Dear Sir. Concerning your issue about the reference standard mix for metabolomics analysis using LC-MSMS. Metabolomics has emerged as the latest of the so-called “omics” disciplines and has great potential to provide deeper understanding of fundamental biochemical processes at the biological system level. Among recent technological developments, LC–HRMS enables determination of hundreds to thousands of metabolites over a wide range of concentrations and has developed into one of the most powerful techniques in non-targeted metabolomics. The analysis of mixtures of in-vivo-stable isotopic-labeled samples or reference substances with un-labeled samples leads to specific LC–MS data patterns which can be systematically exploited in practically all data-processing steps. This includes recognition of true metabolite-derived analytical features in highly complex LC–MS data and characterization of the global biochemical composition of biological samples. In addition, stable-isotopic labeling can be used for more accurate quantification (via internal standardization) and identification of compounds in different organisms. stable isotopic labeling (SIL) is a very promising and increasingly popular technique in metabolomics research, which is perfectly suited to be combined with GC–MS and LC–MS. Stable isotope techniques were preceded by the use of radioactive isotopes in biochemical research. In targeted analysis, SIL reference standards can be used for internal standardization of peak intensities in trace analysis by use of LC–HRMS or LC–(HR)MS–MS, making this the most common use of SIL. Use of authentic, labeled reference standards enables easy, rapid, and absolute quantification of the respective metabolites even in complex matrixes. I think the following below links may help you in your analysis:

http://www.shimadzu.com/an/journal/selection/selection2-2_03.pdf

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1904337/pdf/nihms21280.pdf https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536965/pdf/216_2012_Article_6375.pdf

Thanks

Dear Alejandro,

It depends on what your samples are derived from. In addition to the excellent answer of Dr. Isam Eldin Hussein Elgailani, I would like to bring the following under your attention. Even when using stable isotopes as internal standard, these standards have to be analyzed by conventional protocols to establish the actual composition and content of all the available/ for your research relevant metabolites. For plant derived samples, a good source is the following: https://isolife.nl/. In any other case, you will have to mix your own standards (very expensive) or grow your samples on 13C carbon sources followed by a quantification of all the metabolites and their respective labelling (also very expensive). I hope this is helpful information. Kind regards, Harrie Verhoeven.

Thanks Harrie and Isam,

I am aware of the SIL and the different metabolomic apporaches... maybe I did not pose my question well enough. I was just looking a chemically defined Std mixture to validate my LC-MSMS methods, that is, if I inject a 'known' standard, how good is my chromtography and the MS/bioninformatics methods to detect them before I move into more complex biological samples.

I have received an answer directly through a message of another RG member (thank you Biswapriya Biswavas Misra), which I will reproduce below, which I think best addresses my question, just in case someone else is going through this process:

Hello Alejandro,

Hope you are doing well!

Saw your question on metabolomics standards for your laboratory/ core facility I suppose.

To be very honest, as suggested by a couple of people (not from metabolomics background but analytical chemistry), stable isotope labelled standards (SILs) are truly expensive and only used by top notch core facilities and rarely by research groups. Also, they do not solve any problem- except a better quantification- but by no means reliable!

[1] If you can afford and have got some funds, I would highly recommend the MSML Library

MSMLS Sigma Mass Spectrometry Metabolite Library: https://www.sigmaaldrich.com/catalog/product/sigma/msmls?lang=en®ion=US&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-5which is Supplied by IROA Technologieshttp://iroatech.com/page/Mass%20Spectrometry%20Metabolite%20Library%20of%20Standards and if you get the quote (and can bargain!) would be lot cheaper than that from Sigma. This is MUST have for any facility doing LC-MS (and even GC-MS) based routine metabolomics. With 600+ standards one can easily create a library of all generic pathway wide metabolites. All are VERY RELEVANT to all sample types, starting from sugars amino acids, TCA cycle to phosphates and metabolic intermediates. And currently, there is not other company that provides such a huge list. Do not go for their SIL standards as that would be too expensive and needs further software and specific applications. Just the unlabeled 600+ standards in micro well plates would do the job!

So, there are a few Sigma Kits available that I must point to, and they are grouped by what you will looking into in a typical untargeted/ targeted analysis.

[2] ML0010 Sigma TCA Cycle Metabolite Library : https://www.sigmaaldrich.com/catalog/product/sigma/ml0010?lang=en®ion=US&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-1

[3] CAR10 Sigma Carbohydrates Kit :https://www.sigmaaldrich.com/catalog/product/sigma/car10?lang=en®ion=US&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-2

[4] V1 Sigma Vitamins Kit ~98% (Components, TLC) :https://www.sigmaaldrich.com/catalog/product/sigma/v1?lang=en®ion=US&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-4

[5]47264 Supelco Organic Acids Kit (individually packaged), analytical standard : https://www.sigmaaldrich.com/catalog/product/supelco/47264?lang=en®ion=US&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-4

[6] 47266 Supelco Sugar Alcohol Kit (individually packaged in quantities indicated), analytical standard : https://www.sigmaaldrich.com/catalog/product/supelco/47266?lang=en®ion=US&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-3

[7]A9906 Sigma-Aldrich Amino acid standards, physiological analytical standard, acidics, neutrals, and basics : https://www.sigmaaldrich.com/catalog/product/sial/a9906?lang=en®ion=US

[8] If you plan to get/ use a lot of Human plasma or such samples, then better to have and compare NIST's human plasma pools available from Sigma: Metabolites in human plasma NIST® SRM® 1950 : https://www.sigmaaldrich.com/catalog/product/sial/nist1950?lang=en®ion=US

I mean the choice is yours and depending on the sample types you are expecting. But, I would always go for the IROA kit and se what is missing and then compensate it with the Sigma kits, and for sure have the NIST plasma as QC run always.

Hope the above lists help you focus on a few and go for them. If you got more questions please do not hesitate to ask again.

Thanks and regards, Biswa

Also see attached - this is very handy also together with the previously mentioned MSMLS