HPLC injection volume accuracy acceptance criteria mostly considered as RSD NMT 2.0% of six injections calculated using injection area but this criteria not properly justify volume accuracy of instrument. Specifically in assay test. Because in assay mostly two injections are injected and assay value calculated for individual injections. Criteria for difference between two asssay results are used as not more than 0.5%. I think the calibration procedure not proper with respect to use requirement. So error in injections volume 1 or 2 or 3 microlitre (for 20 microlitre injection volume) gives passing result during volume accuracy checks during calibration but if same error repeated during assay analysis which gives huge difference between two result of set. Which may lead to nonconformity. I need suggestions that is it a limitation of instrument or can be rectify with better calibration procedure.
It is a limitation of instrument.For Waters 2695 model the acceptance criteria is +/- 2.0 microlitre per 50 microlitre of injection volume, determined with balance.
The assumption is that the HPLC has already been commissioned and qualified (IQ/OQ/PQ). The manufacturer’s specifications for injector precision is ‘far’ lower than 2.0% RSD (it’s usually 0.01% or lower). Therefore, your suspicion is wrong.
USP <621> which requires NMT 2.0% RSD for 5 injections (5.0% RSD for 6 injections for an RS method) applies to the assay method, not the instrument. You are demonstrating through SST (system suitability) that the instrument can perform the method as intended.
I am not clear that our instrument manufactur specification is far away from 2.0 % for injector precision. As I had discussed this issue with instrument supplier even they had not commented about acceptance criteria. I had reviewed instrument qualification which was performed by instrument provider. They did qualification with NMT 2.0% criteria for injection precision during qualification.
Further I would like to comment that during routine calibration; injector precision checked by five inections and calibration successfully completed if RSD of five injection area found Less than 2.0%. Similarly during Related substance analysis system suitability confirms the injection volume precision. Additional RSD is statistical tool where the negative error and positive error are neutralized and gives centralized result.
But during assay analysis by HPLC the procedure suggested only two injections instead of five. Also the result calculated for each injection area not by “averaging”. So the area obtained with individual injection is more important in assay determination.
So minor error in volume during assay analysis may fluctuate result higher.
In short measuring device must be more precise and accurate than user requirement.
It would be more explanable for understand my query.
Check specifications for injector precision online. The manufacturer does a FAT and SAT (which includes injector precision) on your site when he sets up a new instrument. Otherwise, his DQ (design qualification) would not be valid. This applies to a ‘new’ instrument, not a ‘used’ instrument.
USP <621> REQUIRES 5 (or 6) injections, as well as the calculation of the %RSD. NOT the average! It’s the law! HPLC is either capable of meeting these requirements OR you call a service technician for a repair (replacement of parts).
Despite the USP advise to inject five times and meet a RSD of 2%, I have seen system suitabilities that ask to prepare two set of standards and inject each of them three times (six inyections in total). They compare the respective RF and the deviation between them must be less than 1%. Is This way of SST in harmony with what the UPS advise?
Not really. Six injections are used when the SST requirement is >2.0% which is common for determining the concentration of the ‘related substance’ in the drug product (in the sub ug/mL or ng/mL range). A ‘check’ standard can be used in the assay (but is not required) and it should be within 1.0% of the 1st standard.