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Bioanalytical Method Development - Science topic

A place for discussion and collaboration over topics such as bioanalysis, method validation, guidelines and application of bioanalytical assays in life sciences and translational research.
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I am a rookie bioanalytical scientist for a biopharmaceutical company. I develop ligand-binding assays for preclinical PK/PD studies and am trying to understand a fundamental principle:
As opposed to clinical development, pre-clinical development is conducted using matrix in-vitro. So how does quantitating drug in naïve matrix (serum, urine, CSF, etc.) in-vitro accurately describe the ADME of the drug, if it didn’t go through natural absorption, distribution, metabolism, and excretion processes?
PK refers to what the body does to the drug.
(Conc x Time). In the 1 PK assay I developed, we quantitated drug concentration in CSF (a matrix devoid of the target protein). How does a PK method for drug quantitation in matrix describe Concentration over Time? I understand during sample analysis samples treated for various intervals are used for sample analysis, but during method development and validation, a naive sample is used..?
PD refers to what the drug does to the body.
(Response x Conc). In the 1 PD assay I developed, we quantitated target protein concentration in human serum. I understand the concept of PD since the response is directly proportional to the concentration of the target protein, but I don’t really understand how the PK asses I develop help describe the ADME of the drug candidate.
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Hi
First, pre-clinical development of biotechnological drugs is not conducted using matrix in-vitro only. This is a big simplification of the very specific part of the drug development process. In vitro, in silico, ex vivo, and finally in vivo, relevant animal models make it possible collection all necessary data which makes possible entry into FIH studies. In the case of biotechnological drugs, this part of the drug development process is only defined in outline. Health Authorities (FDA/EMA guideline/guidance) apply the "case-by-case" principle to each drug or drug candidate separately. There is no one pattern of how studies should be conducted here. Almost always, any type of research can answer only certain questions. Therefore, different types of studies and research environments (in vitro, in silico, ex vivo, in vivo) are to complete the puzzle as far as possible.
Moreover, it must be remembered that in the case of biotechnological drugs which are also antigens, pharmacokinetics cannot be separated from pharmacodynamics. Both of these elements influence each other. Moreover, these two elements are influenced by suppression or activation of the immune system (A:S ratio). In addition, the situation is complicated by the fact that A:S may be the result of the drug's mechanism of action (highly immunosuppressive drugs). It may also be the result of a response by producing anti-drug antibodies or mode of action (downstream effects). So matrix of observations could be very complicated. Preclinical development collects all necessary data which can be helpful in prediction translation and safety risk analysis before FIH. This step always has some gaps ....
Please find below some documents which may better explain this specific landscape ....
Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers
Guideline on strategies to identify and mitigate risks for first-in-human and early clinical trials with investigational medicinal products
ICH guideline S6 (R1) – preclinical safety evaluation of biotechnology-derived pharmaceuticals
S6 Addendum to Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals
In conclusion, preclinical development related to PK/PD and immunogenicity etc is not a simple hierarchical scheme. If you really would like to understand the topic my advice is to study basic mechanisms in general pharmacology immunology and pathophysiology. Such compilation could be very helpful,
Best regards,
Tomasz
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My lab is about to buy TapeStation but I had never listened about it, only about Bioanalyzer. 
Could anyone provide a brief opinion if is better or worst than Bioanalyzer? 
Thanks.
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Hi Carla, what did you decide at the end, TapeStation?
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I have an analyte that exhibits concentration dependent ion enhancement (ionization), i.e. the response factor (analyte peak area/nominal concentration) increases as the concentration of the analyte increases. Any suggestions on how to mitigate this issue?
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1. We don't have SIL and that complicates the issue.
2. Ion enhancement is in neat solution as well as matrix.
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Hello ,
I am currently developing a new LC-MS method for an analyte. However, I am facing some issue with the ph.
Knowing that my analyte has two pka (3.7 and 7.1), I tried running samples at both low and mid ph.
Bellow the process followed:
Step 1:
Ph 2.7 (Ammonium formate - formic acid)
Ph 5.4 (Ammonium acetate in - formic acid)
The peak obtained at ph 2.7 was better in term of tailing but still showed a slight tailing.
Step 2:
Ph 2.5 and ph 5.1
Knowing that phosphoric acid is not recommended for Mass spectrometry, I prepared the mobile phase at ph 2.5 using formic acid in pure water.
Both ph 2.5 and ph 5.1 showed the same results (the tailing was less than the peak obtained at ph2.7 but both peaks at ph 2.5 and ph 5.1 showed co-elution).
Step 3:
Ph 2.6 and ph 5.2.
Both peaks showed tailing and late elution.
The results showed that my analyte is affected by even a small ph change. This suggests that it would be better to run at a lower ph using a buffer to avoid impact of ph fluctuations.
However, phosphoric acid buffer can’t be used with mass spectrometry and there is no other buffer that can be used at this range.
I have two questions:
1) what is the best approach to choose the mobile phase ph and optimize chromatography conditions?
2) Considering the second pka of my analyte (7.1), would ph 5.1 be a good choice of mobile phase ph ? And can the co-elution obtained, be suppressed by changing the elution gradient?
Thank you very much for your answers.
Supplementary information:
- Acetonitrile and methanol were both used as organic solvents and didn’t show any significant difference.
- Software used : Empower 3 ( Not provided with system suitability option )
- An elution gradient was used
- Column : BEH C18
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** Unless you desire to damage your LC-MS instrument, please ignore Kou Hayakawa's advice to use phosphate buffer . We do not use or "run" phosphate buffer through LC-MS systems. Phosphate buffer is non-volatile and will result in clogging and source contamination of your MS. The damage caused may result in long periods of down-time and expensive source cleaning services.
Also do not use pH 2.0 for your method unless there is a reason to do so. As analytical scientists we should first focus on acquiring relevant information about the sample and goals first, then choose a method to start with based on the information provided. Since we have no information about the sample type or structure, any such method development suggestions are purely speculative.
I am sorry to report that Kou does not read the questions posted, but instead replies to all RG requests with the same exact response (his old Lysozyme paper) which does not address the question(s) posed.
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I am working on Bioanalytical Method development of Amphotericin liposomal drug , and in the development following issues are faced
a) Stability of Amphotericin in organic solvent ( methanol, DMSO, DMF ect.), Does any one have stability data of amphotericin in solvents.
b) Precision and accuracy batches are found within acceptance criteria but when QC having liposomal drug spiked, QC at the higher concentration shows accuracy at 88-92% range however all other QC concentration does not show such pattern.
What could be the reason for this?
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Just adding an update, got the solution and Sodium laurayl sulphate worked for me when added in appropriate amount to sample.
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Analytes: phosphorylated carbohydrate (inositol phosphate)
These analytes having little pka difference and different number of negetive charge functional groups. ( we have only two standsrds, IP3 and IP6). These two are eluting almost at same retention time (chromatographic conditions: scherzo multimode c18 column, mobile phase A is water+methanol 80:20 and B is 50 mM ammoinum formate +50mM ammonium hydroxide + methanol 40:40:20,ph8.4, detector ELSD). With minimum flow rate (0.150 ), we got resolution about only 0.35 min only.
In literature, It was developed by using ion pairing agent such as tetra butyl ammonium hydroxide and C18 column. Here, column is modifing into SAX property by mobile phase layer.
Multi mode column is also works as similar without use of ion pairing agent. But we are not able to get the separation as achived earlier from the literature.
Please give me the suggestions for  the development of this method.
And alsothe appropriate retention mechanism and chromatographic conditions to separate these compounds.
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My experience with the mixed mode column is that pH of the mobile phase has a vital role to retain or elute the ionized analyte.. I would try the Mobile phase at different pH isocratically. Please stay away from pH gradient method as you may have retention reproducibility issue. Keep organic solvent fraction constant and very only pH first the vary the solvent concentration later.
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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?.
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Hi Dr Chibuike Udenigwe,
Can you help me for the calculation part. I am stucked..
For example, my sample has a FRAP value og 1.53 mM FE.. Then, I want to express it in mM Fe/g DW.. So how?
Thank you.
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LogP <0 means very polar and vice versa is for moderately polar.
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"c18 polymeric column" ?  think you are again referring to a silica based column.
It helps when we discuss chromatography to include all of the details (column brand, name, support, particle size, dimensions... flow, mobile phase, %composition (iso or grad), inj vol, conc, detection settings....).
Column equilibration is a function of the support type (available volume), surface type (coated vs. bound), flow rate relative to column volume, temperature and mobile phase properties. In HILIC mode (not column), it can take a long time because the column support is having trouble maintaining the thin water layer (maybe 2-3%) on the surface and ANY change in mobile phase composition (and all of the items I noted earlier) can result in instability in that layer resulting in very slow equilibration. For more conventional modes of liquid chromatography we sometimes say that a column is often equilbrated after passing 10 column volumes of mobile phase through it. However, this is just based on diffusion and a generalization. In real life, because there are so many variables (and the time needed for the detector to stabilize is another one), it will take as long as it takes.
We have one LC method that we run in our lab where the total run time is 3 minutes and the column is re-equilbrated and ready to go in 5 minutes (on a 2.1 x 30 mm, C18 column). Other methods, with larger volume columns may take 30 minutes to run, 20 minutes to wash and 30 minutes to equilibrate again (or even 3x times that). Too many variables to state "this is how long it takes". I think I said once before that "it will take as long as it takes". I have been professionally teaching chromatography for almost 30 years and that answer applies to all columns and modes of chromatography.
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Hi experts,
Can we use MRM transition using higher m/z value using QTRAP4000 (for example 1300.3>350.2)? Just curious. QTRAP4000 can scan maximum 1200 m/z then how can it scan for >1200 m/z  compound in MRM mode? Please let me know if this is possible?
Thank you for your anzwers
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Dear Sangita,
In case you´re working with peptides these will preferentially ionise as doubly and triply charged ions and this way you´ll be able to monitor compounds with Mw>1200 (using your own example you may scan the transition 650.6>350.5).
Hope this helps,
Ana
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Dear Peter Hastwell, is the genedisc reader commercially available? How the DNA assays are performed on the consumable discs? genedisc project
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Thanks for the information. Raustech’s platform 3rd generation electrophotography sounds very promissing.
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I don't have kit for molecular work. So is there any simple method specifically for the purification of this fungus? Any references can be provided? Thanks in advanced!
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hi
i hope this file help you .
DNA extraction
The samples were frozen in liquid nitrogen in 0.5 ml tubes
and crushed thoroughly using a micropestle. To each sample
100 ul of lysis buffer (200 mM TrisHCl pH 7á5, 250 mM
NaCl, 1 mM EDTA and 1% SDS, w/v) was added and
mixed thoroughly. The samples were subjected to three
alternate cycles of freezing in liquid nitrogen and incubation
at 100°C for 1 min, followed by freezing and a ®nal
incubation at 100°C for 10 min. The contents of each tube
were centrifuged at 11 000 g for 3 min and the supernatant
collected carefully. The supernatants were passed through
DNA puri®cation columns (QIAquick, Qiagen) according to
the manufacturer's instructions. The puri®ed DNA was
eluted in 50 ul 1 mM TrisHCl pH 8.5 prewarmed to 55°C.
good luck
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Hello experts,
I want to build a calibration curve by purchasing the lipid mixture containing several group of lipids with different concentration (For example: PC, PE, CE, SM with concentration 100ug/ml, 20ug/ml, 5ug/ml, 40ug/ml). My concern is- Do I need to use individual lipid to make a calibration curve or use mixture of lipid at different dilution to make calibration curve for different lipid in the mixture?
Also does anyone can suggest me if I can use any matrix for my internal standard to make the matrix closer to plasma or serum? I want to make calibration curve by extracting lipid from the  matrix rather than just the organic solvent mixture.
Please let me know if my question is not clear.
Thank you.
Sangita
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If the PC, PE CE and SM are mixtures, the mixture would have to be thoroughly  characterized (%14:0-18:1, %16:0-18:1, %18:0-18:1, %14:0-18:2, %16:0-18:2, %18:0-18:2, and etc.) to be of any use as an external standard. It would be better to use a pure lipid like DPPC and quantitate all the other PC's against this pure standard; then use DPPE for PE's and etc. You can always do relative response experiments to show the accuracy of this method, if pure standards for the other lipids with different side chains are available.
As for matrix we simulated plasma by using phosphate buffered saline with fatty acid free BSA (or HSA) at 20mg/ml.
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I make ESI emitters for LC-MS nano-column packing with P2000 Sutter puller. The problem is that I cannot find a parameter set which will provide me with a large orifice for the emitter (in the manual they promise an orifice from 2 to 15 um). So far I get emitters with the cone ID around 3-5 um for 75 um ID capillaries and ~1 um for 20 um ID capillaries more or less independently of the parameters I use. I want to get 10 um for both of them. Parameters I tried so far ranged in temperatures from 240 to 330oC (no principal difference), pull force 10-20 (similarly, no principal difference) and time delay 128 everywhere. It is possible to go very far from recommended parameters, but it will take time.
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Dear Murat, the task is to make emitters which add as little post-column dead volume as possible. The column ID is 75 um and the flow rate is 300 nl/min. Addition of large ID capillaries as emitters will end up with peak broadening. That is why it is necessary to make emitters from as small as possible ID capillaries (well, in reasonable limits). But it is also good to keep the opening of the emitter as large as possible (ultimately, as large as ID of the capillary - like in TaperTips). Otherwise small opening leads to higher clogging probability and higher backpressure. NewObjective somehow make such capillaries. The question is  how....
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Dear friends, is there any guielines for bioanalytical method validation for endogenious substances?
For example:
I have clinical trial with drug that contains caffeine. How to show specificity of the method for endogenous substance that presents in blank plasma? 
May I use water solutions of caffeine as the blank samples? 
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Dear Pavel,
Using a valid HPLC method you can quantify the amount of caffeine in blood samples.
As I am recently working on it, I use the buffer(pH 2.5 to 4.00) -acetonitrile combination as a mobile phase and PDA column as a stationary phase. First validate the method using different concentration of caffeine solution by using the same ratio of buffer-acetonitirle as a solvent and make a standard curve. Then run the blank serum sample from volunteer using  exclusion criteria like not taking any medicine or food that my contain caffeine. After that run the spiked serum sample of caffeine to specify the retention time. Lastly run the samples of your clinical trial and quantify the amount with the help of standard curve.
Regards
Md. Mazharul Islam Chowdhury
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While performing recovery test with an extraction method of protein precipitation, the recovery results for all QC levels are always above 100% , the area's of Bio-analytical samples are higher than the aqueous samples . what can be the explanation ?
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Hi,
If the actual recovery is 100% then maximum acceptable +15% method error could be a reason. Out of 15% means you have ion enhancement/suppression.
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To analyze the terpenoids or compounds of fungal extracts which is a suitable way like UV Vis Spec,GCMS ,FTIR ? How to quantify the terpenoids from fungal extracts?
If we use UV Vis Spec for fungal compound characterization how to analyse by using online sofwares? If there are free softawares please provide
also provide the same information about GCMS ,FTIR of fungal compounds
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 Dear Sir thank you for suggestions.
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I have seen that some people had recorded at 570 nm whereas some people had done it at 590 or 595 nm. People keep reference wavelength as 625 nm. 
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Hi Mukesh
The optimal wavelength for absorbance is 570 nm, but any filter that absorbs between 550 and 600 nm may be used.
good luck
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Hi, everyone
Rencently I need to collect Standard Raman Spectra Data to test the measured quality/precision of my optical system, especially for the case of Human skin/nail (Laser: 785 nm wavelength). I am not really sure about the range of the Raman Peaks, although I already readed some articles. Can anyone suggest some typical or widely-accepted published works for reference?
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If you're trying to assess the quality/precision of your equipment, as you say, the more appropriate sample to use is an atactic polystyrene standard (often used by pharmaceutical companies to "calibrate" their infrared spectrometers for regulatory purposes, but also good for Raman).  Renishaw's webpage (publically viewable) happens to have a spectrum available, although if you search "polystyrene raman" you'll find several publications that would have the peak frequency information.
Although human skin and fingernails are not standards and really not great for calibration, you might still want to know for the purpose of interpreting your data.  There are papers on Raman spectroscopy on human skin and fingernail (links attached).
Also, I believe keratins will feature prominently (and there may be experts in this forum who know better).  There was a paper a while back on Raman vibrations of bird feather keratins, so that might be a place to start (attached as link, below), so that you might have a cleaner spectrum to examine.
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Facing difficulty in extraction of Ergocalciferol and hence in the Assay.
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The following German reference procedure is available:
TECHNICAL RULE
BVL L 49.00-1:1991-06
Title (german) Untersuchung von Lebensmitteln; Bestimmung von Vitamin D in diätetischen Lebensmitteln
See also this article:
Abstract: A standardized method to determine the vitamin D content of food by means of HPLC is described. After the test material was homogenized and saponified with ethanolic aqueous potassium hydroxide solution, vitamin D was extracted usingn-hexane. Using HPLC on a silica gel column, the fraction containing vitamin D is separated from the nonsaponifiable residue. After the fraction was reduced, the residue was dissolved in methanol and the vitamin D content determined after HPLC separation on an RP-C18 column. For evaluation, either a conventional external standard method using laboratory and matrix specific recovery rates as correction factors, or an internal standard method using vitamin D2, and D3, respectively as internal standards were employed. The method was developed and standardized by the working group “Vitamin Analysis” in accordance with § 35 LMBG (German Food Act). Repeatability and comparability of the results were checked in collaborative studies (14 laboratories) in milk powder and gruel that had been enriched with vitamin D3. Applicability of the method to other food (eggs, milk, fish, margarine) was checked separately. The statistical evalution of the results of the collaborative studies has shown that the method is reliable enough to be included into the “Amtliche Sammlung” (official collection of analytical methods) according to § 35 LMBG. The present method may be used to determine the content of vitamin D2 and D3 in natural and vitaminized food. It is specific for the vitamins D2 and D3 released during saponification of food, but does not allow separate determination of the pre-vitamins D already present in the sample.
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We are looking for an appropriate internal standard for the analysis of toxins in human plasma. Our sample preparation consist of a heating step to denaturate proteins, followed by ultrafiltration to determine the total toxin concentration. However, we are also interested in the free toxin concentration. Therefore, plasma is ultrafiltered in a first step.
An ideal internal standard would be a compound that is not present in healthy subjects nor in patients, doesn't bind to proteins (for the free toxin concentration analysis), is stable during heating at 95°C and is detectable by fluorescence.
Thank you for your suggestions!
Olivier
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For that toxins ? Mycotoxins ?
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There have been few tests that I have seen done, and the processes are out of date and quite extensive. I was hoping there may be an easier process (if you know a way to do it, please provide as much detail as possible) so that I may perform the experiment at my university for my senior project. Thank you.
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 There are many publications describing the quantitational methods for histamine in many different sources using LC-MS/MS MRM detection.
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Hi everyone
Can I ask whether MALDI can detect organism straight from the positive bottle without first growing the colony/colonies?
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Barnini, S., Ghelardi, E., Brucculeri, V., Morici, P., and Lupetti, A. (2015). Rapid and reliable identification of Gram-negative bacteria and Gram-positive cocci by deposition of bacteria harvested from blood cultures onto the MALDI-TOF plate. BMC microbiology 15, 124.
Carbonnelle, E., Mesquita, C., Bille, E., Day, N., Dauphin, B., Beretti, J.-L., Ferroni, A., Gutmann, L., and Nassif, X. (2011). MALDI-TOF mass spectrometry tools for bacterial identification in clinical microbiology laboratory. Clin Biochem 44, 104-109.
Deak, E., Charlton, C.L., Bobenchik, A.M., Miller, S.A., Pollett, S., McHardy, I.H., Wu, M.T., and Garner, O.B. (2015). Comparison of the Vitek MS and Bruker Microflex LT MALDI-TOF MS platforms for routine identification of commonly isolated bacteria and yeast in the clinical microbiology laboratory. Diagn Microbiol Infect Dis 81, 27-33.
Kohlmann, R., Hoffmann, A., Geis, G., and Gatermann, S. (2015). MALDI-TOF mass spectrometry following short incubation on a solid medium is a valuable tool for rapid pathogen identification from positive blood cultures. Int J Med Microbiol 305, 469-479.
Schneiderhan, W., Grundt, A., Wörner, S., Findeisen, P., and Neumaier, M. (2013). Work flow analysis of around-the-clock processing of blood culture samples and integrated MALDI-TOF mass spectrometry analysis for the diagnosis of bloodstream infections. Clin Chem 59, 1649-1656.
Singhal, N., Kumar, M., Kanaujia, P.K., and Virdi, J.S. (2015). MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis. Front Microbiol 6, 791.
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For GMP application, I have to validate the test for specificity , reproducibility and sensitivity.
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Hey Mariangela,
I hope this Website might help you:
For GMP-compliance, you would need to meet all criteria. I would recommend you to perform a spiking experiment. Besides specificity, this might give you an idea about your upper and lower limit of detection.
For further information, just search for the USP (U.S. Pharmacopeial Convention) 1225 paragraph online.
Much of success!
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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 ?
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Now, "Thermo" (the manufacturer of "Nanodrop" UV/Vis spectrophotometers) also offers the NanoDrop 3300 fluorospectrometer.
According to the (Thermo) company:
"Utilizing a patented sample retention system it minimizes the mass detection limit while increasing sensitivity, allowing measurement of samples as low as
1 picogram/µl of dsDNA. The flexibility of the NanoDrop 3300 allows users to work with multiple brands of assays, giving them the freedom to select the best assay for a specific workflow."
I didn't use the above instrument, so I can't tell you about any actual performance and/or price.
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Can anyone have method/reference to quantify epsilon-aminocaproic acid by using HPLC specially without derivitization method.
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Hello Uma:
See the link below:
Praveen
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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
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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).
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I am conducting field trials in Australia of different cultivation methods of high artemisinin yielding Artemisia annua hybrids.
Australian bioanalytical mass spectrometry facilities using liquid chromatography tandem mass spectrometry (LCMS/ MS) for the quantification of artemisinin in plant material, are beyond the budget of my self-funded research. I am searching for valid, accessible and affordable international alternatives. Any advice that may help with my project is also appreciated. 
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For high yielding plants it's not necessary to use MS,  HPLC or UPLC with a Light Scattering Detector works well. Unfortunately I'm in South Africa and getting plant material to me would be difficult but not impossible, I did a similar set of analysis for a local grower. 
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I've tried increasing the concentration of both capture and reporter agents to increase binding capacity on both ends of the sandwich, however the peak of the 'hook effect' hasn't shifted.  However, the hook effect is now more of a plateau.  Perhaps the secondary ab is limiting.  Either way, I'm wondering if anyone has feedback on this as I investigate.
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There will be a maximum amount of capture attainable.  Perhaps you are at that maximum?  Not knowing what your antibodies are, I would suggest that the coating Ab be polyclonal  And the detecting Ab monoclonal, or if poly, then a different species of course.  The polyclonal coat will give you the best coat coverage with the highest epitopic hold of analyte.  I would also suggest running a multi-row test of different coating antibody dilutions, with a range of analyte that goes as high as you think you'd ever want to go, while also having a low end that is solidly in the linear range of what you have now.  That way you can tell if the higher attainable analyte is destroying your ability to see changes at the lower end.
The most direct method of getting a higher range in a sandwich will be getting the highest coat possible.  But at some point that is going to prevent you from seeing the dilution effects at the low end of the dilution profile.
I would doubt the secondary Ab is limiting anything.  As long as it is a normal secondary, and you're not into the 1:100,000 range or something like that.
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Can we use the plasma of controlled group rats in combine? Or the same rat?
Is there a method data is reliable or not when we used more rats plasma for the development and validation?
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You need to review how many test you'll do, then you can calculate the amount you need. 
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I wish to know which is the appropriate truncation point half of the dosing interval or Tmax for calculating for calculating Partial AUC in bioequivalence studies.
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Hi Nitin,
It depends of the drug!, but always have to be pre-specify and justified in the protocol, in general terms the european guideline recomend to saparate early and terminal using the half of the dosage interval; FDA recomends to truncate the partial area at the population mean of tmax values for reference formulation, but as I say before, it depends of the drug and the state of the art, i.e. recently the FDA published a specific guideline to evaluate BE of Methylphenidate modified release formulation proposing 3 partial areas instead of the usually early and terminal; and sometimes the time to separate the partial areas vary from a fasting state studies to fed state studies.
hope it helps
saludos
Juan Manuel
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I worked in Bioanalytical method development and validation of two drugs, Paracetamol (PCM) and Pamabrom (PAMA), the multiple reaction monitoring (MRM) of the transitions m/z 152.10→110.10 for PCM in positive mode, m/z 256.90→199.90 for PAMA. PCM fragmentation pattern was easily understood, but PAMA fragmentation pattern was not clear and it is also not available in Review of literature. can anybody try and explain???? 
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The m/z 256.90 to 199.90 transition i.e. 57u loss for PAMA is probably due to CH3NCO loss from dimethyluracil ring. This is RDA (retro-Diels-Alder) fragmentation reaction, very common in compounds, which contain uracil moiety.
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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)?
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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-homogenizers
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Is there any LC-UV analytical method available for quantitative determination of Trypsin and/or Chymotrypsin?
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Sir I need to quantitate the content of trypsin in a mixture of composition of trypsin-chymotrypsin powder. The activity ca be determined through UV-vis spectrophotometer kinetics experiment.
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We are developing methods for the quantification of lipids preferable in glycerol, squalene and cholesterol in sebam sample using HPLC method. 
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Unfortunately glycerol is UV-silent so you will not be able to detect free glycerol with a UV detector without some sort of chemical modification.  We has a similar issue several years ago and developed a HPLC/UV-based method to detect benzoylated derivatives of glycerol and other polyols from biological samples.  The method is pretty simple, works well and provides excellent resolution and reproducibility (Journal of Chromatography B (2009) 877: 3667-3672).    At the end of this manuscript we also compare results using this method to other available methods, which may be of help if you decide not to use HPLC.
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Solution preparation.
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I am assuming the serum is none diluted. Add 3 ml of BSA to 97 ml diluent (buffer, saline or Water)
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Talking with colleagues, it seems that the possible usefulness of an HPLC method development software repeatedly comes into the analyst's mind during the development of a tough HPLC/UPLC assay. Could you share your experiences with HPLC method development software? Which one would you choose and why (reliability, ease of use, intuitive usage etc)? With real samples, do they provide information as reliable as stated by the vendors? Are there any drawbacks?
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that's right . the methods in HPLC actually depend on the researcher himself .what is he want ? type of the reaction mixture , the source of the sample and the interference s with it all of these make u select the proper method with proper conditions.
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I have a HPLC system with a UV-detector and I want to determine the drugs in a sample having concentration range 1.0 ng/ml to 100 ng/ml. Is it possible to determine on UV detector? If yes please suggest the best way to determine the sample in this concentration range. Please suggest the best way for analysis on a HPLC system.
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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.
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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.
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Right now I am working on the Liposomal Formulation method of analysis.I face the problem that the exact value of free drug and encapsulated drug vary every time for precision and accuracy batches and do not meet the nominal values.
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The free and encapsulated drug have different microenvironment (microacidity, micropolarity, effective dielectric constant ) inside the liposome in comparison to bulk solution. Depending on the drug and liposome nature the distribution of the drug between bulk solution and liposome exists usually in milli- or microseconds interval. So the ratio between bound and unbound drug can be fixed by different methods for example spectroscopic (molecular absorption or luminescence, or NMR etc...) and electrochemical. The molecules that change their characteristics in different media usually call "spectroscopic probes". If your drug spectra are changed very insignificant you can find the "probe" molecule that have very high absorbtivity (molar extinction coefficient) or luminescence (high quantum yield) that can be added to your system in micromolar or nanomolar concentrations and can change its distribution and characteristics simultaneously with your drug. It can help you to see the changing of drug distribution with time and temperature. These changing can be mesure by using calibration curve.
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I am trying to see if I can analyse amino acids in animal feeds blends and am curious if anyone can assist with finding a sample preparation and HPLC method.
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You can prepare your sample as follows, first hydrolysis of amino acids where amino acids . The Amino Acids are hydrolyzed in order to cleave the peptide bonds in the proteins to release the free amino for analysis using HPLC.
Method: The common method used for Amino acid hydrolysis is by adding 6N HCL and heating at 110ºC for 24hrs or 145ºC for 6hrs.
CAUTION:
Some Amino acids are chemically unstable in physiological fluids (eg.progressive decline of plasmaglutamine and cystine in time) and also in the standard mixtures keeping samples and standard mixtures which have not been stored properly should not be used for instrument calibration, order a fresh one.
Samples for analysis is recommended to prepare a fresh sample, Samples prepare during
the day may be left on the auto sampler tray at room temperature to be analyzed during
the night or the next day.
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If Linearity is performed for determining the minimum and possible maximum strength of formulated product, then is it necessary to perform Linearity separately for LOD and LOQ determination or LOD&LOQ can be calculated indirectly from the one time Linearity?
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Do you really need to calculate LOD and LOQ in method validation related to "Assay" test of formulated drug? Assay method do not require LOD and LOQ parameter as you are not going to get such a low result. As Kar-Weng Chan has written, that LOD value is determined by running low concentration standards. For Assay method, Linearity and Range at the concentration level of 10% to 200% of its initial concentration is ok. If, at all LOD and LOQ parameter are required in AMV of Assay, then it should be determined separately. Generally we do not consider LOD and LOQ parameter in AMV related to Assay of formulated drug.
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Are method development and validation guidelines mandatory for validation of new methods or is it also accepted to just validate the parameters of interest and which are specific importancy for that new method. I like to know other persons experiences and thoughts, because I personally think that full validation practice of new methods should be mandatory.
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Dear Roland, I could infer that:) you were seeking opinion! I think validation depends a lot on our work settings, geographic location, resources available, and a cohort of variables! All that we can do it, try our best, given our limitations!
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Should these guidelines be applied considering there is typically no blank matrix for calibration?
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The analyte free blank matrix has to be prepared by using techniques like charcoal stripping and than validation should be done with anlyte free matrix. Else the baseline value has to be derived and the procedure for deriving baseline is also mentioned in few of OGD (for e.g Progesterone OGD). The impact of stripping also needs to be evaluated by comparing stripped vs unstripped.
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Analytical method
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Dear Haraeendran, please be more specific !
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Does somebody have experience or published a paper discussing pre-method validation of new bioanalytical methods prior to full validation?
I found a discussion about this subject in the Bioanalysis discussion group on LinkedIn.
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Dear Gellert,
another guidelines available for validation of bioanalytical methods are the guidelines from the European Medicines Agency (EMEA) introduced in February this year, so very recent!
Check here for the guideline: