[budget]

Hi. I'm in the process of obtaining a calibration curve for a reference substance by HPLC, but had a some uncertainties regarding what factors to consider when making up a stock solution for the calibration standards.

My supervisor mentioned that it's best to weigh more than 10mg (he said 100mg is preferable but 100mg is all I have) of the substance when making the stock. I'm assuming this is to minimize the error during weighing since this is going to be the stock solution. But is there an accepted error range for this specific procedure? I'm planning to weigh 10mg on a scale that measures down to 3 decimal places (0.001mg) which would mean the error % is (0.001/10)*100=0.01%. I won't be needing a working standard since the stock will only require 1 dilution step to the calibration standards.

The other uncertainty I had was the number of replicates for the calibration standard. I'm planning to run calibration standards on different days and maybe using the mean to quantify the unknown samples that I will be running. Is there a recommended or accepted method for the number of replicates or procedures for obtaining the calibration curve? From what I have read, it seems as though the validation process and determining the linearity would maybe determine how reliable the curve is but I'm surprised that I haven't seen more about the issue in books and other literature.

I appreciate any feedback. Thanks

[enahs]

Unless you have some very unique balance, you can read to 3 decimal places which is 0.001 g, not mg.
0.001 mg is a microgram, those are very expensive and sophisticated balances.
Meaning, you can only read to 1mg accuracy, and so if you take 10 mg, you have 10% uncertainty in your mass.



As for your other question, it depends on your instrumentation as well as how precisely you really need to know your results too.
As for repetition, it is best to do at least 3 times, and if all 3 agree your usually good. But, you really do not want to just average your data points together, you want to do statistical fitting.

[JGK]

USP General Chapter <41>, which specifies that the minimum weight (the minimum permissible load) for a balance is that which will have an error less than 0.10% of that weight.

So for a 3 dp balance, weighing to 0.001 g, the minimum weight you should weigh out is 1 g.

In a commercial GLP environment where we had to prepare stock solutions in duplicate with less than 2% variation, we would be weighing ~10 mg on a 5 dp balance.

ICH guidelines recommend a minimum of 5 non-zero STDs in the calibration, and I would recommend injection in duplicate.

[budget]

I really appreciate the quick response. I had to have another check on the balance that I'll be using and it turns out it only measures to 2 decimal places (0.01mg) instead of 3. So it will be on the 0.10% error of the USP specifications if I do decide to go through with weighing 10mg. The USP is something I haven't looked at too much, so I'll plan to have a read of that soon.

I also plan to have my validation based on Professor Claus Cornett's method (based ICH guidelines) which leads to more questions (I've only recently been exposed to the area of validating and it's taking me some time to get my head around things). From what I've read so far (http://corn12.dk/download/validation/DownloadValidation.html), I'm required to determine the linearity range at a minimum of 5 concentration levels with either the 6 or 2 replicates per concentration level. Is this a one off process during the entire experiment? Does it matter what time in the experiment the linearity is determined? When making up the minimum 5 standards for this process, is it made up from scratch and completely separate to the quality control stock solution that's going to be used to determine the precision?

Thanks again for the help and if you had any further recommendations on readings that may be newbie friendly, please do share.

[JGK]

Having regularly had to validate methods, I can say that you will definately have to perform it more than once. In a typical validation we would perform:

Linearity x3 - one analyst would perform the exercise on two separate occasions fresh STDs each time. A second analyst woould also perform one exercise (with fresh STDs) to assure us that the assay is not dependent on the technician performing the work.

Stock solutions were prepared in duplicate and their peak responses compared prior to STD preparation, a unit response difference of < 2% was required in order to assure that the stds would be prepared from comparable stock solutions.

Fresh STD were used, as until you perform some assessment of STD stability, you don't know how long they can be stored.