3. Responsibilities

• Analyst: Prepares and executes the run sequence as per study design and batch layout.
• Reviewer/Supervisor: Verifies run setup parameters and sequence structure.
• QA Officer: Audits analytical sequences and records for GxP compliance.

4. Accountability

The Head of Bioanalytical Operations is accountable for ensuring adherence to this SOP during all analytical run setups to maintain data integrity and regulatory consistency.

5. Procedure

5.1 Sequence Design and Naming Convention

1. Create the run sequence file using the LC-MS/MS software (e.g., Analyst™, MassLynx™, Empower™).
2. Name each sequence file as per the following convention:
   • Format: [StudyCode]_Seq_[BatchNumber]_YYYYMMDD
   • Example: BE2456_Seq_02_20250417
3. Save the file in a secure, study-specific folder with backup.

5.2 Sequence Structure and Order

1. Include the following components in this exact order:
   • System Suitability Test (blank and system check sample)
   • Double blank sample (no analyte, no internal standard)
   • Single blank (no analyte, with internal standard)
   • Calibrators: STD01 to STDx (minimum 6 non-zero concentrations)
   • Quality Control Samples: LQC, MQC, HQC in duplicate
   • Study Samples (randomized and interspersed with QCs)
   • Anchor QCs (at beginning, middle, and end)
   • Final blank to ensure no carryover
2. Use randomization software to arrange subject samples.

5.3 Instrument Method and Tuning File

1. Select validated instrument method file containing:
   • Retention times
   • MRM transitions
   • Dwell times
2. Load appropriate tuning/calibration file approved during system readiness check.
3. Ensure signal intensity of tuning compound meets system suitability criteria.

5.4 Sample Information and Metadata Entry

1. Enter:
   • Sample ID
   • Sample type (STD, QC, Subject, Blank)
   • Vial position
   • Batch ID
2. Assign correct processing method and integration parameters.

5.5 Final Review and Run Initiation

1. Have sequence reviewed by the study supervisor or scientific reviewer using Annexure-1: Analytical Run Checklist.
2. QA should review run logbook entries, audit trail settings, and lock the sequence file.
3. Begin run after confirmation of:
   • Correct tray loading
   • Run time within 36 hours
   • Environmental conditions within range

5.6 Run Completion and Documentation

1. After run completion, perform:
   • Peak integration (auto/manual)
   • System suitability verification
   • Backup of data
2. Document run performance in Annexure-2: Analytical Run Log.

6. Abbreviations

• BA/BE: Bioavailability/Bioequivalence
• LC-MS/MS: Liquid Chromatography-Tandem Mass Spectrometry
• QC: Quality Control
• STD: Standard
• MRM: Multiple Reaction Monitoring

7. Documents

1. Analytical Run Checklist – Annexure-1
2. Analytical Run Log – Annexure-2

8. References

• ICH M10: Bioanalytical Method Validation
• FDA Guidance for Industry: Bioanalytical Method Validation
• Internal LC-MS/MS Operation SOP

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Analytical Run Checklist

Parameter	Checked	Remarks	Initials
Correct sequence file loaded	Yes	Verified	SR
Sample tray matched with layout	Yes	All vials checked	RK

Annexure-2: Analytical Run Log

Run Date	Batch ID	System Used	Start Time	End Time	Analyst
17/04/2025	BE2456-MET-02	Sciex 6500+	08:30	20:00	Sunita Reddy

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
01/01/2022	1.0	Initial version	New SOP	QA Head
17/04/2025	2.0	Sequence layout structure and verification steps added	Regulatory update	QA Head