HPLC Calibration Scheme

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

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I am currently working on a potency method by HPLC that analyzes for 11 analytes. My target range is 1 µg/mL - 1000 µg/mL for each analyte. The max concentration for standards that are provided by vendors for each analyte is 1000 µg/mL. So the issue here, is that it is impossible to make a calibration point at 1000 µg/mL where all analytes are present in the solution. I have devised a scheme where the low levels contain each analyte in a single solution and the high levels analyze individual solutions of each analyte. Is this a suitable approach to calibration? State licencing officials do not seem to be accepting of the approach citing that there is no published sources saying this is acceptable but also, I can't find anything saying that all analytes need to be present in all solutions. What is justification against calibrating each individual compound using 11 different curve preparations? Any insight would be much appreciated!
Further to your other post, there is no reason why your approach would give the wrong answer. Put it this way, if you imagine your batch analyzing analyte "A", if you treat all the unnecessary standards that you ran during that batch in order to quantify "B" and "C" as mere samples, the process would be fine, wouldn't it? And if your method falls over if an extra sample is inserted in the sequence that happens to contain pure "B" at high concentration, you've got a serious robustness problem!

But (enormous but), if you're working in a regulated environment, you have to do what the regulators say, no matter how utterly unfounded and misguided it might be. They are the regulators, that's what they do. If they say that work is valid only if carried out by a left-handed analyst facing North on a Thursday, then you'd better install a compass and start employing left-handed analysts, because that's a whole lot easier than changing the rules.

Practically, does your method have sufficient sensitivity to yield adequate results if you dilute everything, standards and samples, 11-fold? If so, you can use a conventional mixed standard, and dilute the samples so that they still fall in the range of the calibration curve. This should be regulator-friendly provided you do your limit-of-quantification.

Alternatively, mix all your standards together, dry them down, and redissolve in the volume you want. Of course this process runs the risk of losing standard, so to validate it, quantify a dilution of this dried, mixed standard against a calibration prepared by conventional mixing.
lmh wrote:
Further to your other post, there is no reason why your approach would give the wrong answer. Put it this way, if you imagine your batch analyzing analyte "A", if you treat all the unnecessary standards that you ran during that batch in order to quantify "B" and "C" as mere samples, the process would be fine, wouldn't it? And if your method falls over if an extra sample is inserted in the sequence that happens to contain pure "B" at high concentration, you've got a serious robustness problem!

But (enormous but), if you're working in a regulated environment, you have to do what the regulators say, no matter how utterly unfounded and misguided it might be. They are the regulators, that's what they do. If they say that work is valid only if carried out by a left-handed analyst facing North on a Thursday, then you'd better install a compass and start employing left-handed analysts, because that's a whole lot easier than changing the rules.

Practically, does your method have sufficient sensitivity to yield adequate results if you dilute everything, standards and samples, 11-fold? If so, you can use a conventional mixed standard, and dilute the samples so that they still fall in the range of the calibration curve. This should be regulator-friendly provided you do your limit-of-quantification.

Alternatively, mix all your standards together, dry them down, and redissolve in the volume you want. Of course this process runs the risk of losing standard, so to validate it, quantify a dilution of this dried, mixed standard against a calibration prepared by conventional mixing.


This is what I was thinking would work, run the calibration range from 1-75 instead of 1-1000, that way you satisfy the regulators. Dilute any samples that go over the 75 concentration limit. If most samples will be above 75, then add the dilution step to the sample preparation method.
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