Quality routines for dose response

When requesting compounds for a dose response follow up, an IP (identity and purity) analysis will also be performed on LC-MS.

Exception: Some of the purchased libraries exists only as a few µl on screening plates and IP analysis cannot be performed due to insufficient volumes.

Identity and Purity Analysis Using HPLC‑MS

Abbreviations

ES+ : Electrospray, positive mode
MilliQ : Ultrapure water treated with UV light and particle-filtered
HPLC : High Pressure Liquid Chromatography
TFA : Trifluoroacetic acid

Description

A Dose Response experiment describes how the effect on an organism (e.g., cells) changes when exposed to varying concentrations of a compound during a defined time period.
It is used to determine the activity of compounds previously identified as hits.

The procedure ensures data quality by confirming:

• Identity of each compound
• Purity, which must be >85% to pass quality control

Compounds below purity threshold or lacking detectable mass peaks are flagged as suspects and subjected to further analysis (e.g., alternative LC‑MS methods or NMR).

Procedures

The SciLifeLab Compound Center performs identity and purity analysis using HPLC UV/MS for all completed Dose Response experiments.

Sample preparation

• From each selected compound:
  • Transfer 5 µL of 10 mM DMSO solution to a glass vial or 96‑well polypropylene plate
  • or transfer 500 nL to a 96‑well polypropylene plate
• Dilution:
  • 5 µL samples → add 50 µL methanol or acetonitrile
  • 500 nL samples → add 10 µL methanol or acetonitrile
• Load prepared samples into the HPLC‑MS system

Summary of Method

Full instrument specifications and method validation are documented in:

• ELN (KI): EXP‑22‑DT9001
• ELN (UU): SOP_UU_BK0902

Equipment Needed

• Agilent 1260 Infinity II HPLC UV/MS
• Systems used: “Caesar” and “Cleopatra”

Methods

Primary Method (ES+)

• Mobile phase A: 0.1% TFA in MilliQ water
• Methods: QA1097‑3, A1097‑3

Secondary Method (for insufficient primary results)

• Mobile phase A: 10 mM NH₄HCO₃, pH 10, in MilliQ water
• Method: QX1097‑3

Data Validation

• Automated UV‑response integration and purity calculation are included in the analytical report.
• Manual review is performed if any results deviate from expectations, including:
  • UV chromatogram inspection
  • Mass spectrometry verification

Actions and Future Aim

• All results are stored in Scarab
• Raw data archived at: \storage3.ad.scilifelab.se\NMR400\data\Caesar\Asa IP

Compounds that meet the quality requirements will not undergo any further actions. Compounds not meeting the quality requirements will be reported to the scientist responsible for the experiment. The aim is to assure the quality of the Dose Response experiments to guarantee the generated result as reliable data.