LC Accuracy Issues

[Solowatchman]

I am using HPLC to assay DNase and RNase in solution. My standard is prepared in a buffered solution to stabilize the DNase. All analysis and prep is carried out in plastic. When the standard is prepared four separate times to measure method accuracy I get 100% recovery for the RNase but 107% recovery for the DNase


The column is a C4 3.5um 4.6x150mm and the method uses 2% Phosphoric Acid in water and 2% Phosphoric Acid in ACN as the gradient mobile phases. The pH of the aqueous mobile phase is 2.

Any thoughts as to why the DNAse would be coming out 7% high when it is the same material (just different prep) as used to establish system suitability and calculate results from?

[unmgvar]

apparently what you do differently make the difference
can you give more details of the 2 sample prep and their differences?

[Solowatchman]

The stds are prepared at the sametime similar to check standard preparations. Each chemist prepared four stds and analyzed. No difference existed. The percent RSD for all the RNase and DNase recoveries were below 2% indicating satisfactory prep.

The standards contain both RNase and DNase and the RNase was consistently at 100% give or take a percent whereas regardless of the operator the DNase would be high.

Different LC systems and columns, even different days. Same result.

[Bruce Hamilton]

Can you please tell us a little more about the standard concentrations and calculation. How do you get a 107% error from the standards you use to prepare the calibration curve?. Are all the standards at suitable concentrations to bracket other standards?.

Do you have both DNase and RNase in each standard solution for the calibration, and is their ratio constant?. Do you get the same +7% if you just use a DNase Standard to prepare the calibration curve?. If you vary the DNase / RNase ratio, do you still get the +7%?.

It appears that your calibration standard is different to the working standard, and only you know the differences in preparation. The standards should be prepared to match the sample profile

[Solowatchman]

Thanks for those helping. Here are the requested details:

The standard Prep: 20mg DNase and 20mg RNase quantitatively transferred into a 50mL plastic tube. Using a 20mL volumetric pipette transfer 20mL buffer into the tube. (the buffer is 96mL water and 4mL water containing Ca and Mg). The standard is then mixed by gentle rocking on an automatic rocker for 20 minutes. A 2mL aliqout is then transferred to a plastic 100 mL volumetric flask and brought to volume with buffer.

The above prep was repeated four times. The sample set was

Std 1 (5 injections for system suit)
Std 2 (3 injections)
Std 1 (1 injection for bracketing purposes)
Std 3 (3 injections)
Std 1 (1 injection for bracketing purposes)
Std 4 (3 injections)
Std 1 (1 injection for bracketing purposes)

To calculate std 2 accuracy recovery the calculation is:
(Std 2 avg area/((Inj 5 of system suit + the area of 1st brk std)/2))*(Std1wt/Std2wt)*100%

To calculate std 3 accuracy recovery the calculation is:
(Std 3 avg area/((the area of 2nd brk std + the area of 1st brk std)/2))*(Std1wt/Std2wt)*100%

when this experiment was repeated by four other analysts no matter which standard was used as 'Std 1' the same odd effect of a 7% gain in DNase peak area response for the other three standard preps was noted.

This was never seen for the RNase component which is in the same standard preparations.

[tcay584]

Is your RNAse injection clean at the RT of DNAse? Just asking b/c sometimes we have seen RNAse that's not so clean and you could have co-elution. Try a single RNAse prep and check for interference.

[banala_b]

Hi,
There could be some problem with the DNase standard potency. Did you check how the potency/purity pf the DNase was calculated. I had similar problem with some other compounds. When I investigated, I found that the standards were very hygroscopic. Since we have been using the potency assigned few months ago and not accounting for the moisture increase in the standard material, we we getting assay value about 10% high (110%).
You could look into this. This may not be true to your case, but might help in your investigation.

Good Luck.

Bhaskar

I am using HPLC to assay DNase and RNase in solution. My standard is prepared in a buffered solution to stabilize the DNase. All analysis and prep is carried out in plastic. When the standard is prepared four separate times to measure method accuracy I get 100% recovery for the RNase but 107% recovery for the DNase


The column is a C4 3.5um 4.6x150mm and the method uses 2% Phosphoric Acid in water and 2% Phosphoric Acid in ACN as the gradient mobile phases. The pH of the aqueous mobile phase is 2.

Any thoughts as to why the DNAse would be coming out 7% high when it is the same material (just different prep) as used to establish system suitability and calculate results from?