How to prepare QC for endogenous molecule [Bioanalytics]

posted by chandru – India, 2018-09-10 17:33 (742 d 22:58 ago) – Posting: # 19255
Views: 1,700

We are quantifying Levothyroxine in serum by LC-MS/MS method. Since it is endogenous molecule we are going by Standard addition method.

Calibration range- 35 to 350ng/mL
Typical endogenous level- 40 to 80 ng/mL
LLOQC-35ng/mL
LQC-60ng/mL
IMQC-90ng/mL
MQC-160ng/mL
HQC-270ng/mL

Doubt:
Since we are going by standard addition method, how the quality control samples be prepared?
Assume that before QC preparation the endogenous level was determined and it is 50ng/mL.
1.LQC preparation -Endogenous(50ng/mL)+spiked (10ng/mL) and capturing the nominal concentration as 60ng/mL
or
2.LQC preparation-Endogenous(50ng/mL)+spiked (60ng/mL) and subtracting the endogenous level in QC as well(like subtracting from Calibration standard).

For standard addition method how to prepare quality control samples.

a. In other words the doubt is, while preparation of QC sample the endogenous concentration level should be considered or not.
b. If procedure 1 is correct. Other QC samples can be prepared by additive (E+spiked) but how to prepare LLOQC samples in validation.

If there are any literature regarding standard addition can you please share.

Is there any guideline requirement for LLOQC concentration?
If my LLOQ(CS01) is 35ng/mL
Then my LLOQC should be of what concentration?(eg. between 35-37 or 100-105% of LLOQ)

Regards,
Chandru

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