Science topic

Method Validation - Science topic

Explore the latest questions and answers in Method Validation, and find Method Validation experts.
Questions related to Method Validation
  • asked a question related to Method Validation
Question
9 answers
Hi all,
I have RNA seq data and I wanted to check the relative expression of selected targets based on RNA seq data. To validate this I have isolated RNA from a separate cohort and run the qPCR. However, the trend of my qPCR data is completely against the RNA seq data. The genes which are up-regulated in RNA seq is down-regulated in qPCR and vice versa. I do not know whether I am missing any variable here.
I know I have not written in detail but will be happy to discuss more if need any information.
Thanks
Relevant answer
Answer
What was the N for your RNA seq, and qPCR analysis? Surprisingly, the trend is the opposite, which shouldn't be the case, unless the primer isn't specific and amplifies some other segment.
  • asked a question related to Method Validation
Question
4 answers
How to select mobile phase of HPTLC methods validation if drug pka value id 3.5 which factor we consider salection of mobile phases?
Relevant answer
Answer
All parameters as per ICH Q2(R1) guideline
  • asked a question related to Method Validation
Question
3 answers
We know, that we can use only validated methods for comparative studies. In our lab we have performed the validation of the dissolution method for the specific medicine. In the comparative study we used several different media, these media did not participate in our validation, exept the one for quality control. Does it mean that we have to perform full validation procedures in each media additionally?
Relevant answer
Answer
Interesting question. I agree with Andrei Blasko and Saeed Ayaz Khan . However, you did not mean a regular analytical method validation. You are dealing with dissolution kinetics. The dissolution itself will depend on the physicochemical parameters you used, so the kinetics.
You find in the literature different behavior of the same AFI, for instance losartan, in several mediums, pH, ionic strength, and solvation conditions. Therefore several other surface and chemical interactions may influence the kinetics, not the determination method itself.
For your safety, check the validation with the already used media. The kinetics of significant different media will change. Therefore, the dissolution kinetics may have different parameters that may affect or not the determination method.
Best regards,
WNM
  • asked a question related to Method Validation
Question
3 answers
We are currently validating a method for trace metals quantification in rice samples but during the validation process, we encountered errors such as observed values are far from the true values of CRM samples.
Relevant answer
Answer
Probably not advisable to continue until you know why you don't get good recovery from the CRM
If you continue as you are, you may be making a serious error in the extraction process, or sampling errors might be affecting the calculations, or the measurement technique might be faulty. You won't know what the "true" results are until you have improved the entire process to within the acceptable tolerance limits.
Your question does leave a few gaps for a complete answer though. A few ideas to help:
  • Is the CRM appropriate (rice preferably, or at least a high carbohydrate like wheat flour)? Leafy CRMs are not always appropriate for starchy materials and vice versa.
  • Have you dried the CRM sufficiently? Even an hour in humid conditions will absorb moisture (especially with rice) and alter your dry matter weights. Keep it at 80C right up until you need to weigh it.
  • Are you getting consistent high or low recoveries? Which elements? What are the tolerances for these elements compare to your efforts?
  • What instrument(s) are you using? Are you trying to achieve the unlikely (eg ± 1ppb for Pb using AAS?).
  • Is your blank solution clean enough? can it achieve your required limit of detection? Acid extracts elements from the glass bottles it is supplied in, so if you're trying to measure ultratrace elements with contaminated acid, you will be facing an uphill battle. Maybe switch to a different supplier?
  • Is the extraction procedure appropriate? Are you using enough acid? Is it hot enough, and for long enough? are you losing sample during the digest? Are the tubes re-used or disposed of after digestion? Are they clean?
If you can go through your method and find a few key points to describe where your method is going awry, then we might be able to provide some more helpful suggestions than these generalised responses.
  • asked a question related to Method Validation
Question
3 answers
We are developing an HPTLC method for simultaneous estimation of five antidiabetic drugs. While performing a linearity study, through regression fitting model of WINCATS software, the R-square value for all the drugs are better in polynomial regression equation compared to linear model..and giving 0.99 and above values....Can polynomial regression equation be used in method validation?
What could be the justification?
Relevant answer
Answer
Using polynomial equations is pretty normal in the pharmaceutical field. Especially when you work with biotech/biologics or detectors with limited range of linearity like ELSD. I have never had compliance issues after submitting analytical methods with polynomial curves.
As hinted above though, you could adjust your sample concentrations to fit within the linear portion of the curve if that's possible for you.
As for the second part of the question, how to justify it. Run a method qualification by doing 5 solutions with concentration ranging from at least 80-120% of your target. Include all your analytes. Run those in triplicates and calculate the %RSD and the regression coefficient. Ideally, R squared has to be 0.99 or more, but for many methods 0.98 is fine. After that, perform accuracy from spiked samples: Spike your diluent/placebo/matrix with your analytes in at least 80-120% range from the target and analyze in triplicates. Calculate recovery (expected ~95-105% for assays) and %RSD. If you don't have matrix other than your diluent, than you can calculate the accuracy from your linearity solutions. Have another colleague run the method on a different day to confirm your results (you can run just linearity curve and one sample at 100% target). This will prove that you have data for your curve and that the method is accurate for quantitative purposes. Then you can propose this to a more regulated environment like GDP or GMP lab to do full method validation.
  • asked a question related to Method Validation
Question
3 answers
Hello,
Are there any available guidelines/literature for types of analytical method validation ( complete, partial, and cross-validation) with respect to non-bioanalytical methods?
Can one use validation criteria/rationals of the bioanalytical method validation [ ] for other analytical methods..?
Please advice
Relevant answer
Answer
Analytical method validation is always for a specific purpose and there can be no general rule. Regarding specificity (sometimes called selectivity, a close but not exact synonym), I've seen the two extremes:
Firstly in pharmaceutical quality control, you know what you're analysing because there's a separate identity test. This often has a spectral fingerprint but historically was designed in the negative sense, simply to exclude other compounds likely to be encountered. Validation of the specificity of a subsequent chromatographic (for example) analysis is largely a matter of demonstrating that known impurities and degradation products would be detected.
Secondly, when determining a new metabolite or validating a new method for an old one, it's absolutely vital to prove that you're measuring the target compound and nothing else. This is a totally different game. The best guideline I've seen is by the "metabolomics" people DOI: 10.1007/s11306-007-0082-2 (2007). This doesn't really target the analytical community: a book needs to be written though that won't be by me.
To emphasise the importance of getting this right, I can cite some articles I've written about nonsensical claims dating back three decades and still going strong, that the arrow poison ouabain is a mammalian hormone:
DOI: 10.13140/RG.2.2.24929.84323
Incidentally, that's a reminder that cross contamination can be an important validation criterion; ouabain happens to be used, in almost industrial quantities, as a pharmacological tool in the laboratories concerned...
Finally, there are likely to be other long-running biomedical stories that have been running for along time without adequate challenge regarding specificity. One of these may be about claims that the hormone melatonin is produced by organs other than the pineal gland. This may or may not have something to do with the disquieting acceptance and promotion of melatonin as an ordinary food supplement. I'm not aware of any critical review of this subject.
  • asked a question related to Method Validation
Question
4 answers
We are currently doing a method validation of MP-AES. Upon going through the parameters, we believe there are some errors. For example, during our accuracy testing, we're using CRMs and our measurements are too far from the true value. We're also doing standard additions but the measurements are still high. Our solutions were 50 ml (0.5 g of samples).
Also, how can we use the method of standard addition if the measurements are still high? It should correct the interferences I think.
Relevant answer
Answer
There are no ALTERNATIVES to method validation. You must meet all the parameters of ICH Q2 (for pharmaceuticals). However, AES is very sensitive! Your concentration is at least 10X too high! Perform a dilution to bring your concentration down to at least 1 to 0.1 ppm.
  • asked a question related to Method Validation
Question
3 answers
Please why taling factor is not required in hptlc method validation and number of paper published without mention taling factor and resolution .how we decide peak shape is good or not without calculating taling factor . because software not calulate taling factor so they not mention . please explain me taling factor is not required on HPTLC method if required they why not mention in paper no single paper found they mention taling factor all follow the same.
Relevant answer
Answer
But in selectivity mention taling factor as per ICH guideline for method validation of HPTLC
Then how you decide your peak shape good in which basis?
Without taling factor u can't decide either peak is good or not
NO singal paper of HPTLC not mention taling factor and resolution if combination
  • asked a question related to Method Validation
Question
6 answers
ISO15189 or American guidelines are only rough guidelines for method validation and incomplete.
In general the focus is on (im)precision and trueness, where the latter is often complicated by the absence of a reference method. In addition pre-analytical influence like aging of the sample, storage conditions, aging and quality of reagents is seldom discussed. Finally what good is a method when it cannot be reproduced in other labs. So interlab variation should also be discussed. Some years ago we tried to sum up all the important factors and gave an indication how they could be handled in a white paper (link below). Please comment on that paper or point at alternative and comparable approaches
Relevant answer
Answer
  • asked a question related to Method Validation
Question
5 answers
One of the requirements during method validation is to test parallelism between the standar curve and the sample curve when they are not the same product. I'm trying to find a way to measure this parallelism on Minitab and to get a proper test that provides a single p-value that states whether two curves are parallel or not.
Relevant answer
Answer
You can do it with Prism Graphpad
  • asked a question related to Method Validation
Question
1 answer
I am doing a FACS method validation using cell lysate and came across this issue of need to validate the long term stability if temperature changes during storage. Since cell lysate are kept in liquid nitrogen, the baseline is to keep samples in LN2. But the samples need to shipped abroad and during shipment it will be stored at -80 degrees celcius. After shipment when arrived to the lab, if the samples are to be stored in the liquid nitrogen tank, then would this course of temperature change need to be validated? I am planning to validated the long-term stability at -80 degrees celcius and in LN2 tank for at least 6 months. Would it be necessary to validate LN2 tank 1month then -80 degrees celcius 1 week then LN2 tank 1 month to properly validate the method? Or would it be ok just to validated -80 and LN2 tank each at 1 month and 6 months?
Relevant answer
Answer
Yes, I think so, it is necessary to validate LN2 tank 1month then -80 degrees celcius 1 week then LN2 tank.
  • asked a question related to Method Validation
Question
2 answers
Dear all,
I am currently studying on the transition state of a dimer. The IRC results have not worked well for my system as it is a charged open-shell one. Therefore, I am going to do Atom Centered Density Matrix Propagation molecular dynamics (ADMP) and Born-Oppenheimer molecular dynamics (BOMD) calculations using Gaussian in order to validate my TS results. Now, I have three questions regarding the subject:
1- Are these calculation methods valid and reliable in reviewers' point of view?
2- Would anyone please provide me with an example input file for both the aforementioned methods?
3- Any guess of the calculation run time for a 40-atom system?
Many thanks in advance to all the researchers who provide me with their valuable answers.
Regards,
Ramin Eradeh
Relevant answer
Answer
Thanks dear Fernando Aguilar-Galindo for the answer.
  • asked a question related to Method Validation
Question
6 answers
Hello experts,
I'm a beginner in structural modelling and MD simulation of DNA. Kindly share your opinion on what should I do to ensure that my structural prediction and simulation is right? Do I need to validate the methodology of published article (repeat the same method as published and then compare the output) and later I run my own sample using the same parameter ? Is it compulsory to run method validation prior to perform our own sample?
Thank you in advance.
Relevant answer
Answer
Method validation is a good practice. It is like a positive control and any control experiment is better than no control. However, the conclusion from a positive control can not always be extended to the test system under investigation. Here is an example with molecular docking. In molecular docking, you can take a known ligand-bound crystal structure and then do a self-docking to check if the docking method can reproduce the binding pose of the ligand as observed in the crystal structure (within a reasonable limit of deviation). If the docking method can reproduce the binding pose of the ligand as observed in the crystal structure, you can say that the method is valid and is working for your system. Then you take an unknown ligand for docking and follow the same protocol and find a binding pose with a comparable or maybe even better binding energy. But this does not guarantee that the result is not a false positive. Afterall, it is just a simulation. The only way to validate the docking result is to show at least the binding is actually there. A simple spectroscopy or calorimetry can confirm if there is actually any binding. When there is this experimental confirmation, you can conclude from the in silico result that the simulation did not produce a false positive; the binding is there and the computed binding pose is the best possible binding pose. The same goes for the MD simulation. Now coming to your question, if you try to reproduce a published result keeping all the conditions similar (software, parameters etc.), you should get a similar result. But that is not method validation. Method validation should also be tied to experimental observation. Say you take a solution NMR structure of a DNA fragment from a nucleotide database and then run MD following the protocol that you want to use for your experiment and see if the structure remains intact. If the equilibrium structure remains the same as the input, you can say that the method worked for such a system (a known DNA fragment from database) and if your system is similar (e.g. a DNA fragment with different length and composition), it probably should work for your system as well.
  • asked a question related to Method Validation
Question
5 answers
Hi everyone,
We need to detect the nanoscaled geometric defects of a dense cylinder array using optical methods. However, the period of the array is so small that only 0th diffractive light exist via the bright field microscopy, making it difficult to get any information except the contrast. The nanoscaled defects are submerged by the contrast, since the intensity of the array is nearly consistent.
Well, is there any other optical method valid for distinguish the geometric information of a dense periodic array?
Relevant answer
Answer
Scatterometry of sub-wavelength periodic structures is a highly-developed technique that for the most part relies on zero'th order diffraction. At Zygo we have in past years developed interferometric scatterometers for some specialized applications; but there are many other designs. Just web search "scatterometry semiconductor" to get started.
  • asked a question related to Method Validation
Question
4 answers
Hello All,
The calibration standards were 10, 20, 30, 40, 50, 60, 80 and 100 ng/mL), Concentrations of QC standard were 10 (lower limit of quantification, LLOQ), 30 (QC1), 50 (QC2) and 100 ng/mL (QC3).
Is it correct the selection QCS, or do we need to select concentrations different from calibration standards.
Thank you
Relevant answer
Answer
It depends on the analytes, on the matrices and how the validation study was carried out.
In general the selection of these QCSs is correct and it is possible to choose calibration standard levels as QC levels.
From Guide to Quality in Analytical Chemistry - EURACHEM:
"Standards or materials similar to those used for calibration, placed at intervals in an analytical batch, enable checks to be made that the response of the analytical process to the analyte is stable. It is the responsibility of the laboratory management to set and justify an appropriate level of QC, based on risk assessment, taking into account the reliability of the method, the criticality of the work, and the feasibility of repeating the analysis if the QC sample result is unacceptable."
Here is the link of full text:
Also, consulting other guidelines (e.g. ICH, SANTE, IUPAC) is a good suggestion.
KRs
  • asked a question related to Method Validation
Question
3 answers
I have 10 spectrums from a sample to assess FTIR-ATR's repeatability. I have the spectrums in excel with an average, STDEV, and %CV of each transmittance, over the 400-4000cm-1 wavenumber range. which is a lot of values. To determine the repeatability of the FTIR-ATR, do i need to talk about the %CV of every peak, or just 1 peak - which is acceptable, and what %CV is acceptable? I can't find any guidelines online that are free to access for method validation: repeatability. This is for my research project about PET plastic fogging after heating.
Thank you
Relevant answer
Answer
To calculate the reproducibility, it is sufficient to take one peak. It should be intense, but not exceed the maximum recorded value of the optical density. The suitable peak will be determined by the film thickness and spectral resolution, it can be 725 (720 + 730) or 1467.
  • asked a question related to Method Validation
Question
1 answer
Hey all
Is it difficult to work with acetaldehyde? I'm about to start an acetaldehyde method validation on an Enzyme Robot Arena 20XT instrument for wine samples using Megazyme kits. Any tips? Suggestions? Experience?
Thank you in advance.
Relevant answer
Answer
Headspace gas chromatography (HS-GC) and reverse phase high-performance liquid chromatography (RP-HPLC) are the two main methods used for measurement of acetaldehyde. The HPLC method entails labeling with a derivatization reagent for stabilization and selective determination of acetaldehyde.
For more info, have a look:
Kind Regards
Qamar Ul Islam
  • asked a question related to Method Validation
Question
15 answers
Is is legal to use phosphate buffer saline as surrogate matrix for bioanalytical method validation?
P.S. I want something amino acid free and have the same matrix of plasma to do the validation.
I tried bovine albumin serum but it has amino acids in it and peaks appeared
Relevant answer
Answer
I hope its meet your needs
  • asked a question related to Method Validation
Question
5 answers
I want to do validation but I need the same matrix of human plasma cleared from amino acids and other additives.
I actually I thought of using buffer, what do think? Is it legal to use buffer for validation of biological samples?
Relevant answer
  • asked a question related to Method Validation
Question
4 answers
1- any studies can support this opinion, that I used tool in Arabic from Jordan study which its the same language ,region and culture without the opinion of our local experts in the field.
2- if alpha cronbach was in pilot study 0.918 for a tool of 30 items , but in internal constancy there is four items not significant but I don't want to delete those items . any studies can support this thing.
Are there any studies that support this proposition to cite those in the methodology chapter?
mention those studies
Relevant answer
Answer
YES!!: Of course;Why not??
  • asked a question related to Method Validation
Question
13 answers
Hello everyone. i want to seek a suggestion regarding simulated results on any research topic. Most of the work on Distillation columns have been carried out using simulated softwares like Aspen Plus, Aspen Hysys etc. there is lack of Experimental data on these topics. how can anyone validate his/her results.
Relevant answer
Answer
Via thermodynamic analysis, concentration profiles can be determined based on Gibb's free energy minimisation or equilibrium (stoichiometric) balances at specified conditions (temperature, pressures)
Likewise, adiabatic flame temperature calculations involving energy balances can be carried out to evaluate temperature profiles on basis of variant inlet oxidant, fuel temperatures and air enrichment ratios.
Engineering analysis of thermal/chemical reacting/non-reacting systems can result in values for metrics such as thermal efficiency, chemical efficiency and so on and so forth that can also be calculated from the simulation results and compared
  • asked a question related to Method Validation
Question
2 answers
As a 2nd-year PhD researcher, I have been curious about the number of simulation-only PhD theses that exist in fields such as Engineering as well as unrelated fields as well. There is of course value in lab-testing, given how a simulation cannot always account for every boundary condition or factor. Usually, from my limited but growing experience, simulation, in the case of Additive Manufacturing and mechanical testing, is normally used as a validating tool; to help prove in a non-virtual environment what may be seen and tested in the lab, before any kind of scaling or future work is done using simulation (for cost-effectiveness and resource-saving etc.).
But in fields where in-situ testing is normally done, are there PhD theses that "Jump the gun" as it were, and go straight to simulation? Any information on this will be most appreciated given how, in some locations/countries where interaction and access to tools on campus and labs are minimal still due to Covid, improvisations must be made.
Relevant answer
Answer
Ezekiel Yorke I believe you partly answered your question in the last sentence of the first paragraph-"for cost-effectiveness and resource-saving".
  • asked a question related to Method Validation
Question
7 answers
Recently, I read that we do not validate the questionnaire, but the scores obtained through this questionnaire. So is it wrong the papers with the title"Validation of the XXXXXX questionnaire"?
Relevant answer
Answer
Early in my career I encountered Anne Anastasi's book "Psychological Testing". I was struck, and have remained impressed all my life, by her thought that a "questionnaire is a sample of behaviour". That is, a questionnaire is a *sample* from which the analyst makes predictions about the *ensemble* of behaviours the respondent may be said to exhibit. It makes complete sense therefore to investigate the ability of a questionnaire to allow the prediction to be made with validity (does the sample really measure the ensemble of interest?) and reliability (does the sample fluctuate at random?) Using unvalidated questionnaires is, as I used to drone to my dear students till they knew this lesson, pseudo-science and snake-oil. As in all inferential statistics, the actual *data* (in the original question, "the scores") is an accident that has just taken place. What the investigator wants to know is: what does the accident tell us ("the questionnaire")? Welcome to the wonderful world of Psychometrics!
  • asked a question related to Method Validation
Question
8 answers
We have been using HPGe detectors for evaluating the level of natural and artificial radionuclides in different kinds of environmental samples. Parallelly, we also provide radioactivity testing services to ensure that there are no consequences triggered due to the consumption of highly radioactive food materials. We usually use certified reference material (CRM) collected from IAEA for calibration, performance evaluation (PE), method validation (MV), and QC. We are planning to collect a CRM of milk powder spiked with Cs-137...but could not find it anywhere? Could anyone of you please help me find it? It is urgent for us as we are working on getting ISO17025 accreditation by this year.
Relevant answer
Answer
Bergonie-Tribondeau's law based on experimental observations in rat testes applies at cellular levels (albeit with various exceptions such as lymphocytes, some gonadal cells, AT cells, and SV40-transformed cells), but less generally at tissue or organism levels. Younger people are not necessarily radiosensitive than older people where dependence of radiosensitivity on age (e.g., age at exposure, attained age) greatly differs by endpoints (health outcomes). For instance, children are more radiosensitive than adults for about 25% of cancers (e.g, leukemia, thyroid, skin, breast and brain cancers), as detailed in
  • asked a question related to Method Validation
Question
2 answers
I would like to do method validation for co-trimoxazole tablet but couldn't find the proper neutralizer to deactivate co-trimoxazole active.
Without deactivating i cant get bacteria,yeast and mould recovery.
Relevant answer
Answer
Hello, the certain group of microorganisms
has cotrimixazole degradation activity such as Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas putida, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus rhodocrous, and Rhodococcus zopfii.
The most active is Rhodococcus equi.
If you want to validate cotrimoxazole tablet look for Pharmacopoeia method.
  • asked a question related to Method Validation
Question
7 answers
We work with a method validated for one thermocycler. We have purchased new equipment and would like to use the same method on it. How to validate it properly to make sure that the results from both equipment are consistent?
Relevant answer
Answer
Thank you Dear Amaury Borges Miranda for your comment.
In our laboratory routine, we always work with periodically calibrated equipment. Protocol validation and optimization steps are always necessary. Mainly because sometimes we are unable to buy reagents of the same brand that we often use (due to lack of stock). So whenever there is a change in the PCR reagents, we have to optimize the protocols and check the amplification fidelity of the gene with those new reagents.
  • asked a question related to Method Validation
Question
5 answers
One of senior analysts told me I could report the recovery as following.
1. Calculate % Result with obtained peak area
2. ( % Result / 100) x (Actual amount added) = Amount recovered.
3. Report the % Result, Actual amount and Amount recovered and that's it.
But I personally think the calculation below is more acceptable.
1. (Area - Intercept) / Slope = Obtained amount
2. Obtained amount / Actual amount = Recovery
Are the 2 methods correct? or incorrect?
What is the most recommended calculation for recovery?
Relevant answer
Answer
Dear Kim,
Are different parameters:
Accuracy (bias):
This is a measure of the difference between the expectation of the test result and the accepted reference value due to systematic method and laboratory error. It is usually expressed as a percentage. Accuracy and precision together determine the total error of the analysis. Accuracy is ideally determined using Certified Reference Materials (CRMs), if available, reference methods, collaborative studies or by comparison with other methods;
Recovery :
The recovery of an analyte in an assay is the detector response obtained from an amount of the analyte added to and extracted from the matrix, compared to the detector response for the true concentration of the pure authentic standard (seized materials). It may also be understood as the percentage of the drug, metabolite, or internal standard originally in the specimen that reaches the end of the procedure.
Best
JSCamara
  • asked a question related to Method Validation
Question
6 answers
Please suggest a suitable reference from I can get information regarding the internal standards and other calculations for bioanalytical method validations for HPLC.
Relevant answer
Answer
Thank you @ Bojidarka B. Ivanova for the inputs and sharing of useful references.
  • asked a question related to Method Validation
Question
5 answers
I would like to develop and validate NMR methods that I would use in the identification of certain chemicals (mainly organophosphorus compounds, amino alcohols, and other fluorinated compounds) according to ISO17025 requirements.
If possible I would like to have some examples of NMR methods validation reports.
Relevant answer
Answer
Mr. Baroudi,
I would like to thank you for the wishes.
Happy New 2021 Year for you, as well.
Successful work!
  • asked a question related to Method Validation
Question
4 answers
Dear Scientists
I am trying to method validation of major phenolic compound from a medicinal plant. My standard curve recovery was an acceptable range (80 to 100%). But, when compound standards are spiking with extract after that recovery % is more than 200%. What types of solutions are needed to solve this problem? Please give me your valuable suggestions.
Thank you
Relevant answer
Answer
Dear Jinadasa
Thank you for your information. I will check and update you.
  • asked a question related to Method Validation
Question
8 answers
We have 2 machines that test products. Both of these qualify the product to be Good or Bad ( Based on amount of leak ).
The final outcome is attribute variable (Good/ Bad) whereas the way we conclude it is a continuous variable (Amount of leak)
One of them is yet to be qualified for Testing purposes. And the other is something that has been validated.
What is the best statistic to prove if the new machine is working as expected?
What should be the best machine validation protocol and corresponding tolerances?
Quality, Statistics, Machine Validation
Relevant answer
Answer
Mark is pointing you in the right direction. This is a classic MSA opportunity the only twist is that you are using it as a Go/No Go gage which will require an Attribute Agreement Analysis and achieve an >80% agreement level. Although, if you can get continuous (or variable) data from both test systems then it would be prudent to go ahead and perform a Gage R&R (10-parts X 2-systems X 3-replicates) to better understand the repeatability and reproducibility of the two systems in which the two systems are the two appraisers. This would give the best data in the shortest amount of time. The question you would need to answer is what level of error will you accept; <10%?, <20%? or <30%? Oh yeah, before you do any of these analyses please make sure both systems are appropriately calibrated and in good working order.
Longer term conducting a chi-squared on your results may make sense if you believe they are sampling from the same population over an appropriate number of samples.
  • asked a question related to Method Validation
Question
26 answers
Which parameters (among linearity, LOD, LOQ, %RSD, % of recovery etc.) I should must check in HPLC method validation ?
Relevant answer
Answer
Dear Mahmudul, listed parameters are sufficent for the establishment of the method (include specificity) but if you need to submit this method for regulatory agencies or adopt for the compendial monograph, then it require full validation as per ICH Q2R1 guidelines
  • asked a question related to Method Validation
Question
5 answers
According to EMA and FDA there should be one calibration curve for each analyte studied in the method validation. So how do I handle the QC samples? At least two analytes in the sample do not seems to be probleamatic; the concentrations of each substance can be read from the given curve.
Relevant answer
Answer
Yes, if by one method you analysing several compounds.
  • asked a question related to Method Validation
Question
3 answers
I'm trying to quantify the anthocyanins in tomato leaves using LC-MS/MS. The two most common anthocyanins in tomato vegetative tissue are petunidin-3-(p-coumaryl rutinoside)-5-glucoside and malvidin-3-(p-coumaryl rutinoside)-5-glucoside.
Is it possible to quantify these two anthocyanins with the commercially available standard malvidin-3-glucoside using molecular-weight correction factors as in the following article?
( Chandra, A., Rana, J., & Li, Y. (2001). Separation, identification, quantification, and method validation of anthocyanins in botanical supplement raw materials by HPLC and HPLC− MS. Journal of agricultural and food chemistry, 49(8), 3515-3521.)
Or is it better to quantify petunidin-3-(p-coumaryl rutinoside)-5-glucoside using petunidin-3-glucoside as a standard?
Is it possible that malvidin-3-(p-coumaryl rutinoside)-5-glucoside and the standard malvidin-3-glucoside don't have the same retention time and is this a problem for the quantification?
Thank you for the help!
Relevant answer
Answer
Hi, I think it is possible, but at first you should determine relative conversion factor.
  • asked a question related to Method Validation
Question
4 answers
Hi all,
I am interested in performing an untargeted metabolomics analysis on a few hundred human blood plasma samples using LC-ESI-MS (specifically HPLC coupled with Sciex's TripleTof 5600 MS). The purpose is for biomarkers discovery and to gain biological insights into the disease I am studying. The few hundred samples include both disease and control samples.
I am in the midst of coming up with a work plan, and I would be grateful if you can point me to a good textbook/article/guidelines which details all the steps to design and perform an LC-MS untargeted metabolomics experiment. For example, some of the things I am still a bit unsure are:
1) Are there protocols and strict rules on method validation to be done prior to the formal analysis of clinical samples?
2) Do you analyse a large quantity of reference standards from different metabolite classes to optimise the LC-MS parameters?
3) What is the standard practice for including QA/QC samples into the analysis?
4) What is the standard practice for data preprocessing, processing, and data analysis?
5) What other steps can be taken to ensure minimal bias, avoid batch effects, and maximise metabolome coverage?
Thank you in advance!
Relevant answer
Answer
Every lab has its own protocols, which often depend on the type of sample being analyzed. Some labs do these analyses more than others and have honed their techniques a bit better. Fiehn's lab at UC Davis is pretty good on Metabolomics from plants. Here are some general rules to remember:
1. ALL sample preparation methods bias the results. In your case you will undoubtedly be precipitating the blood proteins after centrifuging out the cells. The age of the sample and any previous free-thawing will lyse some of the cells spilling their contents into the plasma. Even differences in the blood collection vials and manufacturers can influence the results you get. I once saw a study where they collected the control samples with one vacutainer and the disease samples with another (two different hospitals) and the 3 month LC-MS study found that lactic acid was the principal difference, which was included by the mfg in tube 2. Talk about a waste of time and resources.
2. The LC conditions will similarly bias the results. Not everything will elute from the column and the column packing will change over time. Some materials won't bind and will elute at the flow front in a jumbled mess. Elution time is one marker for identity and sample alignment.
3. The MS mode (+ ion or - ion) will suppress seeing 30-50% of the metabolites in the sample. Furthermore, some molecules won't ionize by ESI at all and require other modes. So never think that you are seeing everything. Also differences in salt compositions might cause a peak to jump from one mas to another mass because of adduct differences.
4. MS is NOT quantitative unless you are spiking with stable isotopes. The best you can hope for is ratiometric comparisons between samples and metabolites. Scaling all peaks to a common reference peak is a way to deal with this issue. In general, you are looking for 1-log differences in relative abundances to identify a difference. Ionization efficiencies simply vary by an order of magnitude over time and runs and more between different molecules.
5. An open method is just that. It is designed to flag potential real differences between samples, that deserve follow-up with additional hypothesis-driven experiments. It is not a validation study. The issue here is basic chemometrics. You need a minimum of 3 independent samples per feature analyzed to actually make a statistical call. In a typical metabolomics study, you might have 1000 features, which means you can't make any real statistical call on any feature without running 3000 patient samples. No one can afford that, so you use what you can afford to run to try to identify leads that deserve independent followup. You have to remember that the lower the sample to feature ratio, the more false leads that will be generated on an exponential or hyperbolic scale.
6. Spend the time to really understand the math in each step of your analysis. Blindly running the software at its default settings will always lead you astray. Until you understand exactly how each step is working, you can't begin to understand what biases the software is introducing to the analysis. This starts at centroiding and doesn't end until the PCA analysis at the end. I always run in profile mode (not centroid mode) because I don't trust that the MS Mfg has implemented his code correctly for complex samples. In fact, I've proved that on pretty much every MS. Then you have 'hidden' features in software like OpenMS and MZmine that when they flag a missing peak in the sample, they go back to the profile mode spectrum to find the highest point in the neighboring baseline and report that instead. Sorry, just not statistically-valid.
  • asked a question related to Method Validation
Question
5 answers
i divided my data into two sets; training set 90%, validation set 10%, and i used the validation set for cross validation, 'early stopping criteria' but i used the same set 'validation set' for testing set as well. is it possible?or i MUST make another set for testing?
Relevant answer
Answer
It was some time ago, thank you for your advise. I agree with you.
Regards,
Tachi
  • asked a question related to Method Validation
Question
6 answers
I'm conducting a study with Imidacloprid (C9H10ClN5O2) in waters and it's important to know if it's photodegraded by light or not.
>TOPIC CLOSED!
Relevant answer
Answer
Yes it will. You can see the below document.
S. Wagner, "Environmental Fate of Imidacloprid," Sacramento, CA, 2016.
Furthermore, the coupling of UV light increased the degradation of Imidacloprid by ozonation. You can check my paper if you would like to.
Best.
Busra S. Baghirzade
  • asked a question related to Method Validation
Question
3 answers
I have read several papers on change impact analysis and I understand they have validated their results generally based on True Positive (TP) and False Positive (FP) results. Furthermore they support their validation with Precision, Recall and F-measures as well. On the other hand there are some other articles that they use statistical results.
My question is that, I understand the TP and FP results, also the metrics. However, I couldn't understand the process how they obtain the TP and FP results from the open source projects. All I know is they use CVS and their repository. The problem that bugs me is, when you check out repository mostly it is free from bugs and even if there is change the impacted areas in the code are already fixed (changed). Therefore, working on real open source project does make any sense.
In other words, the question might be redirected as: How do we form or find the actual impacted set?
Any guide would be helpful?
Thank you.
Relevant answer
Nice Dear Wiem Khlif
  • asked a question related to Method Validation
Question
16 answers
Countries implementing the EITI can become EITI Candidate Countries when the EITI Board is confident that the country has made a political commitment and has taken a number of preparatory steps. Such EITI candidate countries should further implement the EITI process in their country and complete the Validation within the next two years in order to obtain the status of “EITI Compliant Country”.
Relevant answer
Answer
dear dr Jaydip Datta thank you for link i surf it there is such answers i get from
  • asked a question related to Method Validation
Question
4 answers
Regarding pharmaceutical industry, does anyone have guidelines for in process hold time study?
Relevant answer
Answer
Is there any guidelines for in process hold time study of semi-solid dosage form
  • asked a question related to Method Validation
Question
3 answers
Plan for method validation for salmonella by RTPCR, eg., LOD, LOQ, Robustness, precision etc.
Relevant answer
Answer
Hello, see paper attached which describe all requirements in PCR method validation.
Good luck!
  • asked a question related to Method Validation
Question
6 answers
The main focus of a project is to analyze and suggest to improve the quality of the production line or services system in a co-operative type of business using Six Sigma Approach. The co-operative premise offers several services including, printing, selling instant food, accessories, stationaries and corporate attire, serve as a convenience store and provide parcel delivery.
Relevant answer
Answer
The reference that Noman Mohammad gave you (Lincoln and Guba 1985, Naturalistic Inquiry) is the best known source on validity in qualitative research. Note, however, that if you are working with mixed methods, reviewers will be much more likely to care about reliability and validity with regard to the quantitative rather than the qualitative portions of the overall project.
  • asked a question related to Method Validation
Question
4 answers
I want to do a pre-absorption technique, but I'm having trouble finding pure proteins of the following:
ASIC 2
NFP (2F11)
S46
S100P
If anyone knows a good site I can purchase pure proteins or if anyone has another method, that would be greatly appreciated.
Relevant answer
Answer
To clarify the term validation shortly: in the field of antibodies, validation includes mainly specificity and sensitivity in a certain application. To show specifity, genetic methods like specific knock-down of the target antigen in a cell line would be the method of choice. The sensitivity is important to show that your antibody works in a specific assay e.g. Western Blot, FACS, IHC etc. You could have the most specific antibody for an antigen but that doesn't guarantee that it will work for both Western blot and IHC for a specific cell line, for example. That part of the validation is important and needs to be done for the specific application.
Note that using recombinant protein as a control is not 100% reliable in order to verify that your antibody will work for your cell line or lysate. Post-translational modifications, different isoforms in different cell lines or low expression levels could give negative or unclear results even if your recombinant protein is detected, especially when it was produced in E.coli, you might have differences compared to the protein from eukaryotic cells.
  • asked a question related to Method Validation
Question
3 answers
Thinking in terms of a social setting such as a dance, a concert, a meal, if an experiment were to be designed in such a way, how can the method be validated? Similarly, what role would reliability play in an experiment set in a social setting? How can you recreate social settings for further empirical study?
I would love to read some examples of studies if you are familiar with any!
Many thanks.
Relevant answer
You asked about the validity and reliability. These are the critical points. If you chose your subject from very dissimilar groups and settings there is a greater chance to get reliable data as well as external validity which is important for researcher who want to repeat the experiment. Check this out if it gives you advice: https://pdfs.semanticscholar.org/6462/54a1698a66aacb6eceb66c126309c3311997.pdf
  • asked a question related to Method Validation
Question
8 answers
What is the best way in HPLC method to validate the stability of two stock solutions storaged at 2 - 8 C with stated expiration of a three months and a standard solution made by dilution of these two stock solutions and kept at 2- 8 C, with expiration time of a one month?
Relevant answer
Answer
Hello, you can keep as long as you need for further analysis. Before storage perform at least 6 injection in repeatability conditions and calculate standard deviation (SD) for creation of quality control chart with 2xSD limits than after storage each week perform 1 duplicate analysis. If you receive result within range it mean your sample still stabile.
Good luck!
  • asked a question related to Method Validation
Question
3 answers
Can I use a part of a validated questioner (assessing X) along with another validated, full questioner (assessing Y) for a research study assessing (X and Y)? would this new formulated tool still be validated?
Relevant answer
Answer
Hello if you have method X which already validated and all requirements and performance criteria for the method are fulfilled, so you can use parameters from method X to validate method Y. If parameters from Y method equal to X based on it you can say that Y also validated.
  • asked a question related to Method Validation
Question
8 answers
Could you please anybody explain me how to develop the method and validation the method which relevant to finding Aflatoxin in Peanut relevant to R- Biopharm method?
Relevant answer
Answer
These forums do not provide a means to address such a broad question as posed. The basics of HPLC as a formal analytical technique and method validation basics fill volumes of books and journals. Read the books to research the basics. As HPLC method development and HPLC method validation take many years in the workplace to acquire a basic level of proficiency, I suggest that after researching the topic, you hire a contract laboratory to perform the work for you OR work with a local laboratory whom has experienced liquid chromatographers with a demonstrated history of doing the same for compounds of interest.
Additionally, you may wish to take some time and begin to research the basic of liquid chromatography [e.g. The text, "Introduction to Modern Liquid Chromatography" by: Lloyd R. Snyder, Joseph J. Kirkland, John W. Dolan is excellent] , HPLC methods, the basic concepts and fundamentals of analysis. BTW: Avoid sales oriented HPLC websites such as chromforum or chromacademy which have many inexperienced users who may misdirect you. Additionally, if you wish to be involved in the actual analysis work, then you will need to obtain formal training on the specific HPLC system and software that will be used for the work (Many types of HPLC systems and each one requires training in how to use it).
  • asked a question related to Method Validation
Question
7 answers
Can CDA (critical discourse analysis) be a part of a mixed method? And which quantitative method could combine well with CDA in the mixed method for validation or corroboration of the results obtained through CDA method?
Relevant answer
Answer
The decisions involved in combining any two methods within mixed methods depend on not only the purposes that each separate method will serve but also the ways in which you plan to integrate what you learn from each method.
One possible mixed method approach that you might consider is a sequential Explanatory design. (QUANT --> qual). In this case the qualitative method serves as a follow-up study to help explain the results of the original quantitative study. In particular, the role of the qual study is often to explain how and why the quant study produced the results that it did.
  • asked a question related to Method Validation
Question
4 answers
I'm working on a method validation in a specific matrix on ICP-MS for heavy metal determination. I've measured seven samples spiked at my suspected detection limit for the method. The MDL is being calculated by multiplying the Student's t value at 99% confidence interval for the degrees of freedom (6) by the standard deviation of the measured replicates. My question is, since the measured 7 replicates is my population of data, wouldn't I be using the standard deviation of the population equation (dividing by n) rather than that of a sample of a population (dividing by n-1)? If I am to use the the stdev of a sample of the population, why are my 7 replicates considered a sample of a population rather than the population?
Relevant answer
Answer
First, let me suggest that one might consider not spiking at the MDL even if one could estimate it correctly. Errors can be better estimated close to but not at the MDL. Spike to high and your MDL is too large, spike to low and the measures have so much error again your MDLs will not make sense with observation.
You are correct that an estimate of the population standard deviation is divided by the square root of n. However, dividing by square root of n is used for arriving an estimate of the population standard error (s estimate of u). In this case, one is only interested in using the standard deviation of your measures to arrive at a good estimate of the MDL witthout spiking at it directly. Of course, this is acheived by the variability (sd) of your measures being multiplied by the student's t value (n-1) at 99% probability (typically chosen). I prefer to define a limit of determination (LOD) which is defined as the least amount that can be identified and measured with a given predefined accuracy. The abreviation "LOD" is unfortunately to easily confused with limit of detection which it is not. So were rarely speak of it that way. EU has the same difficulty with this term.
  • asked a question related to Method Validation
Question
6 answers
I need to understand if an antibody I'm using is specific or not.
The antibody is not validated.
In immunoblotting, the antibody detects a prominent band at the right molecular weight and two further nonspecific bands.
In negative control protein lysate, the band at the right molecular weight disappears, while the two nonspeific bands are still detected.
In immunofluorescence, I have an intense staining in the right cell localization.
Unfortunately, the blocking peptide for this antibody is not available.
Could the full lenght recombinant protein targeted by the antibody be suitable as blocking peptide?
PS: no data from litterature are available about the expression of the protein of interest in my cellular system.
Thank you.
Relevant answer
Answer
The non-specific bands detected in WB, often minor, are usually artifacts related to the use of cell lysates (or impure proteins) containing cytochrome C or IgG receptors ... The differential between positive cells and cells negatives makes it possible to guarantee the specificity of marking. In publications one is careful not to show these artifacts in order to avoid having to give explanations. As for the blocking of the labeling by the specific antibody, it is imperative to put very large quantities of target proteins to obtain a specific inhibition: of the order of 10 to 100 times the amount of antibody applied. On the other hand, the antibody having a high affinity for the whole protein as for the synthetic peptide, it is preferable to use the entire protein when it is possessed.
  • asked a question related to Method Validation
Question
9 answers
I have modelled a protein structure using Robetta server. Its templates doesn't have any pdb structure with query coverage more than 25%.Please let me know the method to validate it.
Relevant answer
Answer
Dear Ajay,
This an interesting question and I am afraid that the advice you got form the previous posters is only of very limited value. It is so because of three facts:
1) Protein folding as a science problem (a question) is an ill-defined problem. Ill-defined means that does not have well formulated question nor the answer to this question has very well worked out (robust) methodology. You are welcome to ask why I think so, but that is a separate issue requiring a much longer discussion.
2) The available ab initio methods are all using at least some heuristic arguments or methods what makes them self confirmatory not exploratory. So even the best methods as Rosetta despite all the hoopla is incapable answering a significant number of problems with required accuracy.
3) The existing validation methods are build on the existing databases what assumes minimal errors contained in them. This is contrary to my experience which shows that almost 75% of proteins retrieved form PDB, in my hands, contain small or large errors. Removal of this errors is almost impossible because of resistance of the community at large. So before using them in my projects I reach the experimental limitations myself by redefining them or remodeling them.
The only real validation comes form experimental methods that produce their own errors. So the problem is complex and requires iterative (almost Kuhnan approach) of hypothesis forming and testing by experiment. So use all the methods cited by the previous posters but not be surprised if some experimental results come diverging form expectations. In heuristic methods the closer the template to the target the better but in my hands two cases of very close similarities resulted in diverging answers. This is so because above mentioned ill-defined character of the initial question.
Good Luck.
Bog
  • asked a question related to Method Validation
Question
7 answers
Greetings!
I'm tryingto validate an HPLC method for the determination of phenolic compounds in vegetable matrix. There are many international guidelines available which gives the parameters and criteria for method validation (ICH, FDA, etc.) but when it comes to vegetable matrix method validation, guidelines are not quite clear (many articles doesn't even mention which guideline they used to perform the validation). So my question is:
Is there any international guideline for HPLC method validation for vegetable analysis (food matrix) ?
I will appreciate every answer and recommendations
Thank you very much!
Relevant answer
Answer
  • asked a question related to Method Validation
Question
5 answers
How to calculate Peak purity in method validation?
Relevant answer
Answer
Using certified standards to analyze m / z for both the precursor ion and its fragments.
  • asked a question related to Method Validation
Question
14 answers
My research title is "Assessment of mentoring in clinical learning environment for radiotherapy students in Malaysia". Its a 5 point likert scale questionnaire ranging from strongly agree to strongly disagree. I have collected several questionnaires from previous related papers, combine some of relevant questions from there, and edited some of the question to suit my research as some papers involved nursing student instead of radiotherapy students. What is the best method to validate it and how?
Relevant answer
Answer
As above mentioned answers, all are helping; if questionaire has aready been used in some study and it has been passed through all sorts of validity process, and your research question is similar to the previous one then you can easily use that questionare, but if it is new one and you have developed it, then face validity(show ypour questionaire to some expert in the field or your mentor, they can check for face and content validity) Dont get confused, these things seem complex apparently but they are actually not. Then you can piolt that questionaire before conducting actual study (piloting can be done on the 10% of sample in accordance with your actual sample; but sometimes five percent sample is also used, it is stated differently in many books, but for practical purpose, piloting is good thing as sometimes what questions we have posed are comprehended by the participants differently; and if discrepancy exists what we are asking and others are answering, then questions can be modified). Peer review of your questionaire can also help; purpose of all this practice is simple as our questionaire must measure what it needs to be measured, oranges comparison must be with oranges and this process is simple but lengthy and needs careful, common sense. Expert responses on questionaire can also be halpful to see inter rater and intrarater reliability.....Good luck!
  • asked a question related to Method Validation
Question
1 answer
I have a 789x789 within class covariance matrix from MD simulations and I was attempting to perform Quadratic Discriminant Analysis with the goal of acquiring class separation between 4 class sequence-structures of Beta- lactamase. When multiplying the covariance matrix of class k with the mean-data 789x10000 matrix of class k it appears since the dimensions do not agree this method will not be valid according to the definition of matrix multiplication. I wanted to get some input on a strategy to truncate the columns of the mean data matrix by 2110 making it 789x7890 then applying the covariance matrix multiplication to 10 folds of the data matrix then I could horzcat(1,2,3,...) to reconstruct. In this situation the variables are the rows and the columns are the objects/observations. I am using MATLAB to construct the QDA from the mathematical formulas one line at a time and have come across this issue, which could be an indicator of poorly-posed settings for applying QDA. Any insight is greatly appreciated as I am putting together a thesis that compares the strengths and weaknesses of methods for discriminant analysis such as PCA, FDA, and LDA in contrast with an in-house method. I was unsure about my initial intuition on a workaround that still maintains meaning in the data analytics. The code involved up to the issue is attached as a txt file. The paper I am basing the mathematical approach is attached as well (theory section -short). Thank you for your time.
Relevant answer
Answer
Hello, The most commonly used method is statistical pattern recognition is the Bayes ”plug-in” classifier. The usual kernal that is plugged in is the multivariate Gaussian distribution is attached.
Good luck!
  • asked a question related to Method Validation
Question
2 answers
I am in the process of opening the first molecular lab for patient care in a highly specialized eye hospital. But I am having trouble obtaining validation samples (aqueous- vitreous) due to lack of resources. Do you have any advice in the proper validation method and validation samples collection?
Relevant answer
Answer
Hello see attached info may be useful for you.
Good luck!
  • asked a question related to Method Validation
Question
12 answers
In the OED, epistemology is defined as follows:
epistemology /I%pIstI"mQl@dZi, E-/
· n. Philosophy the theory of knowledge, especially with regard to its methods, validity, and scope.
– DERIVATIVES epistemic adj. epistemically adv. epistemological adj. epistemologically adv. epistemologist n.
– ORIGIN C19: from Gk epistUmU ‘knowledge’.
The issue here is whether and to what extent epistemology reveals the multidimensional character of the mind [brain].
Here are a couple of papers related to this question:
Relevant answer
Answer
Epistemology reveals that the human mind is continually in a process of becoming.
  • asked a question related to Method Validation
Question
6 answers
Is single day 24 hours dietary recall method valid for Indian adolescents to analyse their nutrient intake with total sample size of 320?
Relevant answer
Answer
Although 24HR recall is subjective and a single administration of 24 hr recall is unable to account for day-to-day variation, it is still a valid measure of dietary assessment and can be used for epidemiological purposes.
If a more accurate assessment is required it can be combined with FFQ.
  • asked a question related to Method Validation
Question
5 answers
Unlike the right type of scientific research, it tends to be through experiments, experiments, laboratories, and the like. A person's statement when answering a questionnaire tends to be momentary and can change in the future. Although in social science research there are methods of validation and reliability testing.
Relevant answer
Answer
I think to information literacy of media agency in share information, all regards...
  • asked a question related to Method Validation
Question
3 answers
Working on questionnaire based study ...
Relevant answer
Answer
Hello, send your questionnaire to the competent peoples which have experience in same area for comments and remarks.
  • asked a question related to Method Validation
Question
4 answers
I am post doctoral student conducting a qualitative research. One of my thesis correction requirement was for me to state how I validate the researcher as an instrument since the researcher is the key instrument in my research? I have read many references but unable to find method to validate. Can the qualitative experts or doctoral student help?
Relevant answer
Answer
Try ROC curves using data or examples presented to your researchers.
These data / examples should be (blinded for your researchers) evaluated by an expert or given a consensus result by a group of experts.
  • asked a question related to Method Validation
Question
25 answers
May linear regression show that an method/methods is/are valid even though this method(s) is not valid according to bland-altmand and paired sample t test.
Relevant answer
Answer
  • asked a question related to Method Validation
Question
9 answers
Dear Dr's and researchers
what is the best parameters to make method for a new Schiff base derived from benzocaine?
Relevant answer
Answer
Hello, see attached paper may be helpful for you.
Good luck!
  • asked a question related to Method Validation
Question
11 answers
for the experimental portion of the study, do we need separate samples or not, so nature of the experiment is non destructive or destructive?
Relevant answer
Answer
Common P-EIS[1] is non destructive[2], if you performed a measurement under the conditions:
1) a low Vac(:=1 to 5, mV) amplitudes,
2) a low over-potential stress Vdc(=OCP), and
3) short (stress) measurement time (<near, 1h, or less).
2. you can use the same sample.
  • asked a question related to Method Validation
Question
6 answers
Hi Folks,
I am looking for a validated survey tool to track changes in participants' notions of power and inequality in society. Rather than reinventing the wheel, I am wondering if anyone has, or has used one that I may be able to draw from in an upcoming study.
Relevant answer
Answer
you might find the following helpful
The Gilens and Page Measure of Political Inequality
However, it's more likely that you will find measures if you define your variables of interest more as it's unlikely that one measure can capture all aspects of power and inequality.
  • asked a question related to Method Validation
Question
4 answers
I need to validate a method that determines compound X in human plasma. However, this compound is endogenous to plasma and is present in a wide range of concentrations between "blank" plasma lots.
How do I approach the validation of this method to satisfy the FDA's GLP bioanalytical method validation guidance?
In other words, how do I prepare calibration curves and QC? Usually I would use a "blank matrix". But since I cannot obtain a blank matrix what do I do?
Would it be acceptable to have calibration curves in solvent and use a bulk plasma matrix (with a low analyte concentration) for QCs. I could add a QC0 to the method and perform blank subtraction to calculate QC recoveries?
Anyone have experience with this or can suggest a better approach?
Relevant answer
Answer
Dear Fabiola,
in general you can use different approaches. First of all you could show and validate that external calibration is applicable. Some researchers also use surrogate matrices like e.g. saline buffers + BSA to create an artificial matrix or you can also do charcoal stripping of your sample to remove your analyte. However, all these approaches will not let you determine the true matrix effect. I prefer the surrogate calibrant/analyte approach. Here you prepare calibrants and QCs by spiking isotopic labelled analogues of your analaytes into the real matrix and using these signals for quantification of your target analytes.
Here´s also some literature on the topic
  • asked a question related to Method Validation
Question
20 answers
RID is highly sensitive to Temperature and mobile phase composition.
Column has column oven and even RID detector is constant at 35 degree. Mobile phase is 80% ACN (purged properly)
I could not find the reason behind this shift in RTs.
Can you help?
Relevant answer
Answer
Glad you figured out the hardest part of HILIC, which is the equilibration. It takes 19 forevers to equilibrate properly, and 19 forevers to re-equilibrate after each run. There are better, more stable methods for sugars than HILIC. Take a look at mixed mode columns. I think you will like them.
  • asked a question related to Method Validation
Question
6 answers
Hello all,
This is a slightly strange one.
I need to convince a community of practice (field biologists) that they need to validate their tools and methods before they use them to generate results that are used as the basis for important management decisions.
While QC and validation have penetrated to the benches in biochemistry and microbiology labs, they are completely foreign to field biologists, for reasons that I will not burden you with here.
What I need is a basic introduction to QC and validation that is not specific to the hard sciences (physics and chemistry), and that sets out the fundamental reasons why knowing that a tool and a method work properly are essential steps in doing good science and generating results that can be relied on.
I have googled all the combinations of validation, QC, fit for purpose, etc etc that I can think of, and everything I find is too specific to particular applications - I need something that sets out the basic need for validation in general. Peer reviewed or a standard textbook is preferred, but anything clearly written will be a help.
Thanks.
Peter
Relevant answer
Answer
There are no appropriate texts! ICH validation guidelines apply to pharmaceuticals and biopharmaceuticals. With small molecules it has been relatively easy to validate a method but stem cells will be very hard. Is a recovery of 50% for a fluorescent antigen technique appropriate?
The 'validation biologist' will have to demonstrate that the method is consistent and reliable. This will mean setting the appropriate and relevant acceptance criteria or specifications.
  • asked a question related to Method Validation
Question
8 answers
Is it ok to use Honey sample without hmf(Blank honey sample)for recovery studies?
Relevant answer
Answer
Thank you Richard Odemer Bruce Neagle
Kou Hayakawa
  • asked a question related to Method Validation
Question
5 answers
I just want criteria for
Precision : % RSD
Accuracy : % Recovery
LOQ
Relevant answer
Answer
Thank you all.
@ Mahmoud H El-Maghrabey ,
ICH is a good guideline , but it does not mention any criteria.
  • asked a question related to Method Validation
Question
5 answers
I am getting recovery of a drug from plasma about 50% compare to water which is analysed by HPLC and getting lower concentrations detected nicely.... Is the mathod valid or acceptable?
Relevant answer
Answer
Dear Urvesh,
You may refer to FDA Bioanalytical Method Validation guidance (May 2018): „The sponsor should optimize the recovery of the analyte to ensure that the extraction is efficient and reproducible. Recovery need not be 100 percent, but the extent of the recovery of an analyte and of the ISs should be consistent and reproducible.” Summing up, if the recovery is constant across your calibration range (within +/- 15%), then your method will produce reliable results.
Best regards,
Piotr
  • asked a question related to Method Validation
Question
4 answers
If method validation required on Area% by Area normalization on GC, what Criteria to be fixed for Method Precision (Related Substances in Intermediate of API)
Relevant answer
Answer
If you are performing Method Precision, then it should be 2.0% as per ICH Q2(R1).
  • asked a question related to Method Validation
Question
7 answers
During dissolution method validation accuracy parameter need to be performed in dissolution vessel or this test can be out side the dissolution vessel. What dissolution parameters need to be kept for accuracy if it is to be done in dissolution vessel, if accuracy is failing within the QC parameters. How to perform dissolution accuracy for multi media dissolution?
Relevant answer
Answer
Accuracy and Specificity (placebo interference) can be done by flask method also.
  • asked a question related to Method Validation
Question
40 answers
In mathematics, we prove many theorem by contradiction method.. I want to know this method is applicable in nature or not ....
Relevant answer
Answer
Proof by contradiction is valid only under certain conditions. The main conditions are:
- The problem can be described as a set of (usually two) mutually exclusive propositions;
- These cases are demonstrably exhaustive, in the sense that no other possible proposition exists.
Under these circumstances, if all but one of the cases are proven to be false, the remaining case must be true.
It is useful when the proposition of interest is hard to prove, but the contradictory proposition(s) is/are easy to disprove. These conditions do, of course, apply well to many mathematical problems.
For example the simplest proof that the square root of two is irrational is a proof by contradiction. We can state the problem as two mutually exclusive cases; A: sqrt(2) is irrational; or B: sqrt(2) is rational. And this is an exhuastive set; there is no other possibility. It is quite hard to show that A is true directly; but it is quite easy to show that assuming B generates a contradiction and must therefore be false. (See https://en.wikipedia.org/wiki/Proof_by_contradiction#Irrationality_of_the_square_root_of_2).
In the 'natural' world, this still works when these basic conditions hold. Finding one black swan (or a blue one) is essentially a proof-by-contradiction that "All swans are not white". (observation of a single black swan conclusively contradicts the only possible alternative proposition, that "all swans _are_ white").
But the natural world is often messier than this. First, we are often much more interested in partial generalisations (eg "_most_ swans are white") and that is impossible to prove or disprove conclusively without exhaustive counting (though statistical inference allows the statement to be rejected as improbable after suitable sampling exercises). Further, there are often many alternatives of interest, we cannot always disprove all but one, and even if we can, we cannot rule out the possibility of another, unknown case. Proving something is not a cat does not prove that it is a dog. It may be a mouse. And if we also prove it is not a mouse, there are plenty more furry animals to work through, and even after we have gone through all the known furry animals, we may be still looking at a new, previously unknown, species.
So the principle always works if the basic conditions above hold; but it is not valid if they don't. And in the natural world, if those conditions _do_ hold, we're either very lucky, or we have made some improbably sweeping generalisation that is trivially refutable.
  • asked a question related to Method Validation
Question
10 answers
As for the available literature UV spectroscopic method validation does not include robustness and ruggedness study. Is it really essential to perform these studies for simple UV methods or the method is valid even though not validated for robustness or ruggedness?
Relevant answer
Answer
No, Robustness and Ruggedness studies were not require for UV Spectroscopic method.
  • asked a question related to Method Validation
Question
18 answers
My Column is Shimadzu SHIM PACK VP-ODS (4.6 × 150mm x 5 µm) RP
and Mobile phase is 10% Methanol
Kindly suggest
Thanks in advance
Relevant answer
Answer
If your HPLC procedure is just optimized, you need to perform the following experiments:
- linearity
- LOD and LOQ
- precision and trueness
- recovery
- stability (short and long-term)
For each point you need to perform 6 independent experiments.
For linearity are necessary at least 5 non zero points at different concentration levels.
Additionally for precision and trueness you need to analyse 6 times 3 QC levels (low, medium, and high and the concentrations need to be different respect the concentration levels used in linearity) obtained by spiking (with known amount) blank matrix.
All these procedures are deeply discussed at the following links:
  • asked a question related to Method Validation
Question
5 answers
I am performing a (partial) validation of commercially available ELISA kit. I am using the kit on a previously untested matrix, so I wish to validate the assays Accuracy, intra-assay Precision and Spike-recovery.
To my understanding: Accuracy (AC) is defined as: closeness of mean test results to the true concentration. Precision (intra-assay) (PR) is defined as: closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogenous sample. Spike recovery (SR): used to determine if the assay is affected by the difference between the diluent used to prepare the standard curve and the sample matrix.
Since all these parameters are determined by performing multiple measurements of different concentrations, I am wondering if one can combine these into a single series of samples and use the data to determine the 3 parameters above?
i.e I would perform a series with 4 different concentrations of the analyte spiked into sample matrix, and do 5 determinations for each concentration. Is there any reason that AC, PR and SR determination should be divided into separate series on the validation plate?
I have attached is my planned plate-layout including the other parameters (LOD, LOQ, linearity etc). Am I on the right track here?
I appreciate any thoughts/insight you may have on this