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Thi Nguyen ☆ Australia, 2017-09-18 16:43 (2805 d 23:25 ago) Posting: # 17814 Views: 6,524 |
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Dear all, I tried to look through all related topics in the forum and found some questions very closed with my issue, however, I could not get the final answer. Could you please advise what I should do in my case: My lab is validating a method for a compound in human plasma. Two core runs have been done (by 2 different analysts), QCs in 3 levels low, medium and high (6 replicates at each level) past with flying colours (all around 100% for accuracy). No carry-over observed. BUT: core run 1 has one LLOQ 498% and one LLOQ 727%, the other four LLOQs have good accuracy 〜100%. Core run 2 has five LLOQs with accuracy 〜100% and one LLOQ 3000%. For core run 2, 3000% can be regarded as outlier (from Grubb's test - calculated by Prism) but for core run 1, both values 498% and 727% are not outliers. We couldn't figure it out whether there are any sample processing errors so we could't exclude those values from accuracy and precision calculation for LLOQ, it means the core run 1 failed. We stop validation at this point to have investigation. Much appreciated if you can help with any clues for this situation. Thanks and best regards, Thi Nguyen |
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Ohlbe ★★★ France, 2017-09-18 17:53 (2805 d 22:15 ago) @ Thi Nguyen Posting: # 17815 Views: 5,580 |
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Dear Thi Nguyen, ❝ BUT: core run 1 has one LLOQ 498% and one LLOQ 727%, the other four LLOQs have good accuracy 〜100%. Core run 2 has five LLOQs with accuracy 〜100% and one LLOQ 3000%. For core run 2, 3000% can be regarded as outlier (from Grubb's test - calculated by Prism) but for core run 1, both values 498% and 727% are not outliers. Forget about outlier tests in such situation. The validation is there to demonstrate that your method works. If you exclude those values that may indicate it doesn't, you're losing the plot, whatever the result of the outlier test. Now there are several possible explanations, including contamination, sample spiking error and carry over. It is rather difficult to comment further without more information. I would go back to the data and look at, for instance:
— Regards Ohlbe |
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Thi Nguyen ☆ Australia, 2017-09-19 15:14 (2805 d 00:54 ago) @ Ohlbe Posting: # 17816 Views: 5,527 |
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Thank you so much Ohlbe for your reply! We are trying to identify the cause of the issue and focusing on the contamination. Few tests have been done such as extracting LLOQ at the same time with ULOQ by different analysts. The result of first analyst showed perfect clean, no-contaminated, consistent LLOQs (6 replicates). The second batch from another analyst is running right now. LLOQ and all other QCs (LQC, MQC, HQC) were prepared in bulk by spiking solutions into 10 ml plasma then aliquoted into small vials (enough for 2 replicates in assay) so LLOQ are homogenous, it shouldn't cause one very high, another from the same vial normal. And yes, the concentrations measured match with the nominal concentration (accuracy were quite good, most of them around 100%). The abnormal only happened with LLOQ, but not with any other QCs. No carry-over was observed at all, and the LLOQ with high values were not following any ULOQ in the run. Agree with you that there are plenty of possible reasons and aspects to look at. That is my concern: If we can't identify any causes, what should we conclude about the method? Could you please advise how you do in this case in your lab? Thanks and best regards, Thi Nguyen |
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ElMaestro ★★★ Denmark, 2017-09-19 15:38 (2805 d 00:30 ago) @ Thi Nguyen Posting: # 17817 Views: 5,499 |
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Hello Thi Nguyen, can you: 1. Indicate if the same sample no from run 2 was also one of the affected samples in run 1? (I mean if the affected sample in run 2 was "sample 17" then check if "sample 17" was also funky in run 1 etc) 2. Post bits of the result table, in particular indicate if the aberrant result for certain sample arose out of a fluke in the area of the analyte itself, or due to a low IS response, or possibly both? 3. Tell if the RTs in analyte and IS was more or less the same for affected samples and unaffected samples? 4. Eyeball the integration and check of you feel it was correct? Sometimes, at LLOQs depending on noise and bunching and a ton of factors that I know very little about, the actual interpolated baseline under the analyte chromatogram is visibly off and this in itself can give rise to some seriously deviating areas. 5. Check if the chromatograms are rugged, peaks-on-peaks etc. — Pass or fail! ElMaestro |
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Ohlbe ★★★ France, 2017-09-19 15:49 (2805 d 00:19 ago) @ Thi Nguyen Posting: # 17818 Views: 5,517 |
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Dear Thi Nguyen, In addition to ElMaestro's comments and questions: ❝ LLOQ and all other QCs (LQC, MQC, HQC) were prepared in bulk by spiking solutions into 10 ml plasma then aliquoted into small vials (enough for 2 replicates in assay) so LLOQ are homogenous, it shouldn't cause one very high, another from the same vial normal. OK, so it doesn't look like a spiking error. ❝ And yes, the concentrations measured match with the nominal concentration (accuracy were quite good, most of them around 100%). The abnormal only happened with LLOQ, but not with any other QCs. Right. My question was also: does the measured concentration of these problematic LLOQ samples match with the nominal concentration of e.g. HQC or CC7 or whichever other spiked sample ? It could be simply a labelling error after aliquoting, or taking the wrong sample out of the freezer. ❝ No carry-over was observed at all, and the LLOQ with high values were not following any ULOQ in the run. OK. Could it be a contamination at some point ? How do you process the samples: manually, or in 96-well plates with robotic equipment, or what ? — Regards Ohlbe |
Ing. Helmut Schütz