Manual integration [Bioanalytics]

posted by Helmut Homepage – Vienna, Austria, 2010-07-29 17:30 (4991 d 02:30 ago) – Posting: # 5699
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Dear Sagar!

❝ This is my first post to the forum. I would like to thank HS for running and all for contributing their views into this excellent forum.


Welcome to the club and thanks for the :flower:

❝ I have doubts for the concept of manual integration. In what cases it is done and what are the considerations for it?


For some background have a look at these posts: 2261, 2728, 4447.

Let’s have a look how peak integration in chromatography is performed. The detector may deliver signals at high data rates. But if we would use this raw signal, we would see a lot of noise on top of even high peaks. Therefore the raw signal is bundled to slices (either – rarely – within the detector or by the CDS), based on an appropriate time constant. As a rule of thumb for the narrowest peak the width at half height should be divided by ≈10–20. For a 10s peak we would set the data aquisition rate to 0.5–1s (60–120Hz). For long runtimes it is advisable to increase the bundling rate for late eluting peaks. The data system detects the start end end of peak based on – at least – following parameters (terms may differ!):Once you have set up integration parameters all these calculations are running behind the scenes. It’s always possible that the method’s integration parameters will fail. A tricky issue are peaks in the lower concentration range. But it is also possible that the algo fails for high peaks, mainly by problems with the downward-slope threshold. If you have random noise (±) which by chance is increasing the signal the data system will ‘see’ that as the end of the peak and the baseline is drawn too early. If on the other hand the random noise is lower than expected by the algo, the integration will continue – the baseline will be drawn to a too late time point.
It’s important to realize that there is no ‘right’ integration for any given peak. Even if you export the chroma­to­gram’s raw data (peak slices – detector’s response is not stored!) and try to obtain the same peak area with another data system it will be almost impossible because the internal algorithms are different (and not documented in the manual – just my 2¢).

❝ Is it accepted by USFDA?


Yes. You have to have an SOP in place and report which chromatograms were reintegrated (why, by whom, when: all the usual stuff needed for an audit trail). See also an article by John Dolan.1

❝ Also how does one know if the manual integration done is proper or not.


Our brain is a perfect pattern-recognition-system. IMHO any experienced chromatographer will be able to draw baselines better than the CDS’ ones. An excursion into history: before integrators were used in chromatography, strip chart recorders were used. The baseline was drawn by a ruler and the peak height measured. The first integrator was introduced in 1968 (Hewlett Packard 3370), but manual ‘integration’ was the rule until the mid 1970ies. If an inspector has some problems with manual integration remind him/her on drugs getting approval earlier. Would they question these results?

All chromatograms should be reviewed and the integration corrected if necessary. The only textbook on chromatographic integration methods2 states in the introduction:

No analytical report should be accepted unquestioningly.
An integrator might draw a baseline in the wrong place or separate two peaks by skimming a tangent where a perpendicular would better, or vice versa. It is important to inspect these features on the actual chromatogram and confirm their correctness […]

One thing is important: The review has to be done before () concentrations are calculated. Changing integration of a peak in order to bring a calibrator / QC to the expected value (e.g., make a batch valid which would be rejected otherwise) or a predose concentration <LLOQ would be clear evidence of fraud.

See also this presentation on manual integration by Bob Di Rienzo and Melinda Jacobson from The NELAC Institute's Forum for Laboratory Accreditation, Newport Beach, CA, January 2008.

Below a nice example3 (LC/MS-MS, risperidone, protein precipitation, dilution factor 8, API 4000, software Analyst 1.4.1, n=10); 1 ng/ml and 0.1 ng/ml (LLOQ):

If the employees were well trained the values in the region of the LOQ were rather better than with an automated integration.


Look at the CV ranges! Even ‘bad’ analysts got CVs close to the automated integration, but ‘good’ ones outmanouvered silicon-brains with great ease.


  1. Dolan JW. Integration Problems. LCGC North America, Oct 1, 2009.
    online
  2. Dyson N. Chromatographic Integration Methods. The Royal Society of Chemistry 1998 (2nd ed.):p10.
  3. H Kirchherr H. Data Evaluation in LC-MS. In: Kuss H-J, Kromidas S (eds.): Quantification in LC and GC. Wiley 2009:p243–59.

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