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Method Development - Science topic

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The ideal training would be an emphasis on theory and de novo method development. The person I have in mind for this has years of experience running methods, maintaining and troubleshooting. TIA! Mark
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I am optimizing the method of developing natural deep eutectic solvent by using different methods, molar ratio and water content. For heating and stirring method, I used 1:1 Choline chloride:1,2 propanediol. The combination does not form liquid either direct heating or in water bath up-to 2 hours. The temperature is below 100 degree C. I tried to add minimal amount of water (less than 50%). However, the combination does not form homogeneous solution. It only form clear homogeneous solution when I put more water (160%) and its stable at room temperature. My concern is some journals mention the excessive water content may disturb the hydrogen bonds between Choline chloride and 1,2 propanediol. I tried to rotavap to remove water content and it starts recrystallising again. I repeated the experiment by using total dried Choline chloride (freeze dried) after reading some suggestions from previous researchers that had similar issue that caused by wet Choline chloride. However, I obtained similar results 😢. May I have some other suggestions or opinions that may solve the problem? Thanks in advance
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For those arriving at this question: to properly answer this question you have to measure the solid-liquid phase diagram. It will tell you the maximum solubility of choline chloride in 1,2-propanediol at a certain temperature (e.g. 30 ºC). Probably a choline chloride mole fraction of 0.5 (1:1) is higher than the maximum composition at the temperatures you work.
Adding water increases the solubility (lowers the melting point) and can be a solution. Of course also within the thermodynamic limits.
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If yes, then what are the factors that need to be taken into consideration?
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I am developing a gradient method for the estimation of 3 drugs, two are water-soluble and one is hydrophobic. I am using Dionex HPLC system equipped with P680 HPLC pump, ASI-100 automated sample injector, UVD340U detector. The mobile phase is composed of 10mM KH2PO4 buffer pH 6.8 and ACN. A gradient is applied from 97:3 (or 90:10, depending upon the column used) Buffer: ACN up to 5 mins, followed by 50:50 buffer: ACN up to 16 mins, followed by 97:3 Buffer: ACN up to 20 mins. Columns tried are Inertsil ODS 3, Inertsil ODS 3V, Eclipse XDB c18, Phenomenex Gemini C18. Samples prepared in 50:50 Water: Methanol.
After 30-50 injections, the pressure rises to 170-200 bars (initial pressure 96-106 bars). After back flushing or washing at 50 deg celsius using 60:40 ACN water, the pressure reduces. However, again with increasing the aqueous content beyond 60%, the pressure starts rising and the peak shape is also not good. What could be the reason?
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In my opinion try to use Methanol instead of Acetonitrile.....
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Hi, I need to prepare a cholesterol stock solution to run in RP/LC-MS for identification and method development. I have 100mg of powder in a glass tube. What is the best way to prepare it? #methoddevelopment #cholesterol #lipidomics
Thank for your time!
Cheers.
Cagakan
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Search first for analytical method for cholesterol to analyze the sample through LC-MS.
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Hi,
I need to develop above stated method. But I do not find any specific test method on how to quantify this surfactant in coagulant or latex.
Can you all share any info regarding this method development.
Thank you.
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I found an article that might interest you and the approach used for the quantification of sodium dodecyl benzene sulfonate seem interesting.
Best wishes,
Sabri
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What kind of software or what kind of method could be used to manage the huge amount of paper, so that you could find whatever paper you have read fast.
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I suggest Mendeley and Endnote. But Mendeley is user friendly software.
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I am having baseline issue when following the method outlined in a application note from Agilent. The application note references a method that utilizes a gradient in the attached image.
The whole application note can be found by searching for 5994-3791EN.
I do have a binary pump instead of the quaternary pump, but otherwise there is very little difference between my instrument's config and the reference method that would significantly effect afaik. If Agilent's newer software corrects for the baseline issues seen maybe that could be the answer.
I also have the older Agilent DAD from the 1100 series if that is relevant.
What could possibly be the root cause of the difference in baseline seen here?
The transition dip is seen at other gradients of these same two mobile phases.
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I am trying to develop a method for quantification of cinnarizine (1-trans-Cinnamyl-4-diphenylmethylpiperazine) in various pharmaceutical products using the fluorescence spectroscopy since UV/Vis method I developed isn't sensitive enough for that purpose. Cinnarizine is cinnamic acid derivative and it is already known that cinnamic acid is susceptible to photoisomerization (from trans to cis form).
I prepared sample of cinnarizine (0,5 ug/mL) using 70% methanol : 30% water solvent and set excitation wavelength at 256 nm (Perking Elmer LS55 instrument). After irradiating the same sample every ten minutes at mentioned wavelength, intensity of the fluorescence emission (309 nm) decreased continuously decreased. I tried using other wavelengths for excitation and other solvents for preparation of the samples but this "problem" remains. I assume that photoisomerization from trans to cis form is going on (or maybe photodegradation)? There are several reported methods for quantification of cinnarizine using the fluorescence spectroscopy (or using the HPLC with fluorescence detector) but none mentioned this problem. Any advices for solving this problem?
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I think looking at the overlay spectrum photodegradation is taking place rather than photoisomerization. You may check the fluorescence behavior of cis and trans isomers in isolation if available with you by following their kinetics.
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Hey fellows,
I am getting into LCMS method development. I need help in sample preparation like how to do complete validation. Once calibration range is established, I heard people divide samples into small quantity and store in -80 ( in case of biological matrix), then they do validation and other studies (selectivity, precision and analysis, freeze thaw and other studies).
Can anyone help me with protocol on how much quantity should I store for each study. Say for each run my sample size is 100 microgram/mL solutions.
Also, to test stock solution stability, I have prepared stock solution of 1mg/mL, how do I proceed to test stock solution stability. What concentration should I proceed, is it 1mg/mL or something less, because I cannot use 1mg/ml in LCMS.
As for as system suitability study, what is the best concentration once can choose and what are the parameters one look using system suitability solution, while validating LCMS method.
Any help is appreciated. Thank you.
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Dear Bhimanna Kuppast,
Thanks for your interest. I am working with LC- MS since 2011. My research area is: development and validation of analytical methods for pesticide residue determination in different agricultural commodities. For the development of method, first of all, you have to follow the international guidelines for which compound, you are interested to develop and validate the method. For example, for the development and validation of LCMS method for pesticides and veterinary drugs, you may follow the EU guidelines: SANTE/12682/2019. Additionally, you may read several scientifically sound articles published by a very good quality journals. If you want , you may visit the following LCMS based articles:
DOI 10.1002/jsfa.8716
DOI 10.1007/s12161-016-0537-z
DOI 10.1007/s12161-014-9898-3
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My study involves establishing chronic nicotine addiction in mice and I would to check the cotinine level in mice urine using HPLC but currently protocols I found are done using human urine. Bioassays and GC are expensive, and I would like to use urine as the sample. Thus, how do I modify the HPLC protocol for mice urine?
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I have no practical experience, however, I hope the provided link will be very much helpful for you. Please have a look on the following link:
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I am new to SEC and developing this workflow for primarily separating Abeta oligomer from mice brain. However, there are other proteins of interest down the road. I was wondering if anyone can suggest some must-read literature for a beginner.
Thank you for your time!
Best - Navid
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In addition to SEC methods (some have poor resolution), many methods using a C18 or C8 column with wide pores can also be used to retain and resolve many of the key peptides too. When trying to work with clinical Amyloid Beta peptides, the matrix and "other" compounds which are not of interest will guide your separation goals. Try several different modes of chromatography to survey what you have, then let the data point you in the right direction for what to try next.
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Currently, I am working with RAW BLUE cell lines to check the SEAP expression on addition of TLR ligands? Method developed for different TLR ligands. How experiments can design for validations? (parameters).
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Dear Alexander , Thank you so much for the article.
May I ask, I read some articles about positive controls drugs (tereparatide injection) test with RAW Blue cell line. Will those drugs stimulate cytokines expression or any Optical density in the assay?
My thought, is these drugs should inhibit cytokines release and OD should match with blank? Am I right?. Or high concentration of drugs can kill the Raw Blue cells?
Kindly share your thought....
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Hi,
I am at the end of a method development, and validating the extraction and clean up of PAHs from high organic matrices.
I have had consistent high recoveries of deuterated compounds. Now validating with NIST standards, and suddenly very atypical results, so instead of 20 ug mL of each compound, they quantify at 30, and linearly increase to 130 ug mL-1. I am injecting with 20 ug of deuterated. Has anyone seen this before. GC-MS has been well conditioned.
Appreciate any insight - given expense of the NIST standards.
Thanks for your time in advance.
Best regards,
Cal Leech
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Thanks a lot for your interest to work with GC-MS. I have some experience to work with GC-MS for the quantification of pesticides residues. I think, this variations related to the preparation of standard and the sample as well, it is not related to the instrument. I suggest you to prepare the standard properly, and I advice you , please do not prepare the standard following serial dilutions, prepare the standard from one working standards solution. The another important issue is matrix. If you are using matrix matched standard, then you have to be very careful to mix with the matrix properly, otherwise a large variations may come. Thank you for your interest.
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I run the sample preparation using AOAC Vit A,E,D method, that require saponification, concentration , drying using nitrogen gas, reconstitute with methanol. Since the HPLC method development is on the way, so I keep my sample in -20 degree Celsius. Some researcher suggested 4 degree Celsius storage. What do you recommend?
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According to my findings, the temperature does not have to be more than 20 C.
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Hello,
I am suffering from some carry-over problems with HILIC seperation of amino acids in urine. Specifically, basic amino acids arginine, lysine and cytathionine.
System setup:
Sciex 6500+ LC-MS/MS
MPA: 200mM ammonium formate, adjusted to pH 3.65 with formic acid.
MPB: 0.01% formic acid in MeCN.
Needle wash: 90/10 MeCN/water
Sample prep: dilute and shoot, 1 part urine mixed with 2 parts acidified MeCN with 0.15M HCl.
My major issue here is carryover after the highest calibrator for these analytes. Carryover is ~30%.
Gradient starts at 90% MPB and drops to 0% over 13 minutes and a hold for 5 mins at 0% MPB. Isocratic holds at 10-40% MPB don't help remove the carryover. Longer holds don't seem effective either. I have run "double gradients" to eliminate the injection as the source of carryover, and on the second iteration of the gradient, sans injection, the peaks are still there, so that tells me the carryover is on the column itself.
Adjusting pH from 2.6 to 6 did not make a significant difference. Given the pKa's of these compounds in the 1 or 2 range, I am not surprised by this.
I started with 100mM ammonium formate in MPA and bumped up to 200mM which helped remove some of the carryover, from the assumed increase in ionic strength. I am struggling to go over 200mM without perturbing the other analytes in this method.
I am looking for ways to modify the method, sample diluent, or mobile phase to try and remove the carryover.
Any input to solve this problem is greatly appreciated.
Best regards
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This might be to late but since you are doing HILIC, and the strong solvent is water in HILIC, you have to change your needle wash to contain much more water. Just make sure you have as little water as possible “stored” in the needle for the next injection.
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Basin lag times can be calculated by the CN Lag Method; which is based on the hydraulic length of the sub-basin, CN value, and basin slope. Some paper says,The CN Lag Method was developed for sub-basins less than 2,000 acres (approximately 8 km2 ). It is such a small area. My question is, is it feasible to calculate basin lag times by CN- method for larger areas? if it is, then up to what extent?
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Like all empirical formula, the CN method for calculating lag time should only be used within the parameter range used to derive it. Having said that, when compared to other methods for calculating lag time (all of them empirical), the CN method has a better physical basis - in that it is based on the time of travel of water along various flow paths. So, there appears to be no compelling reason why it could not be used for larger basins. I found that the CN method is very reliable and used it for basins as large as 20 km2.
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Please I need a questionnaire to measure one of my research questions for my thesis.
My study is a descriptive study to analyze to what extent do nurse educators employ inquiry based learning, and i am focusing on the relation between inquiry based learning on the development of students critical thinking.
The population of the study are nurse educators this is why i need a specific questionnaire for them so they can evaluate if their teaching method is enhancing their students critical thinking.
Regards
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I want to develop a method of LCMS for Liraglutide analysis, The problem is with the solubility of Liraglutide at neutral pH, Do anyone of you have a past experience with Liraglutide method development for LCMS. Please help.
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Dear Girdhari,
published isoelectric point of liraglutide is approximately 4.9.
You can also calculate this (https://pepcalc.com/) from it's primary sequence (HAEGTFTSDVSSYLEGQAAKEEFIIAWLVKGRG).
It is should be soluble in neutral water. Solubility decreases dramatically below pH 7. Double-check if the final solution really is neutral.
Adjusting pH to above 10 or below 2 should significantly increase solubility.
You could also use liraglutide prescription as a guide:
3ml solution containing 18mg of liraglutide, 1.42mg disodium hydrogen phosphate dihydrate, 14mg propylene glycol, 5.5mg phenol, dissolved in water for injection, pH 8.15
European patent EP 3 295 952 A1 (p. 4)
You can also add an organic solvent to improve solubility (e.g. 30 % ACN).
But make sure the elution strength of your sample solvent does not exceed the starting conditions of your LC-MS method, as this will lead to irregular peak shapes.
Kind regards,
Lorin
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Recently, the dumping of e-waste to environment due to its short life span and becoming obsolete models in shoter period and more user groups.
In order to dispose in eco-friendly way, any biological method used for recovery of Bioenergy products
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I do not know
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Hi all,
I would ask about ICP-OES system, I am using it for metal detection in mineralized aliquots of soil and waste samples.
I am using an old method developed in my current lab, but I would like to improve it to obtain a better signal to noise ratio and lower LOD. So my question is:
Which are the variables to take into account for the method optimization?
I have considered yet:
- the wavelengths (I will perform proofs at different recommended WLs for every metal)
- the radial and axial mode (I will use axial because I saw that the LODs are lower in such a condition)
Is there any other parameter which I would consider? For example: is the time of acquisition important in the increase of the S/N ratio?
Thanks a lot to everyone who can give me some advice about that.
Matteo
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I used Ordinary least square (OLS), Generalized linear modelling (GLM) and Multivariable fractional polynomials (MFP) methods to develop a prediction model and all 3 models produced identical predictions. Can anyone give an explanation for this?
Thanks
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Sameera -
I'm guessing that for your GLM software to do something other than OLS, you have to instruct it to do so.
Have you tried WLS? You might consider this for regression of form y = y* + e:
The WLS regression coefficients are often very close to those for OLS, but the prediction intervals associated with smaller predictions would be shorter, and the prediction intervals associated with larger predictions would be longer, often much longer.
If the sample size is small, the regression coefficients would be more likely to differ more substantially from the OLS ones, but then we would be less sure of the regression weights. But with smaller samples there is more uncertainty, so that is to be expected. Choosing OLS when that is obviously not correct would not help. Sometimes it can be reasonable though. Model and data issues may cause that to be the case.
Cheers - Jim
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I work on quantitative method development for micro/nanoplastics. I need a series of calibration concentration which is below the amounts of analytical balance. Any idea?
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You might need to look at whether your lab needs a Micro or Ultra Micro Balance. Mettler Toledo do ones that measure down to 0.1ug.
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Dear colleagues
It is necessary to know how they develop EPA Standard 8260, for the detection of BTX and MTBE, in aqueous solutions. Specifically, the pattern preparation part, how are the steps to follow, how do the dilution of the patterns and in what medium. If you can share it with me and if not please let me know. From already very grateful for his time. Regards. Luis.
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For HPLC, what different mobile phases are best to start with for methods development? What is a good approach to trying different buffers/motile phases for methods development?
I've looked through a few papers, but as I'm new to using HPLC, wanted to know if there is a good 'rule of thumb' of different types of buffer solutions to try first in methods development, and why.
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Try this "free" program that you can play what if in mobile phase method development. It is a good tools that I did not have when I was in school 35 years ago.
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I am planning to develop a ordinal scale to assess a motor function like sitting to standing. I like to know method to develop and test its relevant psychometric properties. I will be happy if someone likes to work with me.
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Dear Dr. Ramachandran,
In general, the type of measurement scale does not significantly influence the procedure of psychometric assessment. The assessment of psychometric properties involves the check of reliability (internal consistency, test-retest) and validity (construct validity, criterion validity) of the results obtained with the measure. The type of measurement scale may change statistical procedures that you apply to obtain necessary evidence of reliability and validity, but will not change the procedure of scale development.
Best regards,
Kirill
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Is anyone using LC-TOF-MS to quantify serum steroids like 17-OHP and 11-DOC and willing to share any recommendations for method development? Typically, this is done on LC-MS/MS, but we do not have access to one. WIll liquid-liquid extraction be sufficient sample prep? Is there any reason that quantification will not be possible on TOF?
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good answer Adam Zmysłowski
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Could you please anybody explain me how to develop the method and validation the method which relevant to finding Aflatoxin in Peanut relevant to R- Biopharm method?
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These forums do not provide a means to address such a broad question as posed. The basics of HPLC as a formal analytical technique and method validation basics fill volumes of books and journals. Read the books to research the basics. As HPLC method development and HPLC method validation take many years in the workplace to acquire a basic level of proficiency, I suggest that after researching the topic, you hire a contract laboratory to perform the work for you OR work with a local laboratory whom has experienced liquid chromatographers with a demonstrated history of doing the same for compounds of interest.
Additionally, you may wish to take some time and begin to research the basic of liquid chromatography [e.g. The text, "Introduction to Modern Liquid Chromatography" by: Lloyd R. Snyder, Joseph J. Kirkland, John W. Dolan is excellent] , HPLC methods, the basic concepts and fundamentals of analysis. BTW: Avoid sales oriented HPLC websites such as chromforum or chromacademy which have many inexperienced users who may misdirect you. Additionally, if you wish to be involved in the actual analysis work, then you will need to obtain formal training on the specific HPLC system and software that will be used for the work (Many types of HPLC systems and each one requires training in how to use it).
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As part of a research project we are planning to develop a workshop method. We want to accompany this development scientifically with the goal to publish the results. Hence my question: Are there any established methods for developing workshops and for evaluation?
Do you have any experience in this?
Thank you for your help!
Best,
Konstantin
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I am an investigation methodology teacher and yes there are established methods for devoloping workshops and for the evaluation procedure. To develop a workshop, the methodology is as follows: Fist of all you must define the objective, after that, take into account what are the activities that all persons involved in the workshop are going to develop and for the workshop evaluation, we apply the observation methodology of all the activities developed by the participants, following an observation guide.
In my personal oppinion, once the activities you devoloped are finished, you can apply a survey or interviews, to be able to prove the effect or result obtanied according to the planned objective.
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Hi all,
I am currently working on a project that requires me to isolate plasmids from clinical bacterial samples in order to sequence them using Nanopore MinIon. I have used two methods so far - the Qiagen Midiprep plasmid extraction kit and an alkaline-lysis method developed by Kado & Liu.
I am investigating plasmids that contain ESBL and Plasmid-mediated quinolone resistance genes - I know that I need to linearize my plasmids prior to sequencing. Has anybody extracted plasmid DNA for nanopore sequencing? and/or is there any advice on which restriction enzymes to use or a particular protocol I should investigate?
Thanks in advance!
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I might suggest you consider using the Covaris G-tubes for plasmid DNA fragmentation. However, I agree with
Qin Qi
that there is no need for plasmid linearisation prior the library preparation for Nanopore MinION sequencing.
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I am requesting some suggestions for a problem in my LC/MS/MS method development for Prostagladins. I've optimised a mobile phase, column, lc ms (mrm) parameters which all work fine. I extracted some human plasma with added standard with protein precipitation and reconstituted the samples in a mobile phase. Has somebody experienced the same problem? Thanks in advance for your help LC-MS/MS
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Hello, why you need to split peaks in LCMS, you do not have differences in m/z ?
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Asking respondents to sort items such as pictures, colours, or other visual materials is a well-established practice in the Q-methodology. I'm wondering if anyone can share experience with asking respondents to sort maps. For instance, a series of maps showing scenarios of different land uses. I'd appreciate any input from the community, from published works to ideas for method development -or words of caution.
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Hi Diane,
Could you shed more light on the presenting sorting you used? I'll also wish to know if there are freely available online sorting websites I could use for postgraduate research.
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Hi,
I would like to ask, if we have foreground data of certain process but could not get the background data because there is few study/commercial database does not cover it, can we just use any background data for other similar process in other region?(e.g. I do WtE study in Kenya).
Also, many impact method are developed somewhere, should it be adopted in the region of my study or i would not be much problem to use available impact method?
Thanks.
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Thanks for posting the new question.
I agree with A-B Lauren on both points. I also recommend you include any data you suspect might not be respresentative in a sensitivity analysis to let the reader understand your results better.
It is always up to you (the LCA practitioner) to use your expert judgment on what data can be used and to what extent it is representative. Processes modeled on data from other locations are however often better than no processes at all.
To have recommendations on how to deal with impact methods you would have to give more background info, both on the system, aim and scope, as well as the impacts you want to asses.
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For bio-analysis with LC-MS/MS, a linear calibration curve is preferred over a quadratic. Why is this? Is it OK to continue method development with a quadratic fit? I have to add that the quadratic fit for my analyte is only seen in Opti-MEM (a transport medium for cells) and not when, for example, a non-biological matrix is spiked with my analyte. (the fit is y=x*x)
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Dear Asal Asali,
calibration functions with quadratic fits are allowed according to USP 1736:
"A calibration curve is created by plotting analyte-to-internal standard peak area ratios versus concentration for the reference standards analyzed. For the somewhat narrow calibration range required for Category I and Category II measurements, a simple (nonweighted) linear fit is sufficient to define this response versus concentration function. However, 1/x or 1/x2 weighting also can be used, as well as nonlinear (e.g., quadratic) functions, as supported by appropriate and provided validation data."
Due to my experience, most of the people familiar with HPLC-UV sticks to linear calibration functions due to Lambert-Beer law. However, this law does not apply for LC-MS, since LC-MS is not based on (UV)-light absorbtion.
Best regards
Andre
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kindly suggest the best HPLC method development attempts
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Hi Omer,
Target compounds are inositol phosphates. We have selectd reverse phase anion exchange Chromatography .
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I need to find the research method to develop a methodology for the redesign of production processes.
Thanks for your contributions.
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You could do qualitative interviews with experts in the relevant area. You might also consider a "delphi" design where you collect rounds of interviews with experts via email. Here is a brief introduction to delphi techniques:
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I am searching for a comprehensive protocol for spectrophotometric determination of ornithine from plant tissue, though, there is a method developed by Chinard (1951) for the determination of proline and ornithine together, but its not clear to me.
I will be grateful if anybody share the step-by-step working protocol for the determination of ornithine from plant tissue.
Thanks.
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I am working developing/validating an impurity HPLC method. It’s a pretty straightforward HPLC method, with a main peak (monomer) and an impurity peak (dimer).
To evaluate linearity/accuracy/range per ICH guidelines, the classic approach is to take an ultrapure sample and to spike with known amounts of the dimer or with known amounts of a sample with high dimer content. The sample concentration should remain relatively constant (i.e. monomer peak area is constant). The spike recoveries can then be used to evaluate method linearity/accuracy.
Another approach I’ve heard used in method development is to take a sample with high dimer content (e.g. 2%) and serially dilute this sample until you no longer get accurate + precise dimer quantification. For example, a 1:20 dilution may be the most you can dilute this sample down while still getting accurate + precise dimer quantification. You could then divide 2% by the dilution factor to get the lower end of your assay range (i.e. LLOQ; 2%/20 = 0.1 %).
I’m not used to the second approach (the serial dilution). Can anyone tell me what I might be overlooking with this approach? I know it doesn’t follow ICH guidelines, but from a scientific point of view, why or why is it not appropriate?
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Hello, serial dilution is widely used in HPLC method development and validation. Only thing use the rage with is corresponds the range of analyte in future analyzing samples.
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In my HPLC method development, Active peak cumming negative in sample and normal in standard solution. Can anybody suggest the reason behind this?
Instrument method:
Mobile phase : 1% Aceetic acid in water and Acetonitrile 52:48 % v/v.
Diluent : Methanol
Column: Zorbax Eclipse Pluse
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The method was invalid as shown. The consequences of using instrumentation and techniques without proper training often lead to erroneous data being collected and incorrect conclusions or quantitative results being reported. In your case, there are two reasons for this:
(1) The DAD reference wavelength feature on your Agilent 1260 DAD was turned 'ON'. This must be turned 'OFF', not 'ON'. To learn why, please read the attached article below.
(2) Your signal acquisition wavelength is very close to your Reference Wavelength. This guarantees that your data will be invalid as even the peak of interest will fall under the Reference range you specified (360,10), invalidating the method and data obtained.
Those who use HPLC for the first time and/or who have not had any formal training in liquid chromatography (and especially in the use of a diode array detector (aka: PDA)) often makes these mistakes. Clients often use systems setup just like this one and are unaware of the problems caused until a negative peak shows up. The appearance of the new, negative peak is this hint that all of your previous data may be invalid and the only reason you discovered it now is because this time the system had to subtract-out so much data from your main peak that it ended up being negative!
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Kindly suggest the methods for the development of biosensor for antibiotic detection in food products.
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Nano technology based sensors for different antibiotics may be created....good idea but you have to point out specific group of antibiotics....
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I am using Agilent 1290 Infinity HPLC instrument for my analysis. While generating reports, for few of my samples results going blank. But the peaks generated are perfect and area was calculated with proper integration from the calibration method developed. Anyone here faced similar problem? and if there is any solution to overcome this issue please help. Thanks
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I agree with William.
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Dear Experts,
Head motion is an important confounding variable for fmri studies. I know that a range of different methods is used to correct motion in resting-state functional connectivity studies. One of the simplest methods is to exclude subjects whose head motion exceeds 1-3 mm in translation/rotation. Scrubing, frame-wise displacement are among the others. I will use dynamic causal modelling to investigate effective connectivity during a task. However, I am not sure that I should be sensitive to this topic because some of these methods are developed using resting-state data. I was wondering whether it is uselful to apply motion correction to task fmri data as well.
Best,
Seda
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Interesting project! Have you read Peet Höbedas paper at Eurobitume 2000? Title: "Testing the durability of asphalt mixes for severe winter conditions". Peets proposed method was developed after the worst winter damages ever seen on new highways in Sweden. Maybe it is useful in your work.
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Interesting discussion.
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SCS Curve Number method developed by USDA generally applicable for events based simulation. If we wanted to apply this method for long term continuous rainfall-runoff simulation are there any modifications are needed to the original equations? If so, what are those.
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You are quite welcome!
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Hello, 
I wish to integrate QbD approach or methodology for method development and validation of in vitro dissolution studies of extended release or sustained release of some drug formulation. I want to know the plan of work of experiment, the way we conduct experiment. Please suggest me. Thank You.
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During Dissolution Method Development through QbD, the Main objective is to develop "IN VITRO IN VIVO RELATIONSHIP (IVIVR)". There are basic 5 phases involved in developing in-vitro in-vivo correlationship:
1. PRESUMED IVIVR:
Keeping Product concept in mind Define Target In vitro & in vivo specification (based upon solubility & permeability profile of drug)
2. RETROSPECTIVE IVIVR:
Prototype Formulation Development with level of variation in release rate & its selection by extensive in vitro dissolution testing in MULTI media, Pilot PK study (cross over study on 6 -12 subjects)
3. PROSPECTIVE IVIVR
Define formulation meets in vivo performance (with empirical basis of formulation related rate controlling variables), Define BIO RELEVANT & DISCRIMINATORY media, Confirmatory IVIVR PK study & Define LEVEL
4. FORMULATION OPTIMIZATION & PROCESS VALIDATION Formulation/Process optimization, Scale-up/ Pivotal batch manufacture with Process Validation & Pivotal PK studies (on at least 12 subjects)
5. REGULATORY SUBMISSION PRODUCT REGISTRATION/ REGULATORY APPROVAL SCALE UP & POST APPROVAL CHANGES (SUPAC)
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My new answer:
I repeat-On the 8 months ago I wrote my proposition. Are you interested one experimental method for the development of metallic structural materials?. My technological process aim is the efficient and focused identification of compositions and process chains which result in a specific performance profile of the material. I realized and use one scientific and technologically method to study, analysis, quantifies and qualifies metallic structure of more metallic material roughness and coated. Also, my method gives possibility to analysis surface roughness, surface parameters, and wear volume metallic material lost. My method gives me possibility to correlate compochim, wear material volume lost, roughness parameters with work conditions and corrosive activity. Possibility to recommend solutions to improve quality parameters to industrial fabrication and anti-corrosive applications. My method helps to overcome resource limitations to open new frontiers for experimental materials development. The transformation of determined descriptors towards macroscopic material properties of the performance profile is achieved by a heuristic predictor function, which needs only few macroscopic samples. Maybe collaborate! Thank you.
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Dear Paul,
I think there is a misunderstood here!
At the current time, I am not a PhD student but I am searching and looking for a PhD study opportunity and I thought you have a project and need a PhD student to supervise and work with.
Regards
Ruqaya
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I am working on a project related to method development for related substances of tobramycin.
if anyone can provide me with any kind of literature other then the one given in monographs, it will be of great help.
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Since tobramycin lacks chromophore group its estimation is difficult using uv detection unless derivatization is done which is again a tedious process. Its better to go for LC MS for better results.
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I have cryopreserved vial of BMSC (Bone Marrow derived mesenchymal stem cells). Please suggest suitable method to develop healthy osteoclast/osteoblast lineage from the above mention cell line. Also mention the culture condition and media for osteoclast/osteoblast and THP-1 macrophage co-culture condition. DMEM/F-12 (1:1) medium would be appropriate for it?
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We have differentiated THP-1 cells (cultured in RPMI) using the following differentiation media composition:
RPMI media supplemented with 10 % (v/v) foetal bovine serum (FBS), 25 ng/mL recombinant human sRANKL (Gibco, USA), 200 ng/mL PMA (phorbol 12-myristate 13-acetate
Differentiation time 8-10 days
REFERENCE:
The above differentiation media pushes the monocyte non adherent cells towards osteoclastic adherant phenotype. Slowly you can acclimatize the cells with (50:50 RPMI: DMEM)
For osteogenic differentiation of BMSCs, 10mM Beta-glycerophosphate, 100nM dexamethasome, 0.2mM ascorbic acid in DMEM supplemented with 10% (v/v) FBS.
Differentiation time 14-21 days
For co-culture:
Predifferentiated (day-5) osteoclasts (from THP-1) and predifferentiated (day-7) BMSCs (towards osteogenic lineage) can then be co-cultured with 50:50 RPMI:DMEM (with the osteoclast and osteogenic supplements) till day-21.
Hope this helps.
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Ag:TiO2 thin films
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I mean for Titanium oxide
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The method of separation of temperature and emissivity (TES) developed by Gillespie et al. (1998): this algorithm is easy to use, it requires two multispectral measurements: one on the sample and the other on a reference reflective plate to obtain the contribution of the environment. In addition, no prior knowledge of the surface is necessary. The TES algorithm used for ASTER data has been developed for the five infrared thermal bands of the ASTER infrared sensor. The TES combines three interrelated modules: the Normalized Emissivity Method (NEM) module; The Ratio module (Watson, 1992); The Minimum Maximum Difference (MMD) module.
I want some information and a good interaction of you :)
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Dear Sir. Concerning your issue about the Temperature and Emissivity Separation (TES) method. TES algorithm hybridizes three established algorithms, first estimating the normalized emissivities and then calculating emissivity band ratios. An empirical relationship predicts the minimum emissivity from the spectral contrast of the ratioed values, permitting recovery of the emissivity spectrum. TES uses an iterative approach to remove reflected sky irradiance. Based on numerical simulation, TES should be able to recover temperatures within about /spl plusmn/1.5 K and emissivities within about /spl plusmn/0.015. Validation using airborne simulator images taken over playas and ponds in central Nevada demonstrates that, with proper atmospheric compensation, it is possible to meet the theoretical expectations. The main sources of uncertainty in the output temperature and emissivity images are the empirical relationship between emissivity values and spectral contrast, compensation for reflected sky irradiance, and ASTER's precision, calibration, and atmospheric compensation. I think the following below links and the attached file may help you in your analysis:
Thanks
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Method development
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It depends on what your compounds are?
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I tried different suppliers API.
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Amar,
1. For gradient work use a 5 cm column as 25 cm column is too 'sluggish' in responding to mobile phase changes. In addition never use 100% water in a mobile phase (your mobile phase A). It can cause column collapse (I still believe in 'old' wives tales). Add about 5% ACN.
2. Even put 5% water (Mobile Phase A) in your Mobile Phase B. This will reduce gradient peaks or spikes due to refractive index differences.
3. Inset a sample chromatogram so we all can see it. Use a PDA (scan 200-400 nm) instead of 1 wavelength. Your impurity (related substance) may not absorb at 254 nm.
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We just acquired a LC-MSMS (QExactive) for targeted and non-targeted (discovery) metabolomics. I'm in the process of method development, and I'm wondering if anyone has a used (and approved!) a commercial standard with a premixed compounds? I'm probably refining the metabolomics workflows to metabolite groups that is: aminoacids, TCA cycle metabolites, glucolysis, etc, so any standard that would contain at least a few compounds for each metabolic pathway would work. Why not buy them individually and mix? Unfortunately, time is a valuable resource for me ;-)
Thanks
Alex
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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&region=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.
[6] 47266 Supelco Sugar Alcohol Kit (individually packaged in quantities indicated), analytical standard : https://www.sigmaaldrich.com/catalog/product/supelco/47266?lang=en&region=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&region=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&region=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
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Please could you please explain me below operate parameters of LCMSMS,
1. Taken relavant range of scan in precursor ion.
2. then optimize it, and check whether its accuracy
3. how to check the LC and MS parameters in instrument
4.how to create the calibration curve
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Hello: Your question is far too broad in scope and required time to answer. Are you aware of the fact that operating a LC/MS or LC/MS/MS system is very different than using a pH meter? It takes many years of professional experience and practical hands-on use to learn how to properly operate this type of complex analytical instrumentation and develop methods properly. Additionally, each brand and type of system is different, and this requires even more specialized training.
While some aspects of your question are very basic in nature and easy to solve ("how to create the calibration curve"; This involves developing a proper analytical method ad defining the detection settings, then analyzing many standards and plotting them using the software), this is absolutely NOT something that can be taught in a class or offered in a few paragraphs on a website.
Websites such as Researchgate are best for very specific scientific questions and are not a substitute for long-term professional training. Please seek out the assistance of someone local in your organization (e.g. The scientist in charge of operating and maintaining the LC/MS system) or at a professional laboratory to to assist you with theses matters. You will learn much faster in this way.
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I'm doing determinations of amino acid concentrations (using OPA, Agilent Zorbax Eclipse C18 column & a variation/combination of an Agilent and a Dionex instrumentation methods). I get good peaks in some regions, but some blend together too closely. I don't want to go slower, I'm already running at 40 minutes per sample.
I currently have the HPLC running at 0.72mL/min, but in playing with the method I found decent results if I changed part-way to a 0.48mL/min and back again.
Is this kosher?
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Dear colleague,
I agree with M. Farooq Wahab
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Are you interested, I have/use one experimental method for the development of metallic structural materials? The technological process aim is the efficient and focused identification of compositions and process chains which result in a specific performance profile of the material. Maybe collaborate! Thank you.
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This may be in left field - although not dealing with Metal composition - we are trying to create a measurement tool around Crisis Intervention in the Worplace..... We have developed a validated took..... but don't know how to gain any control data... We would just be utilizing the tool a certain number of days after the incident.... does this relate and have you come across a method that works where you can only gain data one time?
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We are busy with method development and validation for 3 polar analytes (pesticide actives); Ethephon, Fosetyl-Aluminium and phosphonic acid .
We are making use of reference method QuPPe version 9.2 and the method in there is 1.3 (glyphosate& Co) and using a HyperCarb column on a Shimadzu LC-MS-MS 8050.
The problem is that, the intensity responses of these standards are very low even when injecting volumes of 20 microliters. In matrix (citrus and apple) we are unable to detect lower levels of the analytes with the calibration from 5 – 200 ppb.
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check out my publications on researchgate. Hypercarb column is old tech and needs to inject blank matrix to prime it. I used mixed-mode column (Acclaim Q1) for many polar pesticides (glyphosate, paraquat, ethephon, fosetyl Al, ETU, PTU, Chlormequat and more.
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Hi,
I am working on my method for a research project. In that project I will use time series data and need to calculate the abnormal return for stocks over a period of time. The Fama-French model has been used by others but more in event study situations. I intend to use the abnormal return to investigate whether a correlation exists with another set of time series data. Do you know of papers or could give some input to assist with the method development ?
Thanks,
Patrick
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Hi,
You may be interested by this paper related to the Fama French model:
Creative Destruction and Asset Prices.
By: Grammig, Joachim; Jank, Stephan. Journal of Financial & Quantitative Analysis. Dec 2016, Vol. 51 Issue 6, p1739-1768. 30p. DOI: 10.1017/S0022109016000557.
Sujets: Rate of return; Investment risk; Small capitalization stocks; Large capitalization stocks; Assets (Accounting) -- Sales & prices; Creative destruction
Best regards
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Dear all,
I develop a method for my product, everything was perfect but at last there is  peak in BLANK at the component RT. sometime it happen only like a baseline drife. But it comes, where 75 min baseline is going accurate except that.
My effort for that is following:
1. Changed in column for carry over,Also tried new column.
2. changed in reagent for MP preparation(A-OPA:ACN 80:20, B-OPA:ACN 20:80 (Buffer pH 5.00)
3.Changed in System and make also.
4. Changed in solvent make,needle wash,pH meter,water and much more
Please suggest what can i do more?
Thanks
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I would have to say that it is probably contamination in the injector. If you are describing an issue with a mass spec then you could also look to the inlet of the mass spec.
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Chiral separation.
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Chiral method development is an advanced technique best suited to chromatographer's with many years of practical HPLC experience. Your method development skills should be strong as should your understanding of the fundamentals of chromatography (as with all chromatography, understanding Kprime, T0, R and S are very important). The most important key is to first identify the correct column for the sample which will retain it with good peak shape (finding the correct mobile phase is the second step).
Chiral method development by HPLC and/or SFC requires the use of many different chiral columns and mobile phases to find a suitable separation.
If you would like to learn about practical Chiral HPLC (and SFC) screening methods for pharmaceutical drugs (for Racemates and Enantiomers), then I would suggest you consult the following articles :
"Chiral HPLC and SFC Column Screening Strategies for Method Development:" http://hplctips.blogspot.com/2011/06/chiral-hplc-and-sfc-column-screening.html
"CHIRAL HPLC / SFC METHOD DEVELOPMENT (Alcohols):" http://hplctips.blogspot.com/2011/04/chiral-hplc-sfc-method-development.html
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Dear colleagues,
I am a graduate student, I began my Ph.D. some months ago, and I am trying to work with an Eksigent nanoLC 1D plus.
I would like to change the valve position for inject using direct control, but
the software can not recognize my command, it still waiting for the LC method, but I did not put any run in the queue. The valve still in the load position all the time.
However, and I run a sample, the valve works normally and changed for the
inject position at the right time. The problem only happens when I try to use the direct control mode.
I do not know how to works now and the manual is not helping me.
Probably the problem is easy to solve, but I could not recognize the solution.
Do you know what to do for to control the valve position?
thanks 
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Thank you for the answer, I will ask the tech support. 
Thanks
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We're planning to make organic superabsorbent polymers similar to what Google science fair awardee Kiara Nirghin has done in her project, however, we could not find any published studies on development or synthesis of organic SAPs. We would like to know if any of who has made studies or have ideas on studies related to this wherein we can base our methods to? thank you!
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thank you!
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Hi..
What are the steps involved in developing a HPLC Assay or RS method development if there is no Compendia or Literature available?
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This is too general a question for a web forum, because there are dozens of possible directions to go depending on the available information. For example: Is the sample a "known" or "unknown" compound? Do you know its structure and/or which detection techniques are applicable? Is it a pure sample or mixture? The more information you have, the better the initial screening will be. So first try and obtain information about the sample before using HPLC.
Some initial guidelines for unknowns that I teach professionally would include (these suggestions try and utilize inexpensive and common types of columns and solvents found in most HPLC labs):
  • Determine the solubility of the compound first. This will provide valuable information as to the initial polarity for NP vs RP method development.We do not analyze samples in liquids that they do not dissolve in (seems straightforward, yet I sometimes see scientists ignore this point).
  • If possible, place a sample dissolved in pure solvent in a spectrophotometer and run a full scan (e.g. 200 - 900 nm). The information obtained might give you an idea of which wavelengths to start with (if you see a peak at 254, but run the sample and do not see it, then it may still be on the column). *If sample is limited, this can be done using the flow cell of a DAD in stopped flow mode or fast scan mode too (see link).
  • If RP HPLC seems applicable and the sample will dissolve 100% in both ACN/Water and MeOH/Water, then start with a general gradient on a 4.6 x 250 mm (or 4.6 x 150 mm) x 3.5 (or 5u) high quality C18 column. Try two different mobile phases in gradient mode: (1) Water vs ACN (95% water / 5% ACN to 95% ACN / 5 % water) at 1.00 ml/min over 20 minutes using a DAD set in full scanning mode; (2) Water vs MeOH (95% water / 5% MeOH to 95% MeOH / 5 % water) at 1.00 ml/min using a DAD set in full scanning mode. *It is very important that you run in both ACN and MeOH as they have very different solubility characteristics and properties (not just polarity). The data obtained should allow you to fine tune one method, with or without additional additives and pH adjustments, to arrive at a good separation. Remember the goal is to scout/screen for acceptable conditions.
  • If NP HPLC seems applicable and the sample is soluble in alcohols (e.g. Ethanol), then set up a similar gradient as described above (for RP) using a high quality NP silica column with 100% ethanol. Set the gradient to run from pure ethanol to 20% ethanol/ 80% n-Hexane (Use high quality 97% n-Hexane or Heptane). Depending on solubility, you could substitute IPA for less polar conditions. The basic idea is the same. Use a gradient to gradually change % composition and find out under what conditions the compound(s) elute. Remember the goal is to scout/screen for acceptable conditions.
These are very general guidelines and follow the 'keep it simple' protocol which relies on running a few simple initial experiments to obtain data, then  add your observations and experience to plan the next steps .
BTW: A two-part HPLC article series on "Modern HPLC Method Development Tips:" (Part 1) can be found at the link below. 
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The modulation period is one of the very important parameter in GCxGC-MS. The un-optimized modulation period yields a very complicated plot using GC image, which looks horrible and interpreting this is extremely complicated. Can someone guide me how to optimize this parameter?
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Please email me, Deepak. Would like to talk there and exchange info. Could you also mail the model and make. Email: bbmisraccb@gmail.com
Thanks,
Biswa
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I have to isolate beta glucan from oats. Isolated beta glucan need to undergo chemical analysis for crude fat,starch, total fiber, insoluble fiber and soluble fiber, and Nitrogen for protein. For this I have to follow the above said methods. If anyone having these methods can share ? 
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Dear Sir, Thanks for  replying. but the papers you sent I already had. And my need is still the same, that is, said numbers of methods in AACC (2003) and AACC (1990).
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I am working on dementia and Depression rat and mice model .And very much keen to estimate the quantity of few Brain markers using HPLC. But now I am having problem in Understanding and Establishing A protocol for few brain markers like: 5HT, NE, DOPAC, DA, MHPG, 5HIAA. In this experiment I am going to Use:
"UHPLC
ultimate 3000
THERMOscientific"
"Can anyone please share the protocol to identify such  Biomolecules "
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Sir
Thank you very much for your precised Answer
Thank you very much
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intend to use the method (GLCM) for extracted the texture featurefrom color image, but i need to applied some other texture features, would you pls help me the combinational method to develop a new feature method
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Hello,
Which programming language are you using?
You can use MATLAB built in function graycomatrix if your are using MATLAB as your implementation language.
Details can be found in the following link:
If you are using python, you can first install scipy, numpy, and skimage and then use skimage.feature.greycomatrix and skimage.feature.greycoprops.  
Let me know if you have any specific question about the detailed implementation. 
Thanks.
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I'm thinking about performing an enzymatic assay to oxidize vitamine K4 (Menadiol) into vitamine K3 (Menadione), the thing is that the only method that seems to work is HPLC-MS/MS, I was wondering if there is any spectrophotometric or fluorometric method to quantify these two quinones.
Thanks for your help!
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Dear Sir. Regarding your issue about the method to quantify the oxidation of Menadiol into Menadione other than HPLC-MS/MS. Different analytical methods (spectrophotometric or spectrofluorimetric) have been proposed. The following below links may help you in your analysis:
Thanks
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I tried to detect ddNTP by HPLC-ESI-MS. Buffer A is 50mM ammonium acetate+2% acetonitrile, Buffer B is 70% acetonitrile + 30% Buffer A. Column is C18. Ion mode is negative. I can find ddATP\ddCTP\ddTTP, but no ddGTP. I tried 2 different lots. The concentrations of the 4 ddNTP were supposed to be same. Is there any method to detect ddGTP? I have ammonium acetate and triethylamine, no triethylammonium acetate. Thank you.
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Dear Sir. Regarding your issue about how to detect ddNTP by HPLC-ESI-MS The following below links which may help you in your analysis:
Thanks
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I have the exact same concentration of solute and am making a 1% DMSO solution each time so I cannot figure out why it hasn't worked on the larger scale.
The first time it went into suspension similar to what I have now, but dissolved again when I heated it to 37C. However I've incubated the 5ml at 37C for hours now and no luck...I've even tried heating it to 50C and increasing the pH, neither worked (the increased pH changed the colour of the solution so I assume it's affecting the compound somehow).
I'm not a chemist myself so I can't think of any possible explanation for this, or of any other ways to get it to dissolve :( Any insights would be much appreciated!
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At small scale the precipitation might not be visible clearly. vigorous vortexing and sonication may also help to solubilize the solute.
Precipitation can be avoided by
1. using solvent in which compound is very soluble (it may not be applicable in your case as you might be working with biological system and organic solvent not allowed)
2. use DMSO (you have already tried but you might not be allowed to use higher percentage of DMSO for the experiment)
3. use Cosolvent / low amount of surfactant/ b-cyclodextrin (this might helpful)
below is attached link of the article.
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Organic Carbon Content in NEH Soils are Very High (more than 1.5 %) But Mineralisable N is Generally Low. The Index Developed in India is not Suitable for NEH region Because Organic Matter is Present in Unhumified Form.
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Microbial population and its activities other than fungi is generally low. So organic matter present in acid soils are not fully humified and N content is low in these soils though the organic matter is high in the the soil.
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There are a number of ways of monitoring exercise training intensity. Subjective measures include the Session Rating of Perceived Exertion (RPE) method developed by Foster et al. (1995) whereby participants indicate a global session RPE 30 minutes after completion of an exercise bout. But I wanted to know if this is the best method to quantify global RPE for interval training? Are there alternative, reliable measures (subjective or objective) of global Session RPE suitable for interval training?
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I don't know that there is a perfect answer unfortunately. I grapple with this a lot myself. It's hard to take ratings during the exertion part of interval training, but I would say immediately after the effort bout, as soon as the rest period begins would be best. Its not perfect but it will give you a sense of how RPE develeps during the session. You could also include session RPE as well, it may even be interesting to see how that compares to ratings taken during the actual effort bout.  Good luck :)
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Through the web I could find only 6 to 10 stationary phases like C1 to C18 and cyano amino diol etc. I opt to get a detailed list. Please suggest a reference.
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As our friends mentioned above the best place is hand book of chromatography, or better hand book of stationary phase. Obviously you are trying to separate the components of a sample, and you have chosen a correct method, because the polarity of the analyte must be matched with the stationary phase, if not the retention times would be too short. 
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I think there are available software (not free) for automatic method development for HPLC, which can help users to optimize the separations using a minimum experimenting works. Do you know such software but  can be freely obtainable?
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Hello: I teach HPLC method development and am familiar with most of the commercially sold as well as 'free' chromatography method development application products available.
The software products mentioned above, by previous posters, is not free.
Free software is available. Example: DryLab is one of the most popular chromatography simulator software application available.
NO known SOFTWARE application can perform truly automated HPLC method development. All require high quality input in the form of data and a skilled chromatographer to function. All of these applications rely, to different degrees, on the input from a very skilled chromatographer to provide initial values for the software to use. Even some of the more advanced software applications such as ChromSword require a great deal of skill on the operator's part to define the types and dimensions of columns used, mobile phase combinations tried and specific methods used for the screening protocols (best for only the most technically savvy chromatography users only). The applications can only fine-tune methods or scout for methods based on a user's input OR the data input from pre-run chromatograms (simulated or real). They are a poor choice for teaching people how to develop methods by HPLC. These programs are just a tool, not a solution. In the hands of a skilled operator, they can be useful. For most users, they are serious overkill and I do not recommend them (BTW: I personally use and like some of the programs, but they are neither free nor the best applications for most users).  For basic and intermediate levels users (99% of all chromatographers) I do recommend the simpler programs such as the basic version of the DryLab application to help teach HPLC fundamentals. This is the area where most chromatographers are deficient and if used with real experiments, these types of programs can aid users in understanding the relationships and concepts needed to develop proper HPLC methods (such as learning about T zero, K prime, Resolution, Solvent % effects and so on). This is a great way to fine tune methods based on just a few initial runs. Most people can start using the software with minimal training and it encourages lots of "what - if" experiments and simulations which I like to see.
More info on Dry Lab can be found at this link: http://molnar-institute.com/drylab/
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