"QC's" I do not think that is the correct term. What are you trying to determine?

"UHPLC" is just called HPLC. This is the analytical technique you will be using, not a method.

You must already have an HPLC method developed and verified for your specific samples to be asking this. It is not possible to set up a strategy until you have the method developed, any calibrations worked out and goals established about what you wish to measure (or hope to observe/record). Once your HPLC method and calibration data have been run enough times to determine their reliability and repeatability, you can use that information to determine how many replicates of each sample you will need for measurement (at least 3 replicates). Once you have that info, you can determine the number of different standard levels (Calibration Stds for each sample type you wish to measure), frequency of calibration and times needed between samples (and also if bracketing is needed). All of these things so far are determined by your method (which we know nothing about).

Yes Bill, sorry for these mistakes. I do have a method developed and verified. I will analyze a aqueous plant extract, with a C18 collumn and a gradient of mobile phase ranging from purified water with 0.1% of formic acid to acetonitrile (other specifications I might have to check, but the method is developed for this plant)

QC is the terminology we use here, but I don´t know if it is correct. The QC is used to verify the equipment and it's calibration.

We will use a mathematical procedure called PCA to analyze the data in the end. The QC usually contains all samples and in the PCA graphic it is supposed to be in the middle, while the other samples group with each other, depending on their similarity between them.

I'm sorry if it I can't express myself very well. This is the first time I work with chromatography.

What do you use to verify if the equipment is working correctly and if it is well calibrated?

"What do you use to verify if the equipment is working correctly and if it is well calibrated? "

To verify the correct operation of an HPLC system, we use the recognized term, Instrument "Operational Qualification" or OQ. This verifies that the instrument operates and works to meet the instrument manufacturer's specifications. The manufacturer generally carries out these documented tests at installation, anytime the instrument is moved or repaired or anually.

"Calibrated" is another term we generally do not use for HPLC systems. Calibrated is specific to specific things that can be calibrated like pipettes or balances (you can not "calibrate" an HPLC as it is composed of dozens of sub-controls and instrument modules). For example, you can check the "calibration" of the HPLC' detector's wavelength output or the pump's flow rate using approved SOP's (Standard Operating Procedures), which describe the procedures for doing so. A large number of SOPs used together are used for the OQ. NOTE: These are separate tests to verify instrument operation and have nothing to do with verifying the HPLC method or any samples analyzed.

Each method is usually "Validated" to demonstrate that it is specific for the compound being tested and reliable to provide qualitative or quantitative data about it. *Unfortunately, due to poor quality training of some users, a "validated" method may not be accurate or usable. The intent was to show that the method follows proper scientific principles, but when many of the users who develop them do not actually know what those are, the results are rather unscientific. Generating high quality chromatography methods requires a great deal of practical hands-on experience. My advice is always to seek out local experienced individuals when working with HPLC systems. Failure to do so, often results in poor quality data being generated. HPLC systems are not scales/balances which get calibrated and then give you an answer. They are just complex tools which can be set up to provide a wide range of inaccurate data. This is because they rely on the experience and knowledge of the operator, not the hardware to generate the data.

Thank you very much for the answer Bill!

Hello William and Ravi, the QC term is used in metabolomics, I found articles to help me in this question. If you wish I can poat their links!

Elisa, sorry that is not correct and you appear to be misunderstanding many of these basic terms used in these fields. Perhaps your instructors can better help you?

Metabolomics is simply defined as the study of small molecule metabolites. Your questions are directed at how to create and set up a method such that, when finished, you can show that the techniques used were appropriate. After all, I think it was you who asked us, "What do you use to verify if the equipment is working correctly and if it is well calibrated? " That is not metabolomics.

Hello, yes, when I asked " What do you use to verify if the equipment is working correctly and if it is well calibrated " I was mistaken.

This is the QC I'm talking about "https://www.researchgate.net/post/How_can_one_choose_proper_QC_samples_during_metabolomics_research"

What I want to know is how many should I have, I'm asking here because I was hoping to find someone in the area that could tell me how many of these are recomended. I was not finding the term anywhere in any article, but my instructor said this is common in the metabolomics field of research, not that my research is in the area of metabolomics.

But thank you very much for your answers, I have to learn how to express myself better in english.

Elisa: No one can answer your question as posed. Not enough information. Also, the answer is "not" area specific, but is related to the experiential design and variations observed.

Perhaps most importantly of all, no method. The number of replicates, controls, blanks and standards to use during your analysis will depend 100% on the method developed AND the results obtained using that method on standards and evaluation samples. Without a review of the actual method used and data generated from it, we can not know how many replicates should be used or their frequency. Those are the kinds of things we discover AFTER we develop the specific method for the samples/standards (aka: "The Scientific Method"). This is because there is no such thing as a "std" method of analysis in chromatography, but there are rules and guidelines we follow to develop them.

While it is possible to plan out an entire experimental strategy with many types of experiments, from start to finish, that is not the case in developing a new HPLC or LC/MS method for a specific task. The precise method needed must be initially developed, thoroughly tested, verified and shown to be accurate, reproducible and selective for the compounds of interest. Once that is done, you can run some statistics and address any problems of variation or change over time which occur IN THE METHOD design (or continue to fine-tune the method used). In doing so, with some help, you should then be able to work out what statistically significant number of controls, blanks, standards and samples should be to obtain valid data.

About the only # we can suggest right now is that a minimum of 3 data points (samples/standards) are always used for statistical purposes.

Hi Elisa, I just add another thing about your question: How stable is your samples? Because this will directly influency your number of standards in a batch. I hope that's usefull.

Best regards