< or > LOQ

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

17 posts Page 1 of 2
Hello to all. I have a doubt.
Lately I am implementing in my laboratory the automatic calculation of HPLC impurities.
A question: if my method involves 2 injections and in one of these there is an impurity superior to LOQ and in the other it is inferior LOQ, I evaluate the average according to you?
Medium a lower LOQ value and a higher LOQ value?
Thank you all.
Hello Crimelist,

How can you quantify something which has a value which is less than the limit of quantification (LOQ). You cannot state not detected, but you can state less than or equals LOQ.
Kind regards,
Ade Kujore
Marketing
Cecil Instruments
Cambridge
United Kingdom

email:- ade.kujore@cecilinstruments.com
telephone:- +44 (0) 1223 420821
web site:- www.cecilinstruments.com
Registered Number 909536
Viewing this from a logical, rather than a regulatory perspective:

The LOQ is chosen at a point where your measurements have the largest acceptable random statistical error. Let's assume the LOQ is 1 pmole and the error is 10%. If your sample contains exactly 1pmole, and you measure it twice, it's quite natural that you'll get values in the region 0.9-1.1, and it's just as likely that the value will be less than the LOQ as it that it will be bigger.

If you ignore the smaller value (because you decided you can't quantify it reliably enough) then you bias the result in an upward direction. This may be a safe, cautious thing to do (or not; it depends entirely on the context of the measurement).

If you take the average of both measurements, regardless of their value, then the new estimate should be slightly more reliable than the old estimate, because it's an average, which always reduces the error (you're now looking at a standard error of two measurements; if their standard deviation was 1, the new standard error is 1/root-2).

So the question is what to do with this average? If it's bigger than your LOQ, you're probably happy. But what if it's smaller?

Well, originally in your LOQ you decided you would trust anything that you could measure to +/- 10% or better, and since the new, average value is an average, it is more accurate than the individual measurements - so it might have an error better than 10%.

So the answer is: your LOQ applies to the whole method you use to make measurements, not to individual injections. If you routinely report the amount of X in a single individual based on multiple chromatography runs, you'd be justified (morally, ethically, and scientifically, but not necessarily legally - that's not my domain) to calculate your LOQ based on the same replication, because what matters to you is entirely the accuracy of the result you report, not the accuracy of any intermediate steps in the calculation.

And, critical point: when people start worrying about the behaviour of their samples on a threshold value, I start worrying about whether the method is adequate. If it matters to you whether your sample contains 1 pmole or 0.9pmoles, then you need a method with a LOQ substantially lower than 1pmole. Life should be arranged so that no important contaminant that needs to be quantified is ever anywhere near as low as the LOQ, and no contaminant that is near the LOQ could possibly be considered concentrated enough to be relevant. Measurement Threshold values should be well clear of Decision-point Threshold values.
Your ideas are very correct. Unfortunately I analyze very pure products. LOQ value is given by the pharmacop.
I often find border-line results.

The problem is that often I have to do two injections and I can not find a rational to make the average. It is difficult to attribute the "pejorative case" within a software.

For example:

LOQ = 0.01 %

Result Inj 1 = 0.007 %
Result Inj 2 = 0.011 %
Mean = 0.009% (rounded 0.01%; > LOQ)

OR:

LOQ = 0.0059%

Result Inj 1 = 0.0057
Result Inj 2 = 0.0061

Mean?????
crimelist wrote:
Your ideas are very correct. Unfortunately I analyze very pure products. LOQ value is given by the pharmacop.
I often find border-line results.

The problem is that often I have to do two injections and I can not find a rational to make the average. It is difficult to attribute the "pejorative case" within a software.

For example:

LOQ = 0.01 %

Result Inj 1 = 0.007 %
Result Inj 2 = 0.011 %
Mean = 0.009% (rounded 0.01%; > LOQ)

OR:

LOQ = 0.0059%

Result Inj 1 = 0.0057
Result Inj 2 = 0.0061

Mean?????


The LOQ cannot be "given by the pharmacop", you have to demonstrate it by validating the methods you run in your lab. I suspect that you are confusing LOQ (an analytical parameter) with an Action Level / Maximum Acceptable Level whihc is a production process parameter; e.g. if the level is above the action level the batch has to be scrapped.

As lmh says, your validated LOQ has to be safely lower than the Action Level or you will be passing and failing batches purely on the basis of analytical imprecision.

Peter
Peter Apps
How would you define this? This is not the equivalent of a limit of quntification (0,05%)?

Image
My experience with reporting impurities in related substances test (pharma):

Calculate mean of both injections (one sample, two paralles) and set "Under the limit" if mean is equal or less of the chosen limit.

The yellow marked sentence is not about LOQ but about Maximum Acceptable Level. It's found in specifications of the product (LOQ would be part of the method validation protocol) and sometimes in the AP itself.
These limits are production pass/fail criteria, not analytical performance parameters.

The best (in fact the only) people to ask about what your customer's specifications mean is your customer.

Peter
Peter Apps
DavidHPLC wrote:
The yellow marked sentence is not about LOQ but about Maximum Acceptable Level. It's found in specifications of the product (LOQ would be part of the method validation protocol) and sometimes in the AP itself.


No, the yellow marked sentence is about the "disregard limit". Anything below this limit doesn't need to get reported (Note: it doesn't NEED to get reported. This has nothing to do with LOQ which defines of what CAN get quantified).
The maximum acceptable level is above this level.
The LOQ of the method should be sufficiently below the disregard limit - it is NOT quoted in the pharmacopoeia. With a good reason - as I keep on preaching: LOQ is NOT a fixed property of the method. It is a property of the whole analytic system (how "fit" is the column, detector, etc.). Actual LOQ on the same system might change on a day-to-day basis!
Therefore, although it is a common practice, it doesn't really make sense to include a fixed LOQ in the method description and state anything below this limit as "below LOQ".
In the pharmacopoeial methods, you have this reporting threshold. Anything below should be stated as "below reporting threshold" or just "<0.05%" (if 0.05% is the reporting threshold, for example).
HPLCaddict wrote:
DavidHPLC wrote:
The yellow marked sentence is not about LOQ but about Maximum Acceptable Level. It's found in specifications of the product (LOQ would be part of the method validation protocol) and sometimes in the AP itself.


No, the yellow marked sentence is about the "disregard limit". Anything below this limit doesn't need to get reported (Note: it doesn't NEED to get reported.


I stand corrected (translation issues).
Yes, me too, thanks for the clarification. In summary, I think there are three different threshold values under discussion here:

(1) The value you need to know. The decision limit, maximum acceptance value, or whatever it happens to be called in this application: the customer's limit-value at which they need to take action. This is set by their requirements and has nothing to do with either the method or the results.

(2) The value you ought to be able to know: this is the expected LOQ that you ought to be able to get using this method on this raw material with this instrument, typically assessed during method development. If this value isn't safely lower than the value in (1), then it's time to go back to the drawing-board and develop a new and more sensitive method.

(3) The actual LOQ today: this is genuinely the lowest value for which your real measurements are sufficiently precise. If this value is much worse than the expected LOQ that the method generally achieves, (2), then something has gone wrong today, and it's time to diagnose what.

I'm guessing that many of us in less regulated environments don't routinely assess (3) as rigorously as we ought. We check that (2) is appropriate, and then assume that if our calibration curve today looks much like it did during method development, then probably (3) would be OK too... hence our laziness in distinguishing between the LOQ (3) and the expected sensitivity of the method (2).
I understand the theoretical differences between the threshold value and LOQ. But the result is the same: I do the average and then I apply the threshold value (or LOQ?). Or I take the worst case (if higher than the threshold value?).
That's all.
Are you trying to say what the value is or are you trying to show that the result is less than a value (at some defined degree of confidence)?
crimelist wrote:
I understand the theoretical differences between the threshold value and LOQ. But the result is the same: I do the average and then I apply the threshold value (or LOQ?). Or I take the worst case (if higher than the threshold value?).
That's all.


Which of the two alternatives is specified in the method that you validated ? Whatever it is, do the same with the samples. That's all.

If your method is not validated you have worse problems than deciding whether to take an average.

If I were you I would take the mean of the peak areas and then calculate the content.
Peter Apps
One client we work for has such limits. If we analyze the sample and the result is just above the limit ( limit is 0.010 and our result is 0.011) then we submit that result. The client then requests an OOS ( out of spec) action where the sample is then run again three time and we send them the three re-analysis results. We let the client make the decision from there. I believe if the average is again within spec, they will call it good, if it is not, then they reject the batch.

If you have results that close to the limit and as you demonstrate one is above and one is below, I would give them both to the client and let them decide the final action if they do not have a written procedure for such occurrences.
The past is there to guide us into the future, not to dwell in.
17 posts Page 1 of 2

Who is online

In total there is 1 user online :: 0 registered, 0 hidden and 1 guest (based on users active over the past 5 minutes)
Most users ever online was 1117 on Mon Jan 31, 2022 2:50 pm

Users browsing this forum: No registered users and 1 guest

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food & Beverage, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry