Chromatography Forum

I am not very sure with some of the following terms:

1) Polar
-when you say the sample is polar, what do you mean? Is it the sample will only dissolve in water or aqeuous?

2) Reverse phase
-If you want to run HPLC, what does this term mean?

3) LOD
- Am I right to say that this is the minimum standard concentration that the HPLC machine can detect for a particular method?
- Does this value change if Iwere to apply the exact same method when I run on a different set of HPLC machine?

4) LOQ
- Am I right to say that this is the minimum concentration for the spike standard set that the HPLC machine can detect for that method?
- Assuming my LOD is 0.1 ug/ml for this machine. But if I were to run my samples on another set of the machine, can my LOD value now change? If that's the case, does it mean that I have to re-determine the LOQ value again each time I run on a different set of HPLC machine?

5) Peak
- If you were to manual do a peak integration, do you take the analyte peak that is below LOQ even though it was eluted at the correct retention time? Is there any guidelines?

Is that any course or material on method validation and peak integration?

Thank you for taking the time to read this. I would appreciate if anyone can give me some advice.

Deat tlili


I suggest you check the FAQ page ( upper left side of taskbar on this forum site.


you will find answers to your questions and much more I am sure

succes

philippe

Thank you for the reply. I have went to the FAQ and has found a great information from there. Really thanks a lot.

However, i still like to know if anyone can tell me more about LOQ. Although I know this is often determined during the method development step, but do you need to find out LOQ each time the same method is used on a different HPLC machine?

I appreciate if anyone can give me some advise.

thanks again for all your help.

Tlili,

Traditionally, the LOQ has been determined only during method validation as you noted. However, that may be changing.

The USP had planned to add a system suitability requirement for "Detection Sensitivity" to its chapter <621> Chromatography. This requirement is basically a check of the LOD/LOQ as that value can change from instrument-to-instrument, day-to-day, etc. The requirement would have been applicable to all impurity methods. However, the USP has postponed implementing this requirement as there may be a problem for older methods to meet the requirement. The USP has suggested that the requirement be added to new impurity methods.

The EP section on system suitability/chromatography (sorry, I forget the section number and I don't have the EP on hand) has additional details on LOD, LOQ and the mesaurement of S/N.

I recommend that you check out both the USP and the EP for this issue.

Regards,
Dan

Hi Dan,

Thank you for taking the time to reply and the valuable advise. I really appreciate it. May i know what does USP and EP stands? Do you happen to have the website for this USP and EP?

Once this chapter 621 is introduced, may I know if it is a must that all HPLC experiments must follow this new clause?

Assuming the LOQ is 0.1ug/ml and is determined by HPLC machine A. But when it is run on HPLC machine B, I am still able to get my analyte peak (reasonable peak and not noise) below the LOQ previously determined by machine A. In that case, can I consider this peak? I would appreciate if anyone can give me some advise. Thank you for reading.

Tlili,

My apologies for not giving complete infromation, I forgot that you are new to all of this.

EP = European Pharmacopoeia http://www.edqm.eu/site/page_628.php

USP = United States Pharmacopoeia http://www.usp.org/

A pharacopoeia is a set of standards that provide guidelines that we in the pharmaceutical industry must follow. These standards are for techniques and for test procedures for drug substances and drug products.

The pharmacopoeias are published in book form at various time intervals (yearly for the USP) with smaller journals/magazines containing updates being published more freuqently.

The wesbites are above. However, you cannot read the chapters that I gave in my previous post. You must purchase these pharmacopoeias or use your library.

You may also find the methods validation guideline from the ICH (International Conference on Harmonization) to be useful. You can find that at this link:
http://www.ich.org/LOB/media/MEDIA417.pdf

Regards,
Dan

Hi Dan,

Thank you for all the helpful websites. I really appreciate it. Thanks again. Have a blessed day.

By the way, what sort of training or materials we can read to improve the HPLC knowledge (both theory and practical)? Any advise from anyone? thanks.

LOQ (limit of quantification) has different definitions in different fields. Some folks use it simply as the lowest fortified sample they included in their validation study. I've also seen it as 10 x noise, or noise + 10 x std. dev. of noise. The definition I personally like (right now...) is the lowest concentration (in your matrix) that gives acceptable precision and accuracy for your intended purpose.

LOQ is often dependent on which instrument you are using. LOD is nearly always dependent on instrument.

By the way, what sort of training or materials we can read to improve the HPLC knowledge (both theory and practical)? Any advise from anyone? thanks.

Check the FAQ (Frequently Asked Questions) link near the upper left of the screen. Follow the links down to "Where to obtain additional information". There are a number of on-line tutorials referenced in there.