calibration standard and Quality Control stock solutions
9. Prepare calibration standards using the following table:

 
     Standard name      Amount (ng)      INT STD vol (mL)      STD vol mixture (mL)        0 STD      0      20mL (50ng/mL)      0        0.0500
  STD      0.0500      20mL (50ng/mL)      10 mL (5 ng/mL)        0.100
  STD      0.100      20mL (50ng/mL)      20 mL (5 ng/mL)        0.250
  STD      0.250      20mL (50ng/mL)      5mL (50 ng/mL)        0.500
  STD      0.500      20mL (50ng/mL)      10mL (50 ng/mL)        1.00 STD      1.00      20mL (50ng/mL)      20mL (50 ng/mL)        2.50 STD      2.50      20mL (50ng/mL)      5 mL (500 ng/mL)        4.50 STD      4.50      20mL (50ng/mL)      9 mL (500 ng/mL        5.00 STD      5.00      20mL (50ng/mL)      10mL (500 ng/mL)   

SECTION: Evaluation of LC-MS/MS data
39. Create a new tab and name it 'Summary'. Copy the first three columns from APAP tab, which contain sample details and calibration level details. Paste into the new tab. Then return to APAP tab and copy the '[APAP] ng' column.