How to perform the BioAccord cell culture media system suitability test - WKB231508

1) Introduction

The SST is designed to ensure that the complete LC-MS system is fully operational prior to routine use. This test is designed so that stringent quality criteria (mass accuracy, RT stability, peak separation, peak response reproducibility, and solvent background) are assessed using a Waters-certified standard solution (Vion Test Mix) in a 10-minute run. This will help users to assess the status of their LC-MS instrument prior to performing data acquisition. The method enables screening for nine substances using the mass and retention time as quality criteria. The method can also display and identify matched fragments, in order to further demonstrate the system’s screening capabilities.

Waters recommends that you routinely perform this or a similar test to maintain the highest levels of confidence in your data.

Example of extracted ion chromatogram of the nine injected compounds for positive ion mode.

2)    Checklist

• Check solvents and prime the ACQUITY Premier LC system and wash syringes.
• Create a new analysis.
• Go to initial conditions.
• Inject two blanks and five injections of the Vion Test Mix.
• Review the report to ensure that the system meets performance criteria.

3)    Sample preparation

• Prepare a blank sample by transferring 1 mL of H₂O plus 0.1% FA (mobile phase A) into a Waters vial and place it in the Sample Manager in position 1:A,1.
• Transfer the content of Waters part 186008462 Vion Test Mix ampoule into a Waters vial and place it in the Sample Manager in position 1:A,2.

4)    Detailed procedure

1. Check the ACQUITY Premier UPLC solvent bottles and ensure that there is sufficient volume of mobile phase.
2. Ensure that there is a blank on position 1:A,1 and the Vion Test mix on position 1:A,2.
3. Confirm that there is sufficient lockmass solution (left bottle of RDa fluidics).
4. Open UNIFI software.
5. When performing the SST for the first time, import the UNIFI UEP file.
   • Import the "SST CCM.uep" file to UNIFI (contains sample list, analysis method, example data, and report template).
6. Create a new analysis by selecting "Create analysis by acquiring new data".
7. Select the "CCM SST sample list".
8. Select the "CCM SST Analysis Method", and click Next.
9. Adjust the analysis name and folder as appropriate, and click Next.
10. Select the system, click Next, and then click Finish.
11. Click on the bottom-right green check mark icon to open the "Instrument System" side panel.
12. On the Binary Solvent Manager, select Prime A/B solvents, select start priming, and close the window.
13. On the Sample Manager FTN, select Prime Syringe, perform prime, and close the window when complete.
14. On Summary, select Go to initial conditions.
15. Wait for the delta pressure of the binary solvent manager to be less than 30 psi.
16. Select sample list lines to be injected.
17. Click the Start button (be sure to select "Process samples", "Save results", "Generate a report", and "Save report and do not print".
18. When acquisition is complete, review the report for Pass or Fail. (see example report: "CCM SST Report.pdf")

SST PASS: Complete

The BioAccord LC-MS system is fully operational. The quality criteria for mass accuracy, RT stability, MS response and reproducibility are within expectations across five replicates injections for each positive and negative polarities. The analysis results meets the following criteria: 1). Identified components mass accuracy is within +/- 5 mDa 2). Observed RT %RSD is better than 1.00% 3). MS Response %RSD is better than 10.0% 4). Sulfadimethoxine mean response is greater than 5'000 counts for positive polarity 5). Sulfadimethoxine mean response is greater than 1'000 counts for negative polarity

SST FAIL: Complete with Error

The BioAccord LC-MS system requires user attention before acquiring new data. One or more quality criteria are not meeting expected results across five replicates injections for each positive and negative polarities. The following page of the report will show red flags where passing criteria have not been achieved.

Here are some tips for troubleshooting the system:

1). Mass error flags will require a new RDa detector setup to be performed.
2). Observed RT %RSD and MS Response %RSD flags indicates an issue on the chromatographic system. This requires to check the flow path for leaks, prepare fresh solvents and prime the system.
3). A flag on sulfadimethoxine response indicates a sensitivity issue with the RDa detector. This may require to make a fresh sample or change the aperture disc.
4). The limit status column of the analysis injection list allows to review which specific injection is causing issues

5)    Analysis method

 

Purpose Manage Components
Component name	Expected RT (min)	Expected neutral mass (Da)	Adducts	Formula	Concentration (µg/mL)
Sulfaguanidine	2.40	214.0524	+H, -H	C7H10N4O2S	5.0
Acetaminophen	3.60	151.0633	+H, -H	C8H9NO2	10.0
Val-tyr-val	3.80	379.2107	+H, -H	C19H29N3O5	2.5
Caffeine*	4.00	194.0804	+H	C8H10N4O2	1.5
Leucine enkephalin	4.60	555.2693	+H, -H	C28H37N5O7	2.5
Verapamil*	5.70	454.2832	+H	C27H38N2O4	0.2
Sulfadimethoxine	5.80	310.0736	+H, -H	C12H14N4O4S	1.0
Reserpine*	5.90	608.2734	+H	C33H40N2O9	0.6
Terfenadine*	6.50	471.3137	+H	C32H41NO2	0.2
*The compound does not ionize in negative ion mode.
Purpose Default Amounts
Add custom level	POS	NEG

 

 

Instrument Column Chemistry	ACQUITY Premier HSS T3 2.1 x 150 mm (186009469)
Column temperature	40 °C
Flow rate	250 µL/min
Mobile phase A	H2O / 0.1% FA
Mobile phase B	90% ACN / 10% IPA / 0.1% FA
Wash	40% ACN / 40% H2O / 20% IPA
Purge	90% H2O / 10% ACN
Seal wash	90:10 water/methanol

 

 

Time (min)	Flow Rate (mL/min)	Composition A (%)	Composition B (%)	Curve
0.00	0.250	95.0	5.0	Initial
0.50	0.250	95.0	5.0	6
7.00	0.250	15.0	85.0	6
7.80	0.250	15.0	85.0	6
8.00	0.250	95.0	5.0	6
10.00	0.250	95.0	5.0	6

 

 

ACQUITY RDa Detector
Mode	"Full scan" or "Full scan with fragmentation"
Mass range	Small molecules (50 – 800 m/z)
Polarity	Positive or Negative
Scan rate	5 Hz
Cone voltage	Default (30 V) for positive and default (40 V) for negative
Fragmentation cone voltage	Custom 60 V to 140 V
Events Table	0.00 Divert to waste 1.00 Divert to MS 8.00 Divert to waste
Capillary voltage	Default (1.50 kV) for positive and default (0.80 kV) for negative
Desolvation temperature	Default (550 °C)
Intelligent data capture	On
Lockmass correction mode	Standard

 

 

Sample List Setup
Sample type	Blank or QC
Run Time (min)	10
Injection volume (µL)	0.5
Replicates	1
Polarity (Instrument)	Positive / Negative

 

 

Peak Processing Settings 3D Peak Detection
Low energy intensity threshold	1000 counts
Targeted Screen Settings Target by Retention Time
Enable screen by retention time	Enabled
Identify target peak using rule	Largest in the RT window
Use absolute retention time identification tolerance	0.5 minutes
Targeted Screen Settings Target by Mass
Target mass tolerance	20 ppm
Generate predicted fragments from structure	Enabled
Fragment match tolerance	10.0 mDa
Extract mass chromatogram	Enabled
Tolerance	Manual, 30 mDa
Extract mass chromatogram containing all identified ions	Enabled
Summary Calculations
Field	Sample type	Group by
Observed RT (min)	QC	Sample position
MS Response	QC	Sample position
Limit Checks
Node	Field name	Component	Level	Error minimum	Error maximum
Component	Mass error (mDa)	All	All	-5	5
Summary fields	Observed RT (min):% RSD (%)	All	All		1
Summary fields	MS Response:% RSD (%)	All	POS		10
Summary fields	MS Response:% RSD (%)	All	NEG		10
Summary fields	MS Response:Mean	Sulfadimethoxine	POS	5000
Summary fields	MS Response:Mean	Sulfadimethoxine	NEG	1000

Sample type is QC for all limit checks field