- Maximilian Blum added an answer:11Acetone as an alternate solvent to acetonitrile for small molecule analysis by LC-MS/MSAs you are all aware of the global shortage of acetonitrile a few years ago, i felt it is the high time for everyone of us to look for alternate solvents like methanol or acetone for SMALL MOLECULE ANALYSIS by liquid chromatography tandem mass spectrometry to prevent such shortages ahead. Apart from methanol, acetone possibly could be a suitable alternate solvent for LC-MS/MS analysis. i have read very few articles in which they replaced acetonitrile with acetone for LARGE MOLECULE ANALYSIS by LC-MS/MS. we are planning to develop bioanalytical methods using acetone for small molecules although acetone is non-polar, which is not suitable for RP-HPLC and highly inflammable solvent (LC-MS/MS requires the application of different cone voltages) compared to acetonitrile. i would like to know whether anyone have ever tried acetone for small molecule analysis by RP-HPLC-MS/MS or just share your inputs on this.
Acetone and THF are not compatible with several columns, for examble polysaccharide coated columns. With ACN and MeOH you have a more widespread application.Following
- Vikas Kumar added an answer:3How can I relate FeSO4 curve with standard and extract?
I hav taken absorbance of FESO4 at range of 2.5-200 uM which show linearity with R2=O,98...then i took absorbance of standard/extract at different conc .....how to get FRAP activity...show me calculation?.
Yeah..it works..bt there is a confusion regarding the formula o.d test/o.d standard*FRAP value of standard..which is 2000 for 1000uM..bt i use different concentration of Feso4 from 2.5-20 uM only...then wat FRAP value i use..will it 20...??Following
- Khoirotin Nisak added an answer:3What is the best method for bioanalysis of glimepiride in human plasma?
i want to ask what is the best method for bioanalysis of glimepirid in human plasma. thank you in advanced.
thank you for the help dear Narong chamkasem and prof. Gun.
- Hermann Mascher added an answer:5How can one quantify the levels of neurotransmitters in the rat brain using HPLC or any other method?
I want evaluate the levels of dopamine and possibly other neurotransmitters in the substantia nigra of the brain
Luis gave are fine detailed answer! Supplementing: We used HPLC-MS/MS for about 15 different neurotransmitters from different brain regions with ESI pos or neg measuring to extremely low levels. HPLC-MS/MS has a much faster stop and go time as HPLC-ECD!Following
- Tigran Margaryan added an answer:6Which criteria are to be set for duplicate curve in small molecule bioanalysis by LC-MS/MS?
We are doing small molecule bio-equivalence studies by LC-MS/MS. Currently we are using one calibration curve per each run, but just now we are changing the procedure to have two curves (separate sample extractions) and inject them at the start and end of the run to compensate between differences through run. We currently have criteria for curve required by FDA 2013 guidance for bioanalytical method validation, but which criteria will you suggest to add for difference between two curves, criteria that would be meaningful and at the same time easy to calculate for everyday use.
Also please share your experience if you are currently using 2 calibration curves.
Thanks in advance,
Guanawan, material compatibility guide doesn't fit within this question .
Emea guidance doesn't say anything about acceptance criteria . please read your suggestions before making it!Following
- Nicolas Guillemin added an answer:5Is there a better system to quantify proteins/nucleic acids than the QuBit Fluorometer (LifeSciences) (Nanodrop is too expensive) ?
I need a fast system able to quantify nucleic acids and proteins, in different samples. I was thinking about Nanodrop but it is too expensive. I saw the Fluorometer from LifeSciences, and I would like to know what can be better ?
Thank you for your answer, it is very helpful !Following
- Charandeep Singh added an answer:3How do you measure oxalate and citrate in the blood or urine?
A simple method for the measurement of Oxalate and Citrate in the blood and urine.
I would say use LC-MS, its the simplest method and at max will take 20 mins per sample. You can determine all these metabolites together in one injection.
- Uma Nath Tripathi added an answer:2How might I quantify epsilon-aminocaproic acid by HPLC?
Can anyone have method/reference to quantify epsilon-aminocaproic acid by using HPLC specially without derivitization method.
thank you Praveen.
- Jithu V James asked a question:OpenHow can we measure Labile iron pool in stored tissues?
- Hermann Mascher added an answer:2Measurement of Glucagon using Dried Blood Spots (DBS) on filter paper?
Hi, Does anyone know if it is possible to quantify Glucagon using the DBS technique? Thanks.
From my point of view in principle it should be possible BUT peptides with basic amino acids always have problems in recovery. So the question of a fitting internal standard is important!Following
- Joseph Unsay added an answer:2How do you calculate concentration of fluorophores in a single cell?
We want calculate the absolute amount/concentration of GFP in cells and I wanted to know if anyone has experience with a specific method. The current option we were thinking about are:
1. Quantitatve Western Blot - because this might be simplest and easiest. But this is a bulk measurement and to be as accurate as possible, we were thinking of a method for single cell
2. Flow Cytometry - There are some available calibration beads (from Clontech) that can gives a conversion from fluorescence intensity to molecular equivalents of soluble fluorophores (MESF). I was just wondering if this MESF is equal to molecule number? If this is the case, do I simply need to divide the MESF with an average cell volume to get an estimate of the number of particles per cell?
Any other suggestions?
I forgot to add that the GFP is in the mitochondria and that we are currently using FCS for this, but we wanted to check it with another standard method for looking at protein concentration.Following
- Arezoo Mohajeri added an answer:2What is the unexpected second peak in my Bioanalyzer result of amplified libraries?
The results of quality control for one of my amplified libraries showed two peaks. The one at 200 was what I expected but I don't know what is the second one and if that might cause problem with sequencing?
That was interesting point. I am certain that i didn't use too much DNA for library prep. But that is a good idea to check for the double stranded DNA. Thank you.Following
- Francesco Gai added an answer:1For Aflatoxin analysis, what is the best mobile phase using LC-MRM method to achieve the best sensitivity?
I am trying to develop a method to achieve 1-10 ppt level detection limit using AB SCIEX 4500, but following the published method can not achieve this goal, anyone has any experience on this area?
Dear Lingyun, I am not an expert on mycotoxins but if you contct me by e-mail firstname.lastname@example.org I can give you the contacts of my ISPA CNR colleagues that are worldwide expert on mycotoxins detection! Sincerely FrancescoFollowing
- Mostafa Darwish added an answer:4Does anyone know how I can determine Tetraethylammonium in plasma and urine?
Tetraethylammonium(TEA) substrate of organic cation transporter in proximal tubules in tubules (OCT2)
have you any paper containing the procedure for the derivatization of that compound??Following
- Ethan Den Boer added an answer:3Can anyone help me for the designing of bioanalytical method validation for pre-clinical study?
Bioanalysis for pre-clinical study.
As Fabrizio says, first determine what you want to measure, in what and why.
Then before asking questions, read the FDA guidelines on bioanalytical methods and some papers on method validation so you get an idea of what should be done.
When you have a (partially) formulated plan, come back and we can help you better.Following
- Gerrit Randau added an answer:4I almost can't see any miRNA fraction on the smallRNA-Bioanalyzer-electropherogram, shall I try to construct a miRNA-library (NEB-Kit) anyways?
on the left there is the reference profile from Agilent - on the right there is my sample - I don't know if it's realistic to see a clear miRNA-fraction on the Bioanalyzer.
Thanks for that advice, Belinda - the enhanced gel view did not show much either. I will prepare librarys with different samples now, including one of the critical ones in comparison, maybe it will work as well - like in your case, I will let you know!Following
- Biswapriya Biswavas Misra added an answer:3How should I test SNPs enrichment in a list of loci?
I have a list of SNPs and I want to test their enrichment in a selected loci from human genome. I know it is usual to do fisher exact test (one-sided) or alternatively hyper geometric test to show enrichment, but I would like to use the simulation tests to do this. I suppose simulation tests have greater efficiency in this regards.
I know Monte Carlo test could be implemented for this job, but I dont know how to do this! Is there a tool or R-code for doing that?
Does anybody have experience on performing Monte Carlo on human genome data?
Any comments would be appreciated greatly.Following
- Branislav Šiler added an answer:4Is it possible to perform fragment analysis of SSRs through Agilent Bioanalyzer 2100
I would use DNA 1000 LabChips. I couldn't find articles suggesting the usage of this technology to analyse variation of alleles in populations.
Ramesh, I've done a lot of analyses on this device so far with some dominant, highly variable markers. Aside the claim from the specification sheet about the resolution of 5 bp, I have to say I've obtained highly comparable results with general sizing error of up to 2 bp. Furthermore, Lab-on-a-Chip is also based on capillary electrophoresis. In my research, I'm going to use 3, 5, and 6-nucleotide repeats. Will I improve confidence by putting an internal standard in, tracking its shift and consequent application to the loci peaks?Following
- Arunas Ramanavicius added an answer:4How can we identify presence of diclofenac in Biological Product..?How can we identify presence of diclofenac in Biological Product..?
Mass spectrometry, publications are attached.Following
- Farhana Mokhtar added an answer:5How can I prepare a standard at lower concentration (in ppt level) from ppm level?
How to prepare standard at lower concentration (in ppt level) from ppm level? Is it relevant to do calibration curve at lower range(ppt) or high range(ppm), if our target compound available at lower range(ppt) in environment?
Thank you Ethan Den Boer and Yuan-Yuan Zhao for the brief comment and explanation... it really helpful in my study..i will try to recheck before rerin back the standard calibration.. thank you once again for the information..:)Following
- Sunil Kumar Yadava added an answer:3What is the criteria for selection of internal standards for the bioanalysis of drugs?
I am planning an in-vivo study of my developed new formulation of ciprofloxacine. For this purpose I am little confused about the selection of a suitable internal standard. Please suggest the most suitable internal standard for the ciprofloxacine hydrochloride.
thanx to every one for nice and useful suggestionsFollowing
- Carlton Hoyt added an answer:5How can I homogenize cardiac tissue?I'm trying to develop a method for quantification of drugs (small molecules) in cardiac tissue with LC/MS-MS. The first step is homogenization with ultrapure water with a gentleMAC dissociator (rotor-stator homogenization) followed by a protein precipitation (PP). However, I'm in doubt whether the homogenization and PP adequately disrupts the cells, so I am measuring the total concentration of the drugs. Do anyone have any experience with quantification of drugs in cardiac tissue (or similar tissue)?
The GentleMACS is a tissue dissociator, not a true homogenizer. It's meant for the dissociation of tissue into viable cells, so I'm not surprised that it wouldn't provide good cell lysis.
For a tough tissue like cardiac tissue, I would recommend a bead mill style homogenizer. The most important things to look for are units that would meet your needs for sample size and throughput. You can find a lot of options here: http://homogenizers.net/collections/bead-mill-homogenizersFollowing
- Jess Loh Swee Cheng added an answer:3How stable is RNAstable?
We've tried several times to send RNA samples in RNA stable but with very bad results. Initially we dried samples in laminar hood. It took more than overnight, the samples did not look completely dry and bioanalyzer results were really bad. We then tried drying in a speedvac, which took about one hour, again some samples did not look entirely dry and bioanalyzer results were not sufficiently good. We thought that our mistake was that we didn't pre-run the speedvac. So, the third time we did that, samples were completely dry in about 20 minutes, and bioanalyzer results were better. Last time we did everything "right" but bioanalyzer results were very bad again. Any ideas?
Hi, I would like to suggest you to be careful on these steps:
1) To incubate sample for 5 minutes in RNASTABLE
2) To resuspend the sample (sample is yellowish in the tips of the tube) without producing bubbles
3) To wipe the vacum with RNAse Away or RNase Zap, to dry without the use of breathable membrane (I tried to use the membrane but my 20 uL sample takes forever to dry..)
4) After speed vac without heat, immediately close only 2 tubes and sealed them using parafilm. Other sample tubes continue to be dried (environment contains moisture, while closing and sealing the first 2 tubes, others are to continue to speed vac). Repeat till all samples have been sealed properly
5) Keep all tubes in moisture barrier bag as recommended
6) Keep at room temperature in a dessicator
7) To resuspend the sample, I incubate the sample with DEPC-treated water for 5 minutes and mix without producing bubbles (again, you will see sample in yellowish inside the tip)
Hope these will help, all the best~Following
- Nick Schaum added an answer:6How to test the RNA integrity?
Is there any other ways of testing the RNA integrity without using bioanalyzer or denaturing gel?
Has anyone tried bleach gel before? I tried it but it doesn't work well.
Wondering if anyone is able to help me troubleshoot
Without more details it is hard to troubleshoot, but I can give you my protocol:
I make bleach gels with 1% bleach v/v, 1% agarose w/v, 0.01% v/v SYBR Safe in 1X TBE, and run 1ug RNA from a variety of mouse organs for 15min at 100V in 1X TBE.
I isolate RNA with TRIzol (1ml per 50mg tissue, double for pancreas) typically on whole organs, or in the attached image, 30mg. Phase separate (phase lock gel makes this much faster and easier) with 1/5 volume of chloroform per volume TRIzol. I've found that mixing the aqueous phase with a half volume of isopropanol and transferring to an RNeasy Mini column gives the cleanest RNA with identical integrity, although your total yield will be a little less compared to manually pelleting and washing. After spinning through RNA/isopropanol mixture, 700ul RW1 --> 500ul RPE --> 500ul RPE 1min. Transfer to new collection tube, 1min centrifuge. Elute with an appropriate volume of water based on your sample (incubate water on the membrane for 5min prior to spinning to improve yield). Spin through and optional re-elute with eluate to increase RNA concentration. I found that incubating resuspended RNA at 42C for 15min evaporates any residual EtOH, although this should generally not be required (I used this for white adipose tissue RNA, which was floating out of my gel well).Following
- John Hildyard added an answer:3What can I do to make my qPCR results "solid"?
Hi everyone, I am working on detecting expression levels of genes among sorted tumour samples (by FACS).
I sorted the 1st tumour into PBS. Bioanalyzer showed to me really bad RIN numbers in these 4 RNA samples extracted from sorted fractions. However, I still used them to run a qPCR (Taqman gene expression assay). I got expected results in all 15 selected genes.
After that, I started working on optimising method/protocol to improve sample RIN by using cultured human cancer cell line. 2 months later, I could consistently get RNA samples with RINs between 7.5-9 from sorted cancer cells. Then, i went back to human tumor xenografts. Samples prepared from both the 2nd and 3rd tumours demonstrated very similar story in the expression all these 15 genes. So far, qPCR results from 3 tumours showed me quite constant results in all selected 15 genes.
However, there were no RNA could be detected by Bioanalyzer in all samples prepared from both tumour No.2 and 3. Today, I re-checked these samples by Qubit. It gave me completely different results comparing to NanoDrop. RNA could not be detected in all samples by Qubit either.
I am really wondering how come i can get consistent qPCR results from samples either with bad RIN numbers or with undetectable RNA levels??? (I setup qPCRs by using sample RNA concentrations provided by NanoDrop).
If all results I got came from some kind of random amplification, how come it consistent in all 15 genes? If these qPCR results are usable, what can I do to make these qPCR results solid?
Thank you very much! I look forward any suggestions/advices!!! Thank you :)
How much RNA are you using to prepare cDNA? (ballpark figure is fine)
How much of this cDNA are you using per well in qPCR?
What sort of Cq values are you obtaining?
Are you including no template controls?
Are you perfoming melt curves?
Have you checked your PCR products are of the correct size (say, via gel)?
Have you generated a standard curve?
How are you normalising your data?
Answers to all of these questions would help identify whether there are any problems with your experiments.
When performed correctly, qPCR can be enormously useful, but there are a lot of potential pitfalls to be wary of (and frankly, my first response to getting "exactly the result I was expecting" would be one of extreme suspicion, though I may be getting cynical).Following
- Parul Jain asked a question:OpenTotal RNA extraction?I am extracting total RNA from leaf samples using Purelink Plant RNA Reagent, followed by DNAse treatment and RNA clean-up according to Qiagen RNeasy kit protocol. On analysing my samples on bioanalyzer, the RIN is more than 7 and I get sharp peaks for 18s and 28s, but peak for 18s is higher than for 28s, 28s/18s ratio is less than 1.0.
Has anyone else faced the same issue and any suggestions about how to get over this? Or would this sample be good to make cDNA?Following
- Abhinay Ramaprasad added an answer:2Assessing integrity of plant RNA with Agilent 2100 Bioanalyzer: What is the meaning of RIN = N/A?Quality control tests of total RNA of some of my plant samples revealed no RIN (RNA integrity number) values, i.e. "N/A", while other samples got good RINs (between 8.9-10). The RNA concentrations were between 331-487 ng/µl (measured via NanoDrop and Qubit). What's behind a 'no' RIN value?The Bioanalyser software sometimes makes mistake in recognizing the two rRNA bands either because of very low/high concentration of input RNA or if your samples has many other RNA peaks (not necessarily degraded RNA). This causes it to miscalculate or report RIN as N/A. Check if you are loading within the RNA concentration range as specified in the kit. But I would rather go by visual inspection than the RIN number. If you are getting two neat rRNA peaks ( you could also compare this with the samples where you got RIN 8.9 and 10) and low MW peak of degraded RNA (if any) of very low concentration, you are good to go.Following
- Paola Ibelles added an answer:2Can you help me with identification of proteins in HDL?I have obtained by ultracentrifugation a band of HDL, mainly HDL3 characteristics, with a protein concentration of 10g / l. Can anyone advise me on the steps of how to identify these proteins?Some strategie for characterization could be the chemical and protein composition and the size distribution. You can try this approach with the methodology described in this paper. There is also a lot of methodologys for to analyze their functions as antioaxidant, at the cholesterol metabolism an more. Is up to what you are more interested on the papers that you could read.
Decreased activity of lecithin:cholesterol acyltransferase and hepatic lipase in chronic hypothyroid rats: Implications for reverse cholesterol transport. Molecular and Cellular Biochemistry 246: 51–56, 2003.
Palmitic acid in HDL is associated to low apo A-I fractional catabolic rates in vivo. Clinica Chimica Acta 378 (2007) 53 –58
Enzymatic assessment of cholesterol on electrophoresis gels for estimating HDL size distribution and plasma concentrations of HDL subclasses. Journal of Lipid Research Volume 51, 2010
- Gellert Karvaly added an answer:2What's the optimal approach to assaying 25-hydroxyvitamin D2 and 25-hydroxyvitami D3 in human blood by HPLC-UV?I have found HPLC applications employing normal as well as reversed phase separations for monitoring the blood levels of 25OHD2 and 25OHD3. I wonder what the routine users of such D-vitamin assays think is the optimal approach to developing a suitable method. In addition, I would appreciate having the opinion of colleagues who use commercial HPLC in vitro diagnostic kits for this purpose on the utility and potential disadvantages of these kits.Thank you indeed for your comment. I am aware that LCMS would be the first choice but unfortunately it won't be available for this project. There had been lots of publications describing the use of UV detection prior to the LCMS era, though. I expect some people had been using some of those stuffs and can share their experience.Following
- Krishna rao Dara added an answer:1How can you quantify doxorubacin liposomal formulation by using LCMS/MS?Extraction procedure for liposomal doxorubacin, total doxorubacin,normal doxorubacin and its estimation.The separation was based upon the selective retention of liposomal (encapsulated)
doxorubicin and non-liposomal (free) doxorubicin on the hydrophobic
reversed-phase Thermo Scientific™ SOLA™ HRP solid phase extraction
(SPE) cartridge. The former exhibited no retention, while the latter was
retained on the stationary phase and eluted with acidified methanol.After separation, each individual fraction was quantified by UV (254 nm) also possible. You can contact me further details on +91-8980045413.Following
Estimation of drugs in biological fluids