Jul 19, 2023
Analysis of paracetamol and AT7519 in serum using LC-MS/MS
This protocol is a draft, published without a DOI.
- 1University of Edinburgh
- Joanna Simpson: mass spectrometry, chromatography, drugs
Protocol Citation: Natalie ZM Homer, Joanna Simpson 2023. Analysis of paracetamol and AT7519 in serum using LC-MS/MS. protocols.io https://protocols.io/view/analysis-of-paracetamol-and-at7519-in-serum-using-cua8wshw
Manuscript citation:
Cartwright JA, Simpson JP, Homer NZM, Rossi AG. Analysis of AT7519 as a pro-resolution compound in an acetaminophen-induced mouse model of acute inflammation by UPLC-MS/MS. Journal of Inflammation (London, England). 2023 Jun;20(1):20. DOI: 10.1186/s12950-023-00345-y. PMID: 37291548; PMCID: PMC10251596.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 16, 2023
Last Modified: July 19, 2023
Protocol Integer ID: 81984
Abstract
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)
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.
Materials
The following tables detail the materials, equipment and solutions needed to carry out the protocol.
| Compound | Abbreviation | Supplier | Catalogue Number | Concentration | Storage Conditions | |
| Paracetamol | APAP | Cerilliant/Merck | A-064-1ML | 1 mg/mL in MeOH | -20C | |
| AT7519 | AT7519 | Astex Pharmaceuticals | /// | 1 mg/mL in MeOH | -20C | |
| [2H]4-paracetamol | D4-APAP | Cerilliant/Merck | P-909-1ML | 1 mg/mL in MeOH | -20C | |
| [2H]8-AT16043M | d8-AT7519 | Astex Pharmaceuticals | /// | 1 mg/mL in MeOH | -20C |
Table M1 - Analytical Standards - Name, abbreviation, supplier, catalogue number, concentration and storage conditions
| Item | Model | Manufacturer | |
| Plate Shaker | Grant Scientific TS-DW Deepwell | ThermoFisher | |
| Liquid Handling Robot | Extrahera | Biotage | |
| Evaporator | SPE Dry-96 dual | Biotage | |
| Liquid Chromatography Pump | Acquity Binary Solvent Manager | Waters | |
| Column oven | Acquity Column Manager | Waters | |
| Autosampler | Waters Acquity Sample Manager with Sample Organiser | Waters | |
| Mass spectrometer | QTrap 5500 | AB Sciex |
Table M2 - Equipment list - All laboratory equipment and instrumentation needed for the extraction and analysis
| Item | Supplier | Part number | |
| Methanol (HPLC grade) | VWR | C-20864320-X | |
| Water (HPLC grade) | Fisher Scientific UK Ltd | C-10449380-X | |
| Acetonitrile (HPLC grade) | VWR | C-20060320-X | |
| Water (LC-MS grade) | VWR | 83645.320 | |
| Methanol (LC-MS grade) | VWR | 83638.320 | |
| Acetonitrile (LC-MS grade) | VWR | 83640.320 | |
| 1.75mL glass vials with lids | Scientific Laboratory Supplies Ltd | TUB1200 | |
| 2 mL deep well 96 well collection plate | Biotage | 121-5203 | |
| Biotage PPT+ plate | Biotage | 120-2040-P01 | |
| 96 Extrahera 1000 L pipette tips | Biotage | 414141 | |
| 2 mL deep well 96 well collection plate | Waters | 186002482 | |
| 96 well plate sealing film | VWR | 391-1250 | |
| Zone-free 96 well plate sealing film | Sigma-Aldrich | Z721646-50EA | |
| Waters BEH C18 100 x 2.1 mm, 1.7m LC column | Waters |
Table M3 - Consumables and Chemicals - Item, Supplier, Part number
| Solution concentration | Volume of stock to add | Concentration of Stock solution | Volume of Methanol (uL) | Final volume (uL) | |
| 50 µg/mL | 50 µL | 1 mg/mL | 900 | 1000 | |
| 5 µg/mL | 100 µL | 50 µg/mL | 950 | 1000 | |
| 500 ng/mL | 100 µL | 5 µg/mL | 900 | 1000 | |
| 50 ng/mL | 100 µL | 500 ng/mL | 900 | 1000 | |
| 5 ng/mL | 100 µL | 50 ng/mL | 900 | 1000 |
Table M4 - Preparation of stock solutions - combined AT7519 and APAP - volumes and concentrations
| Standard Name | Amount (ng) | Internal Standard Vol (µL) | Standard Volume mixture (µL) | Standard concentration | |
| 0 Std | 0 | 20 µL | 0 | 0 | |
| 0.05 std | 0.05 | 20 µL | 10 | 5 ng/mL | |
| 0.10 std | 0.50 | 20 µL | 20 | 5 ng/mL | |
| 0.25 std | 4.50 | 20 µL | 5 | 50 ng/mL | |
| 0.50 std | 0.10 | 20 µL | 10 | 50 ng/mL | |
| 1.00 std | 1.00 | 20 µL | 20 | 50 ng/mL | |
| 2.50 std | 5.00 | 20 µL | 5 | 500 ng/mL | |
| 4.50 std | 0.25 | 20 µL | 10 | 500 ng/mL | |
| 5.00 std | 2.50 | 20 µL | 20 | 500 ng/mL |
Table M5 - Preparation of Calibration Standards - aliquot directly into 96-well PPT+ plate. Use solutions prepared as detailed in Table M4.
| QC Level | Standard Volume of APAP and AT7519 (µL) | Volume of 5% BSA | |
| 0.5 ng/mL | 100 µL x 5 ng/mL | 900 | |
| 0.75 ng/mL | 150 µL x 50 ng/mL | 850 | |
| 5 ng/mL | 100 µL x 50 ng/mL | 900 | |
| 40 ng/mL | 80 µL x 500 ng/mL | 920 |
Table M6 - Preparation of Quality Control solutions - prepared in bulk in 2% BSA and frozen in 100 µL aliquots. Use solutions prepared as detailed in Table 4
Protocol materials
LC-MS grade waterVWR InternationalCatalog #83645.320
Step 24.1
LC-MS grade acetonitrileVWR InternationalCatalog #83640.320
Step 24.1
Acetaminophen (Paracetamol) certified reference materialMerck MilliporeSigma (Sigma-Aldrich)Catalog #A-064-1ML
Step 1
d4-acetaminophen (d4-APAP) certified reference materialMerck MilliporeSigma (Sigma-Aldrich)Catalog #P-909-1ML
Step 1
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
Prepare calibration standard and Quality Control stock solutions
Prepare calibration standard and Quality Control stock solutions
The following table details the analytical standards needed for this protocol
| Compound | Abbreviation | Supplier | Catalogue Number | Concentration | Storage Conditions | |
| Paracetamol | APAP | Cerilliant/Merck | A-064-1ML | 1 mg/mL in MeOH | -20C | |
| AT7519 | AT7519 | Astex Pharmaceuticals | /// | Powder | -20C | |
| [2H]4-paracetamol | D4-APAP | Cerilliant/Merck | P-909-1ML | 1 mg/mL in MeOH | -20C | |
| [2H]8-AT16043M | d8-AT7519 | Astex Pharmaceuticals | /// | Powder | -20C |
Table 1- Analytical Standards - Name, abbreviation, supplier, catalogue number, concentration and storage conditions
Acetaminophen (Paracetamol) certified reference materialMerck MilliporeSigma (Sigma-Aldrich)Catalog #A-064-1ML d4-acetaminophen (d4-APAP) certified reference materialMerck MilliporeSigma (Sigma-Aldrich)Catalog #P-909-1ML
Take the1 mg /mL stock solution of APAP in methanol
Prepare a 10 µg /mL 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.
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).
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.
Take the 1 mg/mL stock solution of d4-APAP in LC grade methanol
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.
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).
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.
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.
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
| Solution concentration | Volume of stock to add | Concentration of Stock solution | Volume of Methanol (uL) | Final volume (uL) | |
| 5 µg/mL | 100 µL | 50 µg/mL | 950 | 1000 | |
| 500 ng/mL | 100 µL | 5 µg/mL | 900 | 1000 | |
| 50 ng/mL | 100 µL | 500 ng/mL | 900 | 1000 | |
| 5 ng/mL | 100 µL | 50 ng/mL | 900 | 1000 |
Preparation of stock dilution solutions - combined AT7519 and APAP - volumes and concentrations
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.
| QC Level | Standard Volume of APAP and AT7519 (µL) | Volume of 5% BSA | |
| 0.5 ng/mL | 100 µL x 5 ng/mL | 900 | |
| 0.75 ng/mL | 150 µL x 50 ng/mL | 850 | |
| 5 ng/mL | 100 µL x 500 ng/mL | 900 | |
| 40 ng/mL | 80 µL x 50 ng/mL | 920 |
Table Preparation of Quality Control solutions - prepared in bulk in 2% BSA and frozen in 110 µL aliquots. Use solutions prepared for calibration solutions.
Prepare calibration standards using the following table:
| Standard Name | Amount (ng) | Int Std (uL) | Std Vol (uL) | |
| 0 STD | 0 | 20uL (50ng/mL) | 0 | |
| 0.0500 STD | 0.0500 | 20 uL (50ng/mL) | 10 uL (5 ng/mL) | |
| 0.100 STD | 0.100 | 20 uL (50ng/mL) | 20 uL (5 ng/mL) | |
| 0.250 STD | 0.250 | 20 uL (50ng/mL) | 5 uL (50 ng/mL) | |
| 0.500 STD | 0.500 | 20 uL (50ng/mL) | 10 uL (50 ng/mL) | |
| 1.00 STD | 1.00 | 20 uL (50ng/mL) | 20 uL (50 ng/mL) | |
| 2.50 STD | 2.50 | 20 uL (50ng/mL) | 5 uL (500 ng/mL) | |
| 4.50 STD | 4.50 | 20 uL (50ng/mL) | 9 uL (500 ng/mL) | |
| 5.00 STD | 5.00 | 20 uL (50ng/mL) | 10 uL (500 ng/mL) |
Table M5 - Calibration standard table - Volumes of Int Std and Std stocks
Extract calibration standard & Quality Control and samples by PPT
Extract calibration standard & Quality Control and samples by PPT
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.
20230515_APAP_AT_MockPlateMap_Protocol_NZMH.xlsx
20230515_APAP_AT_MockPlateMap_Protocol_NZMH.xlsx 
Plate Map Design for 96-well extraction including calibration standards, quality controls, double blanks and up to 77 biological samples per batch
15m
Defrost biological Sample and quality control samples (lower limite of quantitation (LLOQ_, low, med and high)
10m
Set up Extrahera liquid handling robot for PPT+ extraction:
10m
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).
Turn on fume cupboard and make sure duct hose is in place in fume cupboard to ensure proper ventilation.
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.
Once the touch screen is active, from the Maintenance menu select Flush Solvent Inlets and Purge line S3 with Acetonitrile
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.
Place a PPT+ plate (Biotage) in deck position 3. Make sure that it is in the correct orientation and is properly clicked in place.
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!).
Take a Biotage 2mL deep well 96 well collection plate and add 100 uL 5% BSA solution to each of the standard wells.
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.
10m
For sample, according to plate map, add 50 uL serum plus 50 uL of water to the appropriate wells.
10m
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)
3m
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
10m
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).
2m
Remove the plate seal and place the sample plate on the deck of the Extrahera in position 4.
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
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.
20m
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.
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.
30m
Once dry, resuspend all the dry standards and samples in a solvent suitable for LC-MS/MS.
Prepare mobile phase solution for resuspension of samples (90:10 Water/Acetonitrile, v/v). Take 90 mL LC-MS grade waterVWR InternationalCatalog #83645.320 and add to 100 mL glass bottle, then add 10 mL LC-MS grade acetonitrileVWR InternationalCatalog #83640.320 Mix well. Label bottle.
5m
Prepare a repeater pipette with 2.5 mL tip and set volume to 100 µL . Pull up full volume into repeater pipette.
3m
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.
5m
Shake the plate for 10 minutes on the ThermoShaker (600 rpm ) to ensure the samples are resolubilised.
10m
Set up LC-MS/MS for analysis of extracts
Set up LC-MS/MS for analysis of extracts
35m
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.
10m
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.
10m
Put freshly prepared mobile phases onto the Acquity UPLC system. Purge lines with mobile phase A and mobile phase B on Acquity UPLC system
5m
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 °C and equilibrate at 95% A, 0.5 mL/min for at least 15 minutes. Ensure pressure is stable and no leaks on the column.
15m
Create an LC-MS/MS method by building the chromatographic method to be as follows:
| A | B | C | D | E | F | |
| 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 |
Table 2 - Chromatographic gradient details
Start with divert valve going to waste, divert to mass spectrometer at 1 minute, then back to waste at 8 minutes.
In Method creation use existing method (or create a method) with the following multiple reaction monitoring (MRM) parameters as mass spectrometry settings.
| Analyte ID | Q1 (Da) | Q3 (Da) | DP (V) | CE (V) | CXP (V) | |
| APAP 1 | 152.0 | 110.1 | 81 | 23 | 12 | |
| APAP 2 | 152.0 | 92.9 | 81 | 31 | 14 | |
| d4APAP 1 | 156.0 | 114.0 | 81 | 23 | 12 | |
| d4APAP 2 | 156.0 | 97.0 | 81 | 23 | 12 | |
| AT7519 1 | 382.1 | 84.1 | 66 | 29 | 10 | |
| AT7519 2 | 382.1 | 136.0 | 66 | 51 | 14 | |
| d8-AT7519 1 | 390.0 | 89.1 | 66 | 29 | 10 | |
| d8-AT7519 2 | 390.0 | 136.0 | 66 | 51 | 12 |
Mass Spectrometry parameters for multiple reaction monitoring on an AB Sciex 6500+ Qtrap; Turbo V Source, ESI. Curtain gas 35 units, Collision gas Medium, Ion Source Gas 1 50 and gas 2 60 units with a temperature of 550oC and an ionspray voltage of 5500 V.
Set up LC-MS/MS and analyse extracts
Set up LC-MS/MS and analyse extracts
35m
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:
Extracted ion chromatograms of APAP, d4-APAP, AT7519 and d8-AT7519 on C18 BEH column
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.
Assess chromatography and peak response while batch is underway.
Evaluation of LC-MS/MS data
Evaluation of LC-MS/MS data
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.
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.
Define sample type - Blanks, Double Blanks, Standards, Unknowns (samples) and Solvents.
Insert standard curve values and QC valules in 'actual amount' according to the calibration table and QC table.
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
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
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'
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
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'
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.