Bioequivalence and Bioavailability Forum

Dear all,

recently I saw a deficiency of a European agency:

The Applicant should specify the integration method (automatic or manual) adopted to calculate chromatograms area for all submitted studies. If manual integration was adopted, the Applicant should specify the percentage of the total chromatograms and specify which samples (QC/calibrator or unknown) have been analysed.

Sure, that’s according to the 2011 guideline stating:

5.5. Integration
Chromatogram integration and re-integration should be described in a SOP. Any deviation from this SOP should be discussed in the analytical report. Chromatogram integration parameters and in case of re-inte­gration, initial and the final integration data should be documented at the laboratory and should be available upon request.

Instead of simply answering the question, i.e., that only automatic integration was used, the CRO additionally quoted from its SOP:

6.6 MANUAL INTEGRATION
Manual integration is PROHIBITED in any circumstance.

Oh dear, that’s bad scientific practice. Trust a silicon-based life-form more than a human brain?
An experienced analyst outperformed the software easily (esp. at low concentrations). Even ‘normal’ ones did so on the average.*
$$\small{CV\;(n=10)}$$$$\small{\begin{array}{lcc}
\text{integration method} & \text{1 ng/mL} & \text{0.1 ng/mL}\\\hline
\text{automated (smoothing 1, bunching 2)} & 6.5\% & 15.1\%\\
\text{manual correction (experienced analyst)} & 6.3\% & 11.1\%\\
\text{manual correction (ten analysts)} &5.2\% & 12.8\%\\
& (3.8\%-6.8\%) & (6.9\%-16.0\%)\\\hline
\end{array}}$$ See also here (slides 26–29) and there (slides 24–31).

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• Kirchherr H. Data Evaluation in LC-MS. In: Kuss H-J, Kromidas S, editors. Quantification in LC and GC. Wiley 2009; p 243–59.