Bioequivalence and Bioavailability Forum

Dear Jaime!

❝ But: there are examples in the literature (please don't ask me where - buried somewhere in my files...), where the IS performed worse.

Agree. To quote Karnes et al. (1991):*

The assumption for use of an internal standard is that partition characteristics of the analyte and the internal standard are very similar. This can be a false assumption, […] the only appropriate uses of nonisotopic analogue internal standards are to serve as qualitative markers, to monitor detector stability, and to correct for errors in dilution and pipetting. […] The results of analysis can […] be adversely affected by the use of an internal standard because of the added variability of the internal standard measurement. This has been experimentally shown through a consideration of the law of propagation of errors. The internal standard technique will not inevitably improve, nor will it always adversly affect, the precision of an analytical method. Internal and external standardization techniques may both be evaluated when this issue is in question.

(my emphases)

See one example from our files (spironolactone and metabolites). IS was epoxyprogesterone; don’t worry about the chromatograms, different wavelengths (240 nm[nb] / [nb]280 nm) were recorded in two channels.

Day-to-day accuracy & precision (3 validation batches)

Spironolactone
conc.      IS method      ES method 
ng/ml   acc.%    CV%    acc.%    CV%
 41.2    99.6   2.77    102.8   5.62
183      99.7   3.35    101.2   1.23
733     100.3   1.12     99.7   1.05

7-alpha-thiomethylspirolactone
conc.      IS method      ES method 
ng/ml   acc.%    CV%    acc.%    CV%
 52.5    98.2   4.25     98.2   2.79
233      99.2   2.36    101.4   2.24
933     100.6   1.24     99.7   1.80

Canrenone
conc.      IS method      ES method 
ng/ml   acc.%    CV%    acc.%    CV%
 45.0   102.8   2.61     98.1   2.06
200     100.5   0.62    101.2   0.65
800     100.4   0.90     99.3   0.21

I don’t think the external standard method performed worse; the sponsor insisted in using the IS method anyhow.
The price we had to pay were runs-times prolonged by 56%…

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• Karnes TH, Shiu G, and VP Shah
  Validation of Bioanalytical Methods
  Pharm Res 8(4), 421–6 (1991)