Manual integration [Bioanalytics]
❝ 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
❝ 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!):
- Noise threshold: changes below this value are considered random noise and do not trigger peak detection.
- Upward- / downward slope detection: the data system fits a couple of data points to a function (Polynomial, smoothing spline, Savitzky-Golay, …) and calculates the first derivative at each time point. If the derivative is positive and above the threshold = start of peak; if the slope is negative and below the threshold = end of peak. For a Gaussian peak upward- / downward thresholds would be the same, but in chromatography peaks are asymmetrical. Some data systems correct for that by using more slices if the slope is negative or even change to a different fitting algorithm.
- Baseline drift: mainly important for gradient elution; nice question, next question.
- Area threshold: values below this value are not followed.
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 […]
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):
- Automated (smoothing factor 1, bunching factor 2):
CV 6.5% (1 ng/ml), 15.1% (0.1 ng/ml)
- Manual correction (one analyst):
CV 6.3% (1 ng/ml), 11.1% (0.1 ng/ml)
- Manual correction (ten analysts):
CV 5.2% [3.8%-6.8%] (1 ng/ml), 12.8% [6.9%-16.0%] (0.1 ng/ml)
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.
- Dolan JW. Integration Problems. LCGC North America, Oct 1, 2009.
online
- Dyson N. Chromatographic Integration Methods. The Royal Society of Chemistry 1998 (2nd ed.):p10.
- H Kirchherr H. Data Evaluation in LC-MS. In: Kuss H-J, Kromidas S (eds.): Quantification in LC and GC. Wiley 2009:p243–59.
Dif-tor heh smusma 🖖🏼 Довге життя Україна!
Helmut Schütz
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Science Quotes
Complete thread:
- Manual integration sagark 2010-07-29 02:26 [Bioanalytics]
- Manual integrationHelmut 2010-07-29 15:30
- Manual integration ElMaestro 2010-07-29 21:07
- History Helmut 2010-07-30 01:30
- LSB - evil, terrible and annoying ElMaestro 2010-07-30 23:46
- Manual integration ElMaestro 2010-07-29 21:07
- Bad integration: Example Helmut 2010-07-30 20:24
- Bad integration: Example sagark 2010-07-31 12:57
- Bad integration: Example Helmut 2010-08-01 02:09
- Better integration? Example Helmut 2010-08-12 20:11
- Better integration? Example ElMaestro 2010-08-12 23:14
- Better algorithms / more awareness of analysts Helmut 2010-08-13 13:43
- Better integration? Example ElMaestro 2010-08-12 23:14
- Better integration? Example Helmut 2010-08-12 20:11
- Bad integration: Example Helmut 2010-08-01 02:09
- Bad integration: Example sagark 2010-07-31 12:57
- Manual integration keshav khude 2010-08-13 09:11
- Manual integrationHelmut 2010-07-29 15:30