3. Responsibilities

• Bioanalytical Analyst: Prepares calibration standards, maintains records, and verifies concentrations.
• Reviewer (Team Lead/QA): Verifies calculations, labels, and storage records of prepared calibration standards.
• Lab Supervisor: Ensures availability of qualified personnel and proper equipment for preparation.

4. Accountability

The Head of the Bioanalytical Laboratory is accountable for the accuracy and traceability of all calibration standards used in analytical runs.

5. Procedure

5.1 Preparation of Stock Solution

1. Weigh the required amount of certified reference standard using an analytical balance (calibrated as per SOP).
2. Dissolve in an appropriate solvent (e.g., methanol, acetonitrile, or buffer) to achieve the desired concentration (e.g., 1 mg/mL).
3. Record all weights, solvents used, lot numbers, and calculation steps in Annexure-1: Calibration Stock Preparation Log.

5.2 Preparation of Working Solution

1. Prepare a working solution from the stock by appropriate dilution using suitable diluent.
2. Ensure accuracy by using calibrated pipettes and volumetric glassware.
3. Calculate final concentrations and verify independently by a second analyst.
4. Document details in Annexure-2: Working Standard Preparation Record.

5.3 Spiking of Calibration Standards

1. Aliquot blank matrix (plasma, serum, etc.) into labeled microcentrifuge or polypropylene tubes.
2. Spike appropriate volumes of working solution into each tube to prepare calibration levels.
3. Typical levels: LLOQ, Low QC, Mid QC, High QC, and ULOQ (e.g., 7 to 9 levels).
4. Mix samples thoroughly by vortexing; record spiking volumes and calculations in Annexure-3.

5.4 Labeling and Storage

1. Label each spiked tube clearly with:
   • Analyte name
   • Concentration
   • Date of preparation
   • Expiry date
2. Store calibration standards at temperatures validated for analyte stability (e.g., -20°C).
3. Track usage and expiration using Annexure-4: Calibration Standard Inventory Log.

5.5 Usage in Analytical Runs

1. Thaw calibration standards at room temperature and vortex before use.
2. Run calibration standards in each batch in duplicate to generate a calibration curve.
3. Acceptance Criteria:
   • ≥75% of non-zero calibrators within ±15% (±20% at LLOQ)
   • Correlation coefficient (r²) ≥0.99

5.6 Documentation and Archival

1. Ensure all preparation, usage, and storage records are completed in ink and verified.
2. Scan and store documentation electronically as per lab data archival policy.

6. Abbreviations

• BA/BE: Bioavailability/Bioequivalence
• LLOQ: Lower Limit of Quantification
• ULOQ: Upper Limit of Quantification
• QC: Quality Control
• r²: Correlation Coefficient

7. Documents

1. Calibration Stock Preparation Log – Annexure-1
2. Working Standard Preparation Record – Annexure-2
3. Spiking Calculation Sheet – Annexure-3
4. Calibration Standard Inventory Log – Annexure-4

8. References

• ICH M10: Bioanalytical Method Validation
• US FDA Guidance for Industry – Bioanalytical Method Validation

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Calibration Stock Preparation Log

Date	Compound	Weight (mg)	Solvent	Final Volume	Prepared By
15/04/2025	XYZ-101	10.0	MeOH	10 mL	Rajesh Kumar

Annexure-2: Working Standard Preparation Record

Stock ID	Dilution Factor	Concentration	Volume	Prepared By
STK-XYZ101	1:10	100 μg/mL	5 mL	Sunita Reddy

Annexure-3: Spiking Calculation Sheet

Level	Target Conc. (ng/mL)	Plasma Volume	Spike Volume	Total Volume
LQC	50	450 μL	50 μL	500 μL

Annexure-4: Calibration Standard Inventory Log

Standard ID	Level	Prep Date	Expiry	Storage Temp	Status
CAL-XYZ-LQC	LQC	15/04/2025	30/04/2025	-20°C	In Use

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
01/01/2022	1.0	Initial Release	New SOP	QA Head
17/04/2025	2.0	Expanded preparation steps and added inventory tracking	Regulatory Update	QA Head