Science method

LC-MS - Science method

Liquid chromatography–mass spectrometry (LC-MS, or alternatively HPLC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high sensitivity and selectivity.
Questions related to LC-MS
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Is LC-MS a good option to calculate plasmid concentration?
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If you have a well-defined standard against which you can measure, it's a good method, if you have none yet, it isn't.
If you don't have any standard to compare with, the most accurate method is probably evaporating the solvent carefully and weighing what's left. If you have a mixture of species, you can also perform preparative LC and the continue with evaporating-weighing.
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Hi, We are purifing meterials form natural product.
We purified two materials from differnt fraction on first step of purification ( linier grdient 5 > 65 % ACN in 0.1% TFA, 60min, 1ml/min) having some extra steps of HPLC.
And purified meterials was measured Q-ToF LC/MS.
The LC/MS data showed that one of meterial have clear mass peak, but another one is compound of 5 meterials though LC peak was celar on last step of LC
Eventhough the mass peak showed 5 meterials, It was oxdidant fragment of clear mass peak's meterial.
I don't know when oxidataion was occured, before purification or before mass measured.
anyway, is it posible same materials have differnt retendtion time on crude condition?
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Sometimes, this might be happend. Following that, it could be interprated using a series of reasons and errores. Take a look to the following links.
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Hi all !
I want to check weather three targeted compounds of raw honey such as chrysin, cape and caffeic acid in the raw honey. Does anyone have experience handling this sample? do i need to extract using methanol or just filtered water? I have standards for all three compounds. Thank you.
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Hi Dr.Haslina
I am interested in this topic please send me a message on my WhatsApp 00249911511696
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I'm looking for a relative easy way to test the activity of CYP51. Are there any way that I can measure it without using LC-MS or gas chromatography. A simple absorbance or fluorescence test will be great.
Thank you.
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Maybe this article helps: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8996309/ It mentioned the method of using molecular docking and molecular dynamics simulations to model, targeting ligands specific proteins.
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I have aqueous ddH2O and organic solvent 10mM NH4Ac in MeOH, 60-100, another one 10-100. if I want to change to ACN or TFA, how can I wash the column to switch between those organic solvents.
the flow rate is 5ml/min for HPLC and 0.5ml/min for LC-MS, with different type of columns.
Thank you
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You have a BUFFER solution with methanol ("10mM NH4Ac in MeOH,") so first you need to run a solution that is the same, but without the buffer present through the HPLC system to wash the buffer away (be sure to first check column compatibility with the solution, for each storage or just washing). Once ALL of the buffer has been flushed from the system to waste, you can begin to switch over to a new, fully miscible solution / mobile phase (e.g. ACN / Water). ***Please, before changing anything by yourself, ask your teacher or the person who has professional training in using the HPLC and/or LC-MS system for direct help.
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i am looking for a detailed protocol of protein extraction from animal tissue that fits suitable for LC-MS analysis. So that i can prepare the sample accordingly and it send it for analysis. Recently i am following two buffer 1. Triton lysis buffer 2. Ripa lysis buffer
Are these two extraction method right for LC-ms sample preparation ?
please suggest
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Dear Najmin,
when you use detergent-based buffers for cell lysis you should make sure to include a clean up step prior LC-MS/MS analysis (e.g. FASP filter, C18 OMIX tips or in gel digestion), as non-ionic detergents may interfere with optimal peptide separation (as they tend to stick to your column). For tissue extraction optimal results can be obtained by freezing the tissue in liquid nitrogen and subsequently pulverizing the tissue using a pistil. The pulverized tissue may be solubilized in a detergent-based buffer as you tried before, or if you want to avoid any clean up steps with a urea-based lysis buffer.
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Some of LCMS instruments in my lab showing M+100 mass for every sample (Buffer: 0.1% FA, 0.1%TFA, Mobile phase B: acetonitrile). It is automatically disappears and again reappearing. But this M+100 m/z not found when we are using ammonium bicarbonate as buffer. Please let me know.
Thanks
Murty
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Get rid of TFA or use mass spec grade TFA or better use DFA. Try to use only FA or AA for positive ESI not to see mentioned adducts. Ambic can be preferred as a negative ESI buffer that triggers deprotonation owing to its alkaline nature. It also produces multiply charged pseudomolecular ions...
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I am currently developing a LCMS method for certain genotoxic impurity in API on a shimadzu 9030 qTOF LCMS. And I had an argument with my coworker about which mode is more sensitive, SIM or MRM?
My coworker said that MRM is more sensitive based on his past experience using LCMS for API analysis.
I am relatively new to LCMS. But the way I see it, MRM is definitely more selective. But it is only more sensitive than SIM when there is background interference.
MRM is a MS/MS method, which should have fewer ions compared to SIM, which is a MS method. So if we have a clean standard or a sample which has no signal at this particular m/z around the retention time of the target, SIM should be the more sensitive one.
Am I wrong?
Thanks in advance for any comments!
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Theoretically, the SIM method could obtain higher abundance peaks due to not losing the ions in the process of MRM transition. For instance, for higher m/z ions SIM could have high abundance with a relatively low noise signal. However, the MS/MS mode has advantages that should lead to MRM usage (noise reduction with the simultaneous confirmation of compound).
Sometimes the use of SIM is the only option. Some sterols-related compounds when using fragmentation give a very characteristic fragmentation pattern with no predominant ions.
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Hello everyone. I want to do trypsin digestion for peptides sample preparation in LC/MS. But my laboratory doesn't have DDT (Dithiothreitol). Can I use b-mercaptoetanol in stead of DDT for reducing disulfide bond of protein before using Trypsin in digestion? And I don't have IAA for alkalinization, too. So what should I do now?
If you have any protocol about using b-mercaptoetanol for peptides sample preparation in LC/MS, please send me that. Thank you!
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BME works fine at similar concentrations to DDT. I don't use it anymore because BME gives me a migrane. You actually don't need to cap the resulting C's. As long as you keep the sample reduced (i.e., away from O2) you can use it directly in MS. I used to add a tiny amount of sulfite to the sample before ESI to preserve the reducing environment. However, as long as you can smell the BME you should be fine.
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Im curious as to whether the software for LCMS (Labsolutions from Shimadzu) already has a quantification feature or do I have to compute and compare the AUCs to the standards manually? If so, what are the steps in the computation/quantification of compounds?
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  1. In your LabSolutions main window, go to Postrun then open the Browser.
  2. In the Browser's main tab, press on Quant Browser, then open the files/samples you want to quantify.
Things to consider: quantifying requires a quantification method, where you assign m/z, retention time, etc. to your analytes/compounds, so they got recognized by the software. In this case, you will get peak areas (or area ratio in case of using an internal standard), and this can be called qualitative analysis. For quantitative analysis, you need to add the concentration of each calibration curve standard in order to quantify.
You can follow LabSolution training or watch tutorial videos on how to quantify using LabSolutions software.
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I got lcms data. I ran MZmine 3. I need to know which range of height/height ratio I need to consider for further approach of determining chemical formula, please?
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I have digested the polysaccharides with HCl. Can I inject digested content (with acid) into the LCMS column for detection of individual mono or disaccharides ?
Any suggestion or advice, those who are working on carbohydrates
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Thank you for your answer
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I am looking to detect C2O2Cl2 in chloroform solvent in HRSM.
I try both negative and positive modes, direct injection with MeOH .
In negative mode i get 160.8414 Da (M+Cl) instead of 160.8964 Da.
In negative mode i get 90.9046 Da (M-Cl) instead of 90.9587 Da.
Same gap of ~ 0.05 Da
The instrument is after calibration and contain internal standard.
Any one have any idea why is that or how can I improve it?
Thanks a lot
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Sorry for not responding until now (busy days).
Can you please explain how it well help?
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I am curious if column bleeding is still "a thing" on modern UHPLC columns (reversed phase and hydrophilic interaction columns), such as the HSS T3 C18 column from waters or the iHILIC column from hilicon (or any other UHPLC colum). Did anyone observe such bleeding? If yes, which masses did you observe for which columns (if you used MS)?
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To answer your question regarding "column bleed". Not if the functional groups on the silica are BONDED to the support, as most are... If the column support consists of material that is not covalently bounded, then it may indeed slowly elute off the support and out of the column with the mobile phase. There are HPLC columns with coated supports (very delicate and require special treatment to use). These "coated" supports will wash off over time and as the coating is washed off, their retention characteristics will change too. However, "coated" phases are not common. Most HPLC column types you are likely to encounter today have fully bonded supports. Any observed "bleed" is likely to be the result of poor method development resulting in material from the flow path, fouled column, sample(s) and/or mobile phase additives being seen at the detector. With LC-MS, adducts are very commonly observed.
*I should also mention that one rare thing that we do sometimes detect when using columns packed with very small particles (~ 2.1 u or less) is the silica itself. The LC column-end fitting has a frit (filter) on it to prevent the silica from leaching out into the flow path, but these extra small particles are often accompanied by super small "fines" which get past the filters and into the flow path (and detector). Silica and the associated chelated metals that they can attract are sometimes observed.
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Hi,
I would like to analyse 8-dihydroxy ergotamine in plasma by LC/MS. I need to increase the LLOQ and for this reason I need to derivatize the molecule. Does anybody have experience with these kind of molecules? I do not know if there are options....
Thanks a lot for your time and help
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Mrs/Miss Enriqueta casal-banciella
There is not needed derivatization-step.
Please, concentrate on the content on the work (ref. [1]) cited in the following discussion, when it is appeared online available:
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Looking for expert advice on storage conditions to preserve cotinine in blood plasma. I will be collecting whole blood in EDTA treated tubes, which will then be centrifuged to obtain the plasma for Liquid chromatography-Mass spectrometry. Which tubes are best to store in (Eppendorf tubes vs cryovials) and is -80 degrees ideal for storage up to 12 months?
Any advice or tips will be appreciated. Thank you!
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Cryo-vials
The cryo-vials is manufactured for stability in extremely cold storage conditions
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I want to know about the sample preparation method.
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You can find what you are looking for, in OEKO-TEX standards.
AS ATTACHED
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I have been experiencing consistent contamination of the pharmaceuticals ciprofloxacin, trimethoprim, carbamazepine, azithromycin and venlafaxine. The peak height remains the same with or without injection. and is predominantly seen when there is an addition of 5 mM ammonium formate. The standards I previously injected didn't exceed 2.5 ppb .
My method is:
(A) Aqueous : H2O + 0.1 % formic acid and 5 mM ammonium formate
(B) Organic: ACN + 0.1 % formic acid
Gradient:
Time % B
0 5
4 50
5 100
9 100
9.5: 5
The steps I have taken so far include;
- Multiple injections of 1:1 H2O: IPA (not going into column)
- Changing mobile phase (A: H2O + 0.1 % formic acid and 5 mM ammonium formate, B: MeOH
+ 0.1 % formic acid and 5 mM ammonium formate)
- Changing to a new column
- Replacing needle seat, injection needle and MS capillary
- Running an acidified cleaning mix (1:1:1, MeOH:ACN:IPA 1% formic acid) in the stead of my
mobile phase.
- Using an acidified multi-wash (1% formic acid) (A: 1:1:1 IPA:MeOH:ACN, B: 1:1 IPA: H2O, C: 1:1 ACN:MeOH)
- Ran the method with no injection
- Direct injection
- Deep source clean
- Injected acidified organic
- Changing capillaries
- Remade mobile phases with brand new formic acid and ammonium formate
Any help or thoughts on what to try would be a great help!
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Hi William, thank you for this! I will have a look into changing the parts you mentioned. The wash bottles I was addressing were the ones that are linked to the multi wash system, but yes I also changed the seal wash solvent. I will additionally contact Agilent in response to your recommendation.
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Dear all,
I am looking for a LC-MS spike-in dataset with :
- two or more classes.
- at least a hundred of samples.
- a list of the spiked peptides (mz and RT).
I found a dataset in Leepika Tuli et al, 2012 (https://www.researchgate.net/publication/221865421_Using_a_spike-in_experiment_to_evaluate_analysis_of_LC-MS_data) which corresponds perfectly to what I am looking for, but it contains only 10 samples.
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Is lcms analysis good for determine the unknown compounds in nitrocellulose lacquer?
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what kind of compound groups are you targeting to analyze? I assume it's a kind of hydrophobic matrix, therefore GC-MS profiling would be better. Indeed you may prefer LC-MS for a non-targeted approach. You need to narrow down the purpose first in terms of molecule polarity and size to find the best match...
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I want to deglycosylate proteins and analyse them by LC-MS as control. How can I chemically deglycosylate the proteome total of cells ?
Thanks a lot
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N-linked glycosylation can be removed by treatment with PNGase F enzyme.
NEB also supplies a protein deglycosylation kit:
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I am Janu Newar, Ph.D. Scholar from School of Biotechnology, KIIT University, Bhubaneswar, Odisha. Our lab is basically working on proteins from biological sources. This work involves the identification of some proteins. I would like to request some help with the Agilent 6530 Q-TOF instrument.
For identification of the protein, we are using a glass insert of 0.2ml volume (Part no: 5188-5390). My query is can we use 0.025ml of the sample volume in this insert and can inject 0.02ml of sample in the column. If not, then how much lower volume can we use in this 0.2ml insert?
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You may need to play around with the needle depth setting in the method, so the needle would go fairly close to the bottom without actually touching the bottom. Also reduce the draw rate so the liquid on the side wall has the opportunity to coalesce.
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Hello everyone,
I'm planning on running my peptide samples on a high-resolution LC-MS instrument. I'm going to use a ZipTip C18 tip for the extraction of peptides and desalting of the sample. However, I'll be sending these samples overseas and it might take 2-3 days for them to reach their destination. I can potentially keep them in dry ice throughout their delivery, but it is very costly and we had few issues before where the dry ice evaporated until it reached the destination.
If I free-dry my peptide samples, do you think they are going to be stable for couple of days? Considering there won't be any humidity where the enzymes can work on the peptides, but I just wanted to get the opinion of people who has lots of proteomics experience.
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Selam Bora,
leave your peptides on the zip tips (after washing step without elution)and send them in an envelope at RT. The MS-facility can than elute the peptides from the C18 tips and start LC-MS analysis. I have done this several times with custom made C18 stage tips (much cheaper than ZipTips) in a collaboration with colleagues from Acibadem.
Let me know if you need further assistance.
Good Lick,
Murat
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Dear all,
What is the criteria for selecting the positive mode or negative mode in LC-MS/MS analysis?
In brief, when is positive and negative mode of ionizations are used inn LC-MS analysis?
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Hi,
the best way to select the polarity in LC-MS analysis is to try to inject your compound of interest in both polarities and observe which of the two ones gives the most intense signal and the highest S/N.
There are general rules to select the polarity:
NEGATIVE POLARITY --> molecules already charged negatively, molecules containing phosphates, acidic moieties, molecules with many Oxygen atoms, molecules contaning phenolic groups ecc.
POSITIVE POLARITY --> proteins, amines, molecules already charged positively, e.g. anthocyanins...
BUT the best way to select polarity still remains to try both polarities and select the most suitable to you.
BTW, if you are interest in a specific compound, look at the literature, there will be surely information about the experimental conditions, including polarity
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Dear all,
I am doing Vitamin D derivatization with PTAD like described in this paper (reaction mechanism is in Figure 3) to enhance sensitivity in LC-MS.
Normally this reaction is done in absence of water and works great, but completely drying it is a pain, so the ability to do the reaction in presence of 10% water (90% ACN) would be great.
So I tried and could observe close to complete disapperance of the unmodified vitamin D species in the presence of PTAD (not in the absence of PTAD). But, the normal modified form is only present in very minor amount, so there must be another reaction. Does anyone has a clue, what could be this side-reaction? Unfortunately, I have only a Triple-Quad, so I cannot really look unbiased with HR-MS.
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Hi, Florian!
How are you?
I'm from Brazil and trying use PTAD to vitamin D derivatization. Do you have some news about your question?
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Hello,
I'm attempting an LC-MS analysis for a plant methanolic extract for the sake of polyphenol and terpenes quantification. I'm following a protocol used by Nivea M. et al., (2019) in which they used Ribitol as an internal standard.
I was wondering if I can substitute Ribitol with another standard and if I can then what are the possible choices I can use?
Thank you
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Hi.
I see that many papers are analyzed using external standard methods.
Are you concerned about the recovery rate from the analyte?
I think you have a few compounds to choose from following articles.
baicalin
catechin and kaempferol.
quercetin
biochanin A
Anyway, of the polyphenols that would not be in your analyte, you can use those that are relatively close in nature and available in high purity. I believe you can solve this problem by selecting the appropriate one that is not contained in the analyte.
And I believe the topic someone else posted earlier may be helpful...
Good luck!
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I'm currently researching a couple questions related to N-fixing microbes for a project I'm working. I was wondering if anyone could give me any advice and/or point me in the direction of relevant publications for either or both of the following questions.
1) How would you screen, in a high-throughput manner (important), putative associative nitrogen-fixing microbes for their ability to fix and provide nitrogen to a host plant? Ideally in a greenhouse / growth chamber context.
2) How would you do the same thing as the above, but in a system where plants are not present (e.g. in a 96-well plate format)? For example, growing microbes in liquid medium and measuring evolution of fixed nitrogen in the culture medium through LC-MS. But I'm sure there are plenty of other approaches I'm not aware of.
Thanks for any insight you can provide!
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Most photosynthetic microbes can also fix nitrogen (e.g., cyanobacteria). Nitrogen fixation requires energy, which is generally obtained in the form of sugars from the roots of a plant. Therefore, the easiest way to screen is to use a nitrogen-free media (e.g., minimal media with no organic amines or ammonium salts) with sugars present to provide the energy. Growth means the cells must be able to fix N2 from the air. Of course, if the organism needs some other nutrient to grow, you won't know if it could fix N2 in symbiosis with a plant root.
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Hi,
I want to extract amino acids and nucleosides from samples with high salt/buffer, protein, fat and cellular debris. Amino acids and nucleosides will then be analyzed by LC-MS.
Do you know how I can isolate amino acids from my sample? And how I can isolate the nucleosides? (two different analyses).
We are modelling the digestive process so there is digested food stuff (blended before mixing with digestive enzymes) in the substrate that varies depending on the experiment and the substrate used.
The digested substrate is in 40 mL of liquid.
I would like to avoid MWCO filtration if possible.
Thanks
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Hi, u can check this link its will help
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I am planning to start an experiment to measure my samples in 384 well plate.
The samples are in 50% methanol and it will be used in three different LCMS machine (Agilent, Bruker and Orbitrap).
So I am looking for sealer to store my sample. The sealer should be without conventional adhesive and should not be strong enough to damage the needle!
Does anyone have any recommendations for such films or sealer for 384 well plates?
Thank you in advance
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Thank you so much Vishma Pratap Sur and Rob Darrington for the suggestion.
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There are certain drugs exist in liquid form available in the Sigma-Aldrich website, where they have mentioned the concentration of those drugs in "ml" rather than "mg" or "g". How one will find the concentration of such drugs?
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Actually, reading the question with very strict meanings, he might be looking for a conversion to grams. If the density is known, multiply the volume by the density to get the weight. The concentration of a pure liquid compound is 100%.
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Recently I am doing the meatbolic profiling of flavonoid in ginkgo biloba. When selecting the positive mode in MS, i found there is a huge peak split in my BPC with 70% Ethanol as the extraction liquid.Here is my LC-MS condition.
Column: ZIC-pHILIC (4.6 x 150 mm, 5 µm particle size, Merck Sequant, Watford, UK).
Mobile phase: (A) 20 mM ammonium carbonate (pH 9.1), (B) acetonitrile.
Injection volume: 10 µL, flow rate:300 µL/min, Column temperature: 45°C, autosampler temperature: 4 °C.
Gradient: (1) 1-15 min: 20% to 95%
MS parameters: spray voltage (kV): 4.5 (ESI+), 3.5 (ESI-), capillary voltage (V): 20 (ESI+), -15 (ESI-), capillary temperature: 275 °C, heater temperature: 150 °C.
Resolution: 70,000, MS range: m/z 70-1050, full scan mode.
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Mobile phase pH, analyte purity, mobile phase combination can cause the peak splitting
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I want to evaluate chemical screening of some test samples using LCMS . Please suggest any lab in India, where I can get it done
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You can contact ---- ICAR-Central Institute for Research on Cotton Technology, Adenwala Road, Matunga(East), Mumbai-400 019.  E-mail: director.circot@icar.gov.in
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I have an Agilent LC/MS Ulitvo TripleQuad and overnight it seems as if I have lost complete signal of my peaks. Sensitivity has dramatically fallen. A technician confirmed that our MS was fine and that the background was extremely high when just running normal water and methanol (MS grade) through the instrument. They are hypothesizing that there might be salt build up throughout the instrument. I normally run my method on a gradient of 10mM Ammonium Acetate in UPW (Mobile Phase A) and 10mM Ammonium Acetate in MeOH (Mobile Phase B). All solvents used are MS grade and run on ESI + mode.
I have tried flushing the system but still see the high background. Does anyone have a suggestion for flushing out Ammonium Acetate or reducing high background in LC/MS?
Or any possible hypothesis for what the underlying issue could be? It seems to have dropped ins sensitivity overnight. I would think the signal would slowly get worse if there was a buildup of salts overtime.
Thanks in Advance!
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A few misc. comments :
  • Are you evaluating the system with the HPLC column inline? If so, remove the COLUMN and flush the system, then re-evaluate the noise and background levels using a KNOWN Standard (measure S/N ratio of the peak as "high noise" means nothing by itself, only when compared to a benchmark established for your specific system.
  • Have you had the system professionally cleaned (esp. the ESI source, but the HPLC system too, including changing the solvent pickup bottle frits and other maintenance items)? Cleaning is part of routine maintenance. Be sure to run a full tune afterwards.
  • Is your HPLC system running with a fully functional and clean degasser? Is the pump operating properly, without cavitation or bubbles? What is the pressure "ripple" (noise level, as a percentage)? **An unstable pump/flow will generate a lot of MS noise.
  • Flush the system with water and formic acid (0.1%), at a normal flow rate for your system, without the column in line (*be sure to install a restriction capillary to generate the required back-pressure or your pump will not operate properly resulting in even more noise). https://hplctips.blogspot.com/2018/04/the-hplc-restriction-capillary.html
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Looking for a high-reliability lab that can run plant samples for metabolomic analysis using untargeted approaches byeither NMR or LC-MS with follow up with data analysis (PCA/PLSDA).
If possible ideally also annotate discriminatory compounds
any suggestions?
Already came across a few amongst which creative-proteomics/metabolomics in USA although many focus more on biomedical sciences (BioAnalytix Inc, Charles River Laboratories, or KBI Biopharma).
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The Metabolomics Innovation Centre (Canada) - www.metabolomicscentre.ca
Over 50 targeted assays (with absolute quantitation) and 6 global/ untargeted assays. Both NMR and MS based services are available, including assays specifically for plants studies. All prices/ assays are available online. Fell free to contact me directly.
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I have 2 samples extracted with different solvents. One is water and one is methanol. Can I directly inject the methanol extract to LC MS analyses?
And since I have to lyophilize the sample from water extraction, journals says that I should resuspend it in methanol:water solvent prior to lc ms. On the other hand, I plan to inject the methanol extract directly to the LC MS. I am confused if it would be ok. Would having different sample preparation of the same sample be ok?
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Sample should be filtrated 0.45 or 0.2 um.
The rule is to inject in eluent or solvent that has lower or equal elution strenght that primary composition in the system.
regards,
GB
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I need to develop a method for the detection of oligonucleotide (2-5 mers in length) in liquid chromatography-mass spectrometry. It shouldn't have any salted buffers, TEA, TFA phosphates as solvents. Those are the requirements of my system. Could anyone suggest any article for such a method?
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I have performed LCMS for my tumor and normal samples. I have to use PLSDA on my data. XLSTAT has the module for performing the same, but I dont have access to the software as trial version is expired. I also need help arranging the data and selecting the variables. Can anyone provide me license for the software and help me in data arrangement.
Thanks in advance
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I have done LC-MS analysis of crude plant samples. Now I need to identify compounds from the LC-MS chromatogram. Need your help to identify compounds.
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You do not need software or have a "software" solution for your request of: "Now I need to identify compounds from the LC-MS chromatogram". The web will not provide you with answers.
LC-MS chromatograms alone are not reliable or useful to answer such questions posed or suggest next steps. The full method details, sample details, plus additional orthogonal data from other detectors would be needed for evaluation first, then the running of actual standards to make any qualitative ID's might be possible. This can not be done from the chromatograms. MS detection does not "detect" all compounds present and erroneous data can easily be generated when condition are not optimized and/or methods do not follow good chromatography practices (MS is NOT a universal detector and is completely dependent on the actual method used and the training of the instrument operator who developed it).
Please seek out some professional, local help with your project. A proper method of analysis can then be developed, optimized, data collected, standards run, data analyzed and conclusions reached.
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I would like to estimate estrogen and its metabolites using serum samples by LC-MS method. Kindly suggest me a standard protocol for estimation with literature evidence.
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Hello everyone.
Does anyone know how to eliminate ractopamine contamination into LCMS system? I have conducted several clean up procedures and ractopamine still presenting peak intsity even in a no injection mode.
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Better to call an LCMS support engineer
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Is there any software available to estimate estrogen and its metabolites using LC-MS in serum sample . Can you suggest the methodology to standardize the estimation of above mentioned hormone
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I am analysing LC-MS data. I have combined all NEG and POS mode peak intensities in a single file retaining all metabolites with higher peak intensities (appearing in either modes). Now, when I go for analysis in MetaboAnalyst, which ones should I select among:
1. Sampe normalization
2. Data transformation
3. Data Scaling
Thank you for guidance!
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Liquid Chromatography Mass Spectrometry (LC-MS/MS) is an exceedingly sensitive and specific analytical technique that can precisely determine the identities and concentration of compounds within your sample. what about your answers
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what is the difference between HPLC water and LCMS water? For what purpose are both of them used? Which is better?
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my peptide molecule having free acid and gunidine group ,How can we crystallize and what are the best solvents for crystallization ?
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( preferably Maharashtra or Rajasthan)
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Hi I am looking for some online courses to analyse data from targeted LC MS. if someone could tell me some good advice of where I could one one please.
(some pca for example etc)
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No (free or cheap) courses of which I aware. The best advice is to find a paper doing something similar to what you are doing that presents the data in a way that you find compelling. In terms of statistics, THE issue is convincing others at a glance that of the statistical range on the value(s) in question (i.e., variance or standard deviation about a mean). In terms of presentation quality plotting software, I recommend Kaliedagraph, which isn't all that expensive and has some good statistical graphic layouts. KG is limited to 2D, however. R is free, and does pretty well too, but has a steep learning curve to make presentation quality graphics. The nature of the data will dictate the presentation format you will use. Bar charts, box plots, ven diagrams, x-y-z linear, log-linear, or log-log, scatter plots (all with meaningful error bars), statistical plots. The big issues you face is that almost all the stats tools and graphing software is set up for Gaussian (parametric data). No one ever seems to check that their data is actually parametric,. however, which leads to lots of really bad conclusions. If your data is not parametric, you will need to look at non-parametric methods, and adapting the software to reflect that reality.
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Hi All,
I am using zeba desalting column to purify my protein + plasma sample in the beginning step of protein enrichment procedure to get rid of all unwanted stuff. After performing desalting process, I am going through the enrichment and digestion procedure but on LC-MS my peptide peak shape getting poor as the more number of injections injected. Initially peak shape is perfect but after injecting about 30 to 40 samples, chromatography getting poor. Earlier, samples prepared without zeba desalting have not shown any poor chromatography. Anyone have any idea about troubleshooting?
Thanks.
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Kindly, Check the estimated yield of sample in the eluate of desalting column.
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As I'm a newcomer to metabolomic data processing from the very raw data, I was wondering how to combine pos and neg mode data together, especially in the condition of acquiring from separate two-year samples.
I have two separate data acquired from two years in the same untargeted LC/MS method but different samples involved, and I want to combine them. Things turned difficult for me when I found there are some metabolites detected in different mode between the two years and not consistent in the two modes. For example, compound A was detected in year 1's pos mode but in year 2's neg mode , not in pos mode.
I'm not sure whether I can combine such compounds directly into one column and then normalize.
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Hi all,
I was wondering if anyone knows-
which statistical test I should use in order to find whether a sample is an outlier in my proteomic data? It's obvious when looking at the PCA, but how should one calculate this?
Many thanks for your help!
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The standard for excluding a single data point is typically 95% (or 98%) confidence that it is beyond the standard deviation of the other cohort samples. The problem with most LC-MS/MS studies is that the standard deviationis so wide (because n is so low) that almost everything is within 95% confidence. PCA analysis is merely suggestive of what has the most variation between the cohorts. It is rarely statistically-valid at a given confidence level and always requires focused experimental follow up with more single biomarker-focused validation effort. When this is done, all the PCA targets typically disappear as aberrations. The basic issue is that biomarker concentrations cover a range of values for each cohort, these ranges typically overlap and you ultimately need to perform an ROC analysis to identify a useful biomarker, that requires 100's of patient samples.
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There are many antibodies available now for single amino acid detection. How specific are they? Of course the company will claim high specificity, but has anyone used these antibodies in the lab and confirmed that they detect only that amino acid? Will they differentiate between related amino acids, glutamine/glutamate and asparagine/aspartate as examples? Do results using one of these antibodies and a concentration curve to quantify amino acid abundance compare to LC-MS determined levels?
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Antibody generation for a single amino acid is a huge chanllenge indeed, how to obtain acceptable specificity and sensitivity for the generated antibody is a big issue. Here is also a paper that I was ever involved to the hapten design, where the the aim is to descriminate two similiar amino acids M from A, the difference for these two is alomost only half part of a amino. Fortunately, the hapten design strategy made important role, and proved to be ok finally. Maybe this paper could be referenced for your question. Good luck!
Suarez-Pantaleon, C; Huet, A. C.; Kavanagh, O.; Lei, H.; Dervilly-Pinel, G.; Le Bizec, B.; Situ, C. Delahaut, P.Production of polyclonal antibodies directed to recombinant methionyl bovine somatotropin. Analytica Chimica Acta, 2013. 761(1), 186-93
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For doing LCMS analysis of metabolites from crude samples of seeds, what is the procedure for fixing internal standards? Do we do multiple screenings until we get a detectable concentration peak of the standard and then use that for further analysis?
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Please, cosider, also the following attachments, associated with reference [1], shown in my preceding posting:
(i) MTZ-urine-equ.tiff: It contains our innovative basic equations (1) and (2);
equation (3) is according to Arrhenius's theory;
and
(ii) MTZ-urine-dqc.tiff: It contains diffusion coefficients of MTZ in urine according to reference [1] and equations (2) and (3). As can be seen, there is excellent correlation between our theory and Arrenius's one.
Once, when there are assigned MS ions to corresponding 3D molecular structures, there is not needed employment in internal standard.
There is, in actual fact, excellent-to-exact liner correlation between two completely independent theories: Our stochastic dynamic formulas, mentioned above, and the Arrhenius's one.
Thus, 172.072 belongs to N3+-cation of MTZ, while MS peak at m/z 172.041 is assigned to N+O-cation. MS peak at m/z 171 belongs to cation-radical of MTZ.
The shown equations describe completely and absolutely mass spectrometric phenomena toward (a) quantitative and (b) 3D structural analyses of analytes.
In other words, even in complex matrix like urine, there is completely able not only to determine analyte quantitatively, but also to determine its 3D molecular structure; furthermore, exactly in chemometric terms.
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I am looking forward to determining the level of DNA methylation in cell lines and animal tissue samples. The determination of 5mC and 5hmC and DNMT1/3 are considerably important. What are the important differentiating steps for samples processing, for analysis of 5mC, 5hmC through HPLC UV/LCMS.
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Why cont we use negative ESI for peptids
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I agree with Andre Fabris. ESI is a mild/soft ionization technique that performs ionization mainly by transferring or removing hydrogens. This typically ends as detectable hydrogen adducts of the molecule at positive mode as precursor ion. Besides other adducts, this formation needs proton acceptor-bearing molecules such as peptides. Basic amino acids such as lysine arginine histidine and other nitrogen-carrying side chains of peptides make it more prone to accept hydrogens thereby ionization at positive polarization.
Take a look at this for fundamental,
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Dear folks,
I am going to work on untargeted metabolomics with Agilent QToF. Please suggest to me the best software for untargeted metabolomics.
Thank you!!!!
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These softwares may actually help you (MetFrag and XCMS).
Here's their respective links:
Best wishes,
Sabri
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Hi,
I'm looking at MaxQuant Evidence output table for my analysis and have following questions:
1. I'm interested in counting the number of peptides (including those not unique). After filtering peptides associated with the CON and REV proteins, I counted the number of peptides left. This number is way higher than the number of unique peptides reported in Summary table so I'm wondering if I'm doing the right thing?
2. Also, there're a lot of peptides in Evidence table not having MS/MS m/z values reported. What does this mean?
3. Some other peptides contained NaN values for "Calibrated - Uncalibrated m/z", "Mass error", "Uncalibrated Mass error" and "Max Intensity m/z" columns. The "Type" for all these peptides were "MSMS". Is missing precursor the reason why?
4. Similarly, some peptides whose "Type" were "MULTI-MATCH" or "MULTI-MATCH-MSMS" included NaN values from "PIF" column to "Delta score" column. Why?
5. Last but not least, there were many missing values for "Intensity" column. Does that mean that those peptides were too low abundant?
Regards,
Jenna
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As far as I know the "Type" feature only describes the spectrum type. E.g. 'MULTI-MATCH' = MS1 labeling cluster identified by matching between runs. [from: http://www.coxdocs.org/doku.php?id=maxquant:table:evidencetable]
So I assume your 13 RAW files were obtained from multiple measurements of the same sample. Or at least it seams that way if you only obtained a single evidence table. I think MQ returns the MULTI-MATCH type if you use "match between runs" for your search. Either way - I would say these peptides are valid as they were matched between multiple runs.
Are you sure these peptides don't have a precursor ion? What does "Charge", "Retention length" or "Retention time" say for peptides of the type "MULTI-MATCH". If you have values there - you detected a precursor ion for sure.
Again I would highly recommend to a address such a detailed question to the MaxQuant builders.
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I am aware that TiO columns are used to enrich phosphorylated peptides after digestion of proteins, but can they be used to purify full phosphorylated proteins from crude cell extracts? Something around 30 kDa
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Whereas It is possible
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I am a PhD student and am currently working on metabolite profiles of some marine invertebrates.
While analysing some raw data generated from LC-MS, HRMS, NMR and FTIR, I was told by some researchers that these raw data, once submitted to a journal as supporting files, cannot be used further for any other analysis. For each analysis I need to generate the raw data again, otherwise it will be treated as a case of self-plagiarism.
I can see that my raw data has a potential of producing three distinct publications. I can analyse different parts of my raw data differently to present distinct conclusions.
But generating all the raw data again from these analyses, and that too for each publication, does not look sustainable to me. And clubbing all three publications in one also does not seem to be a good option here.
So I would like to know your views on this matter as a researcher and also as an Editor/Reviewer. Also, please share your similar experiences and solutions to it.
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It depends. Much data, for example fisheries records, are published for a given purpose - for example to manage a fishery. Many years after that data was published, it may provide other researchers with other information - for example on understanding "shifting baselines". There are many very good pieces of research using historic datasets. My herbarium vouchers are a form of "data". They are lodged in public Herbaria across Australia. The Herbarium staff make the vouchers, and the associated environmental data, available to researchers around the world. They do not limit the number of times any particular voucher may be used to provide a datapoint in someone's research. Those of us who contribute these data rarely hear about their reuse unless we subscribe to platforms like Bionomia. Over a career collecting environmental data I have much that I may never explore fully. I use platforms like the Atlas of Living Australia's BioCollect platform to host my old datasets. It is possible that one day someone may use the hypersaline lake diatom and physico-chemical data from Australia to develop a "diatom metric" index of water quality for these understudied habitats... or for gnammas, or coastal lagoons...
IAs you know what analyses you plan to subject the dataset to, maybe you would be better served in clubbing a group of papers together that look at the different things you have extracted from the dataset, and then provide the entire group to one journal to publish as a "set".
But I would not like for you to have the opinion that data may only be used once then needs be discarded. What a waste of effort. Well conserved datasets, with excellent metadata relating to the methodologies and data collection, can be valuable into the future, in ways we have no current understandings about.
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If we store the sample at -20 degrees and remove it for cyanotoxin analysis after 2 days, what's the minimum time we need to defrost the sample to get stable results from LC-MS? Also after defrosting the samples we have noticed air bubbles inside the vials. Would that have a great impact on the results of cyanotoxin analysis using LC-MS? If yes then is there any decent way to remove these air bubbles?
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This will be helpful. Thank you Nayan Chandra Mohanto and Udaya Jayasundara
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I am planning to use PCA and OPLS DA for my study in biochemometrics but i quite tight on budget. I am not sure how much is the SIMCA software although they have a trial version, I am worried if I'll be able to maximize the use of the free version in my data. Are there alternatives that is cheaper or free but will give quality data analyses on PCA and OPLS DA?
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You can also use free online tools for multivariate statistics: MetaboAnalyst (https://www.metaboanalyst.ca/) or Biostatflow (http://biostatflow.org).
p.
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I've been doing co-IP to determine which proteins interact with a German cockroach protein known as Bla g 2. However, after preforming LC-MS on the co-immunoprecipitated proteins, I've noticed many of them are housekeeping proteins that are very abundant in cells. Is there software or a technique that can allow me to distinguish interacting partners from proteins that are simply abundant?
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1) Most mAb target more than just one protein.
2) Consider the non-specific binding problem. If you are seeing just high abundance house-keeping proteins, this is likely the culprit.
3) It is possible to add protein crosslinkers to the solution before IP. Note, the diluter the better. Then you can use a specific endoprotease to digest the precipitated protein and look for a high abundance crosslink. Random crosslinking will result is chemical noise in the MS. Jonathan Amster (U Ga) did some work in this area with cleavable crosslinkers so that you could sequence the resulting peptides to ID the proteins. You can use a mass defect tagged crosslinker to help identify the crosslinked peptide pairs, but you will likely have to synthesize that yourself.
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I am doing LCMS data analysis in MetaboAnalyst. Example of a metabolite..
3-Hydroxy-3-methylglutaric acid
Positive ion mode, 0.771 (FC), -0.374 (log2(FC))
Negative ion mode, 1.168 (FC), 0.225 (log2(FC))
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The difference is ionization efficiency. Some ions 'fly' better than others in the same mode in MS. There is lots of speculation about why this is so, which occasionally breaks out in shouting matches (reaching the point of fist-a-cuffs) at ASMS and other MS conferences. Consequentliy, I will leave my speculations out of this response. However, just like some ions will only be seen in positive ion mode and others only in negative ion mode, the differences in relative ionization between molecules in a single mode can easily give you the result you see. If you really want to see if the molecule is upregulated or downregulated, you can spike the sample with a 13C or 18O-analog of the compound and deconvolve the isotopic patterns quantitatively. see reference attached.
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It is my first time analyzing MS data, and I need to compare the abundance of proteins in my samples. However, the data sets I need to compare either have Area (%) or NSAF(%), which doesn’t allow comparison. Is there any way to translate one to another?
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Eef Dirksen Thank you. That's an interesting idea. I have two large data sets so it's impossible to do it for all but maybe for a few chosen ones. I will look into it.
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We are using a C18 reverse-phase column in liquid chromatography for purification and separation of oligonucleotides. However, it seems that they got crystalized inside and the column is stuck now. We have tried elution with acetonitrile, methanol and water but it doesn't work. Is there anything else that could save my column? Thx.
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Back washing of column through Acetonitrile, 0.1% phosphoric acid in water.
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Generally HPLC, we can use it for qualitative and quantitative analysis.
What is the main difference while using it with PDA or with MS detector?
What are the advantages of MS to PDA and vis-versa?
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A photodiode array detector, or PDA, is a type of absorbance detector (such as a UV detector) that is able to detect light-absorbing compounds at the PG level. However, PDA detects an entire spectrum simultaneously. UV and VIS detectors visualize the obtained result in two dimensions ( optical density and time), but PDA adds the third dimension (wavelength). The analyte or related impurities peak can be determined simultaneously during separation at all wavelengths, which is convenient for choosing the most suitable wavelength and determining the purity of the analyte or related impurities.
But you must inject a standard to make a standard curve for qualitative and quantitative analysis (without a standard curve, we can not identify the analyte)
MS Mass Spectrometer
The analytes are detected based on their MW (Mass: charge). The obtained information is especially useful for compound structure and identification (based on the Mass: charge ratio (without standard injection)). By constructing a standard curve, a very low detection limit of analyte can be quantified.
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~85% pure one of unknown impurity by HPLC shows multiple mass by LC-MS.
I run the samples in scan mode as well SIM mode in (+)ve ionization mode to confirm the correct mass but always getting multiple mass.
Retention time of impurity is 16.8 min (HPLC run time 50 min) and during LC-MS in (+)ve ionization mode mass shows at 16.8 min as 615.3 , 276.05.158.05, 146.0 with almost same intensity.
Note:
1. HPLC method is well capable to separate.
2. There is a no mass observed while run the diluent (MeOH:water 50:50) couple of times in LC-MS.
please advise.
Best Regards,
Gaurav.
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Hi Gaurav,
Which ionization source do you use in LCMS?
In LCMS sytems, extra peaks are common and this doesnt always mean that you have a big impurity.
As the ionization efficieny of analytes greatly depends on their structureand the matrix, Seni quantitative approach generally doesnt work well.
There may be several possibilities:
-The impurity may ionize more efficiently than your analyte.
-Dimer, trimer formation may be occured.
To clarify, you can change the gradient and check if retention time of unexpected ion changea or not. Secondly, you can use isotope labelled standard to observe the possible adducts of your analyte.
Regards,
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Hi colleagues. I got trouble in processing LC-MS data for El-maven. Particularly, I am extracting the isotopologues from LC-MS data acquired by SIM mode (2 targets). because I cannot do the smoothing process for the peak, so the peak picking step seems not really reliable. Could you please let me know how we can do smoothing in El-Maven?
Appreciate your help.
Best
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I want to specifically target Flavonoids present in my chloroform plant extract. Is there any specific solvent or method through which I could focus on flavonoids mainly?
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In my humble opinion, it is better to HPLC and LC/MS. The GC/MS does not really favorable results for flavonoids and phenolic compounds. It is essential to prepare the respective standards solutions and draw the calibration curve before starting the test. It is strongly recommended that it was prepared some prototypes and the result obtained were compared with principal samples/analytes.
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I am a PhD student from Nigeria and am working on nutritional interventions against heavy metal toxicities. I enriched polyphenols from some plant extracts and i wish to characterize the polyphenol contents of these extracts using LC/MS or other appropriate technologies. Please can anyone refer any laboratory that accepts international samples for such analysis? I am also open to collaborate with scientist that are expert in doing such types of analysis. Thanks
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Dear Francis, apparently it helps reading you original question carefully. You can e.g. search the internet for terms like "LC/MS service in Nigeria". This provided for example the following address in Lagos:
SGA Analytical Chemistry
I also suggest that you search the various departments of your own institution, the University of Port Harcourt, for researchers who perhaps have LC/MS facilities, e.g. the Department of Pharmaceutics and Pharmaceutical Technology. See e.g. the following potentially useful article:
Gas Chromatography-Mass Spectroscopic (GC-MS) Analysis of n-Hexane Extract of Lentinus tuber-regium (Fr) Fr (Polyporaceae) Syn Pleurotus tuber regium Fr sclerotia
(please see attached pdf file)
I hope this helps. Good luck with your research!
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I will be buying a reference standard fpr my LC MS analyses. Most of he standards in sigma aldrich is available in 1 mg only. I am not sure if this is enough for my LC-MS analyses. At what concentrations should reference standards be injected in the LC MS? I will be using the ff standards: quercetin, apigenin, caffeic acid, pcoumaric acid, syringic acid, isoorientin.
This is included in the procedure :
An injection volume of 100 μl will be used for all standards and samples (prepared with 1.8 mg of extract dissolved in 1 ml of methanol-water 1:1 ratio).
It is not clear to me whether the concentration of the standards be the same wit the sample extract. Thank you!
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Can collision gas be used in regular LC-MS (not any tandem MS) ?
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MS is a technology that identify substances by their molecular mass. To do so, the substance is first ionized and then fragmented into small fragments by collision with an inert gas. The mass detector at the last chamber of the MS will detect the mass of each fragment that arrives at the detector.
This is an introduction to MS, from which we understand that for sure we need collision to happen in every MS (tandem or single).
I am not aware of an MS that detects only molecular ions (parent ions) and identify substances accordingly. Many substances share a similar Molecular mass which makes it not differentiable in the MS without another criterion for identification, which is why fragmentation is used to make unique fragments for the purpose of identification of different substances in one sample.
Who said there is no collision in single quad MS??
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When removing the outlet check valve from the Thermo Horizon LC pump head about 1 mm of the tip stayed in the LC pumpe head. It sits very tight due to the peak seal sticking the fragment to the pump head. Any ideas on how to remove this?
Thanks in advance.
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Thanks, Alexey! I got it now with a tiny drill. Silly thing though! Take care!
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Hello every one
I'm trying to quantitate essential amino acids in serum or DBS by Lc-MSMS(Quadrupole ABsciex 4500 ) .
by searching the net for trusted protocol , all protocols that published employ labeled amino acids internal standard (which I don't have) .
please any one can share a trusted FIA- or LC- MSMS protocol that employ external amino acids standards.
thank for any suggestions
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Hi Hayder,
The answer depends on what amino acids you are wanting to analyse. As a general method might not be applicable to all amino acids, if you have found methods that meet most of your needs but it is only the internal standard that you are stuck on then that is different.
I would suggest using a method you have found and using a generic internal standard. Labelled IS can be quite expensive but generally are much better as it goes through the same extraction.
Amino acids are generally polar and a couple of generic ISs that I would suggest are gemcitabine (cas number: 122111-03-9) and indomethacin (cas: 53-86-1). These may be a good starting point but they do come with some drawbacks. I would use a concentration around your mid QC level to start and adjust it from there.
You will need to make sure you perform a matrix effect experiment comparing your IS to all your analytes. This will allow you to check that the IS is suitable. A good IS will pass through an extraction in the same way as your analytes of interest. So this step is crucial to ensure that you dont have excessive variability and problems reproducing data.
I hope this helps
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I want to extract the T6P (Tre6P) from plant leaves. We have LC-MS-QTOF in our lab instead of Q1 or Q3, so could anyone please help guide me about the method used for the T6P metabolite content via the QTOF only?
thanks in advance
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I need to study about LC MS since I will be using it for my thesis. I know nothing beats practical application but physical classes are not going to start anytime soon i need to equip myself with proper knowledge. Please recommend me any books that i can study as a beginner to be able to grasp the basics. thank you!
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Dear Dr Duri Eun,
I highly recommend you to read this interesting book (in attached), since it summarizes all what you have to know, to improve your knowledge about Liquid chromatography-mass spectrometry, which will help you to perform this technique correctly.
Best wishes,
Sabri