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Hi All,
What are the ways to remove saturation from Calibration Curve (CC) from LC-MS/MS system, we tried
1. Increase dilution in Reconstitution Solution
2. Decrease response of Drug by decreasing CE (Collision Energy)
3. Reduce injection Volume
Although we are using Deuterated IS, still we are not able to remove saturation through area ratio,
Please suggest...
--
Regards
Kanchan Soni
Associate Scientist-BioAnalytical
Veeda Clinical Research
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Kanchan,
You may you quadratic regression type rather than line. It may solve your problem by fitting the
calibration curve.
Abhisheak Sharma,
Research Fellow
Pharmacokinetics & Metabolism Division,
Central Drug Research Institute, Lucknow (UP)
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Another way is to choose the ion pair for the analyst with less signal intensity.
Thanks,
Lea Huang
DMPK, Preclinical Development
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Try narrowing down your calibration curve. May be the highest CC is way too much that your machine
can handle. What is your current CC range? What are your expected concentration range in in vivo
studies?
Manish
Manish Issar, Ph.D
Assistant Professor of Pharmacology
Western University of Health Sciences
College of Osteopathic Medicine of the Pacific
Pomona, CA 91766
Email: missar.-a-.westernu.edu
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I think you are using ESI-ionization technique, try with APCI, you can go upto much higher upper
limit of quantification without any saturation.
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Kanchan:
In regards to using a quadratic regression type , please keep in mind the FDA's "Guidance for
Industry: Bioanalytical Method Validation" available on the CDER website, which says ""The simplest
model that adequately describes the concentration-response relationship should be used. Selection of
weighting and use of a complex regression equation should be justified.". This has always been good
advice. And above all, keep it consistent.
Christopher J. Kemper, Ph.D.
chris_kemper.aaa.pharmanavigatorsllc.com
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Hello Kanchan,
You are on the right path of thinking for reducing saturation on your detector. Did you try
increasing the CE instead of decreasing it? Use your LLOQ and make multiple injections stepping up
the CE to determine where the response is still acceptable. If that does not work another
possibility may be to try and add a split to waste post column before the detector. If you continue
to have saturation problems after attempting to dilute the injection samples then it may be time to
consider reducing the linear range. Not all molecules will have to same linear range on a mass
spectrometer.
Hope that helps!
Lee
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1. Where does the curve depart from linearity?
2. What is the mass and the m/z?
3.The two most common sources of non linearity in LC-MS are incomplete ionization and incomplete
detection.
4. Have you investigated the matrix for impact on ionization/detection?
5. Another impact may be that at high concentrations of unknown, the unknown reduces the the
ionization of the IS and that the IS can reduce the response of low concentrations of the unknown.
6. Use of a quadratic can be more complicated than other regression since, by definition, you can
have two solutions and you can be lulled into complacency rather than compliancy [SIC]. If you need
to, use something other than quadratic, log-linear, logit, etc. Math can do a lot but...
7. Have you tuned the MS for your ULOQ and LLOQ (should be the same or very close.)
8. Where are you with saturation of the column?
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Kanchan,
Apart from using quardatic regression. You can use triveter valve followed by a drainage tube of
specific size (lesser length tube higher drain). It will assure the reduction in analyte quantity
per injection reaches to detector.
Regards
Abhisheak
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Dear Kanchan,
Kindly check solubility of drug, it may be one of the factor for non-linearity.
JigneshBhatt.-at-.torrentpharma.com
--
Hi All,
Thanks for your wonderful Suggestions
To Abhishek Sir & Christopher J. Kemper Sir: As per Literature survey we found that this method is
in Linear although we have good results in quadratic regression type, so we have more emphasis on
Linear rather than quadratic.
To Murari Pal Sir: Yes we are using ESI-ionization technique, we will check on APCI mode.
To Manish Issar Sir: We have decided this range on the basis of Literature survey this is 5000 to
10ng/mL, the Cmax we found approx 2.8µg/mL.
To Lee Winchester Sir: Yes sir we tried by increase of CE value & we are in practice split to waste
post column before the detector but unfortunately we unable to get good results.
To Edward O'connor Sir: Always STD1 Showing % accuracy 88-89 and HQC 90
Mass is 358 and m/z is 359/280 and we are observing same in neat solutions. we are using D compound
for IS, saturation is coming from source not from column, yes we will tune ULOQ and LLOQ.
kanchan
kanchan soni
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Hi Kanchan,
There are many possible causes of saturation, among all many a times, we miss to optimize source
parameters through FIA and another suggestion is findout column saturation.
Good luck
P Pavan Kumar
Pavan Kumar Prathipati
--
My two cents:
Use flow splitting (e.g. 1:4 ). It may decrease saturation without compromising sensitivity.
Cheers,
Rob ter Heine
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Kanchan. Did you change your Mobile phase , it may some time happen that ionisation is not linear.
May be change of mobile phase solve your problem.
vishal shah
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Dear kanchan,
If you find saturation from mass and not from column there is only two possibilities that either
source or detector.
First check source parameter that your mobile phase give complete ionization to analyte and if
problem persists check out CEM.
Hope this will help you and correct if I am wrong.
Please share your experience.
Regards,
Dipak Harwani
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Please check the solubility of the drug. It is one of the main factor for saturation, we general
miss. You can check the ionizations using different mobile phases. Additionally you can also check
the column loading.
Hope this will help.
Regards,
Sudipta Basu
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Kanchan: try calculating curve without the IS and let us know what happened no added wet work just
remove the ratio
Edward O'Connor
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Dear Kanchan,
Before you start the analyis, try for FIA (Flow injection analysis) to
optimize the reponse. Secondly your LLOQ should be around 80ng/ml. Please
recontruct the calibration curve to minimize the saturation.
Regards
Tushar, Salil & Harshal
Bioanalytoical
AnaZeal Analytical & Research Pvt. Ltd.
Manisha
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Dear Kanchan
I feel usage of Deutrated IS does not have any role to play with saturation. It will not help.
Few things can be tried
1. Use splitter to split the flow, the ratio of split can be optimized based on the response you are
getting.
2. Select some other daughter ion in MRM method, this will really help.
3. Narrow down the CC range and establish dilution integrity for the higher concentrations.
4. If don’t want to do dilution integrity than last thing is make 2 CC ranges accordingly.
Hope this works for you,
Regards,
Dr Pradeep Sharma on behalf of Tajinder Pal singh
Pradeep Sharma
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Dear kanchana,
May be you kept broad linearity range, try in APCI
Regards,
Sreekanth Dittakavi
Sreekanth Dittakavi
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Hi,
is it a curve from neat solution (acn/meoh)? If it involves biomatrices\ buffers, please try
parallel dilution instead of serial..hope it helps!
AB Rao
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Other than good suggestions already given;
Increase DP & CE; reduce aliquot vol, use APCI, reconstitute in higher volume.
Regards,
Ravi Trivedi
PS: Alternatively go back to HPLC-UV; as required LLOQ is on higher side !
Good luck !!
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Saturation could be due to......
1. Selection of appropriate mobile phase as the buffer or organic part is insufficient to ionize the
higher concentration standards.
2. Alteration in source dependent parameters like Temperature, Ion spray voltage may sort out.
3. Change system to lower sensitivity i.e. 4000 to 3200 or so on.
Hope these may work
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Hi
Thanks everyone,
Finally we optimize the method, not at 100% accuracy for STD1 but about 96-97%.
We tried all the suggestion which were provide by you all.
Finally we went with the factor solubility and 95% splitting.
once again thanks to you all.
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