Please help me resolving the following:

A sample solution, $\pu{0.24mg/mL}$ Compound A, is exposed to heat to form Compound B. From HPLC data report I have peak area for Compound A and Compound B. The STD concentration is 0.3 Compound A. The concentration of Compound A in sample solution is $\pu{0.20mg/mL}$ Compound A calculated using STD Compound A information. Which is the correct way to calculate concentration of Compound B?

My advisor in the project I'm participating insist I must calculate the concentration of Compound B since is a degradation of Compound A in term of the amount lost of Compound A is the amount gained by Compound B (she call it a indirect method for calculating the concentration). I'm at lost! Originally I performed the calculation using a STD Compound A at the LOQ level of the impurity: $$\text{[Compound B]} = \frac{\text{Area Compound B} \times \text{[STD Compound A]}}{\text{Area STD Compound A}}$$

The new approach is calculate the [compound A] using a STD at the level of the sample and then indirectly calculate the [Compound B].

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    $\begingroup$ There is no way! You need to construct calibration curve for compound B or have a single standard injection for compound B. $\endgroup$ – M. Farooq Aug 30 at 20:55
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    $\begingroup$ @M. Farooq: I agree with you about calibration curves. Also, may need a calibration curve for compound B, if two compounds are structurally different (based on detecting method, usually an UV detector). $\endgroup$ – Mathew Mahindaratne Aug 30 at 23:32
  • $\begingroup$ I think my advisor bold approach is based in Coumpond B is the enantiomer of Compound A (Doxycycline and 4-EPI-Doxycycline). $\endgroup$ – Ada J. Guzman Sep 1 at 10:52
  • $\begingroup$ Okay, then his assumption is valid. The UV spectrum of enantiomers is identical. $\endgroup$ – M. Farooq Sep 1 at 21:29

Your advisor is making a pretty dangerous assumption (although sometimes pharma people do it). The assumption is that the impurity is structurally similar to the main compound and its UV-Vis spectrum is almost similar i.e., their extinction coefficients are the same.

The idea in your formula is that the calibration curve for A will be same as B. Well that might be a first order approximation when the impurity is known to be structurally similar.

If B is a known compound, you should buy a separate standard, and make a calibration curve.

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