Also can you please give a brief explanation of the principles underlying the analytical procedure described below for the determination of amphetamine in plasma by gas chromatography:
To 1 mL of the plasma sample, 3 mL of diethyl ether and 100 µL of 5 M sodium hydroxide were added. This mixture was shaken in tubes for 5 minutes and then centrifuged. The diethyl ether layer was transferred to a clean tube containing 1 mL of 0.1 M sulphuric acid. This tube was then shaken for 2 minutes and then centrifuged. The diethyl ether layer was removed and discarded.
To the acidic layer was added 3 mL of clean diethyl ether and 200 µL of 5 M NaOH. The tube was shaken for 5 minutes and centrifuged. The diethyl ether layer was transferred to a clean tube and evaporated to dryness under nitrogen. The dried extract was derivatized using heptafluorobutyric anhydride and the derivative was injected onto a gas chromatograph using a CP-Sil 5 CB (fused silica) capillary (10m x 0.53mm), oven temperature 125OC, and electron capture detection.
With an external standard you make a calibration line for quantification with different concentrations. Then you should also determine the recovery rate to know how much of your analyte is lost during sample preparation.
With an internal standard (IST) your determine the amount of your analyte by the response factor (intensity ratio of IST and analyte). The IST must be very similiar to the analyte, eg one methyl-group more. But your chromograhic conditions must be able to seperate analyte and IST. Additionally your sample must be free of this compound. You can add the IST before sample preparation. Due to the similiar structure and polarity loss of your analyte and IST during sample preparation should be the same. You can also quantify more analytes with the same IST, if the chemical and physical similarity is high enough.
Generally speaking, internal standard is required when you need to cope with large fluctuations which occur at different stages of the analytical method used. That's why, the EI structure must be as close as possible to your product of interest, capable of similar behaviour for instance during the extraction stage, the derivatization, the injection on GC and so on. In most cases, one uses as EI, the product of interest modified by means of stable isotopes (13C, 15N).
To answer your question 1:
The use of internal standard in bioanalytical work is almost mandatory. It is usually not used to solve simpler analytical tasks.
Question 2.
The explanation of the analytical protocol for the determination of amphetamine. Amphetamine is transformed to the free base for by adding NaOH and the base is extrated by ether which is immiscible with water. The acidic constituents of plasma remain in the aquoeous phase. The thereal layer is extrated by aqueous sulfuric acid. Ampthetamine is transformed to its water-soluble sulfate and is present in the aqueous layer. The ether layer contains neutral constituents not extractable with aqueous sulfuric acid. In the course of the second alkalinization step amphetamine is again transformed to the free base form and extracted by ether.
The derivatization by heptafluorobutyric anhydride is on the one hand increasing the volatility of amphetamine and on th other hand increase the sensitivity of the detection by ECD.
You can use an external standard when your method is stable and the recovery is sufficient (about 80% or more). You can compensate variations in your analytical procedure by adding an internal standard at the start of the extraction protocol. If you decide to do so, you need a standard which is chemical similar to amphetamine. Internal standards can be added prior to HPLC to improve the quantification step only. Usually this improvement is smaller for HPLC than for GC.
If you are unsure if your method is good enough for an external standard, you have to add amphetamine to blank matrix (plasma without amphetamine) and process this sample e.g. 10 times with your protocol and quantify with external standard. Then look at the results and decide if the results are good enough for your purpose.
With an external standard you make a calibration line for quantification with different concentrations. Then you should also determine the recovery rate to know how much of your analyte is lost during sample preparation.
With an internal standard (IST) your determine the amount of your analyte by the response factor (intensity ratio of IST and analyte). The IST must be very similiar to the analyte, eg one methyl-group more. But your chromograhic conditions must be able to seperate analyte and IST. Additionally your sample must be free of this compound. You can add the IST before sample preparation. Due to the similiar structure and polarity loss of your analyte and IST during sample preparation should be the same. You can also quantify more analytes with the same IST, if the chemical and physical similarity is high enough.
An internal standard should always be used in order to compensate for all losses involved in the sample preparation. The internal standard compound should behave the same as the compound of interest. The best is to use the same compound but labeled with stable isotopes, for instance a deuterated compound. It behaves analytically the same. But then you need LC/MS as the detection system.
Some clarification on the use of internal and external standards. Both are used for quantification depending on the condition of the analysis. Choice of an internal standard is usually based on similarity of properties to those of the analytes. For this reason quantification is based on ratio of peak height/area of the analyte to that of the internal standard. Use of internal standard also compensates for matrix/measurement effects. On the other hand, external standard is often used where matrix/ measurement effects are minimal, e.g. less complex sample matrices.
Sam and others provide a clear explanation for the difference between external and internal standard and their use. I would like to add from my experience from a laboratory carrying out routine samples for control of food safety (where we had to avoid errors by all reasonable means),that it appears to be good practice to use both an internal standard (with similar properties concerning sample clean up and chromatography as the analyte) and an external standard (which should have similar chromatographic behaviour as the analyte). The internal standard will help to detect and account for issues related to samle extraction/derivatisation/clean up and the externa standard will control the quality and stability of the chromatographic system. During method evaluation you will see which of the standards is better suitable for quantification. However, you may want to keep the second standard for control purpose during further analses.
I agree with the collegues the use of internal or external standard depends of the method of analysis
I agree with all. Another approach that can be useful with or without external standards is the classical standard addition method whereby the analyte itself is used as a standard for a portion of the sample.
I use internal standards if I am concerned about matrix effects; if I need to do an extraction stage and need to confirm extraction efficiency; if I am developing a new method; if I am submitting samples to a new lab; and occassionally for routine quality control. "Blind" standards are also a good way to verify outside labs are doing a good job.
Basically, Internal standard need to be used to take care of errors in the sample injection volumes. The two injections in external standard method, if differed slightly volumwise (e.g. 1 microltre of reference std against 0.95 microlitres of sample or 1.05 microlitres of samples) gives sustantial error in the final calculations. Use of internal standard takes into account the ratio of area of sample to that of internal standard. Ratio never changes. Hence the errors get minimised and the expected answer if much near to reality.
Basically, as Dr balkrishna mentioned correctly that internal standard is used for minimizing the error, when we are using manual injections. But now a days auto sampler will give the very low % RSD of replicate injections. Even many of the official method used external standard.If u are doing manual injection, please choose the internal standard.
Internal standard are generally used when you are developing a method and validating the same. External standards are more likely to be used in comparison and quantification of compounds.
I do not agree with some of the colleagues. Analysis of a compound in a sample with a certain matrix involves different kinds of error. One of them is the injection error which is determined by the injection system. Another one is the extraction efficiency to extract the compound of interest out of the sample matrix. Both the injection volume and the extraction efficiency vary from sample to sample. They are not constant within a batch of samples and not between batches. Therefore each sample, including calibration samples needs its own internal standard. Adding an internal standard to a sample matrix requires knowledge about the uptake of the standard into the matrix. The internal standard must be present in the sample in the same format as the compound of interest. Is it just solubilized or bound to for instance proteins? Is the compound present in the free form or is it present in a conjugated form?
As a conclusion: an internal standard must be added to each sample in tge right format.
Both Internal and external standards can be used for quali and quantitative purposes. external standards (often a standard of the analyte of interest) is employed for comparing retention times (ID) or peak areas (assay). The internal standards MUST be compounds not present in the regular sample and are employed in order to increase accuracy and precision of the determinations.
If you have the possibility to use internal standard methods because you have a good surrogate or stabile isotope labeled inyternal standard, results will be more reliable than with external standard methods. Remember that internal standard are used for analyte loss during sample preparation and sample matrix effects!
My personal preference is to use a reference which is labelled with a stable isotope, for example Deuterium, or whatever. I fully agree with Frans and Roland in this and their other remarks. But what I would advise: use both internal and external standards, plot them against each other per sample and look for outliers in this graph. These will be the samples with a problematic standardization. Most of the samples will just correlate nicely, and pose no problem.
Mr Peter Kissinger can you please enlighten me more on classical standard addition or direct me to a link where I can get more information?
I've used that method long time ago: injecting the unknown quantity of analyte, you'll obtain an area A; adding a known quantity of the same analyte you will obtain an area A+B; a simple calculation you'll obtain the unknown quantity. Anyway Internal Standard is always useful to compensate little changes in the apparatus (pressure, Temperature, time and quantity of injections... aren't always perfectly equal injection after injection).
i am completely agree with Frans due to the practical argument because extraction is easy but difficult process and even simple traces of compounds put major impact on the final results. i already passed through the same problem and the error was in the system.
I proposed external standard calibration for simple matrix sample and internal standard calibration for complex matrix sample. about determination of amphetamine in plasma by gas chromatography, I am agree with Peter Blanckaert.
External standard can be used for quantification, by obtaining a calibration curve . The internal standard is used to correct the analyte loss during all the analytical procedure, and is strongly recommended when you have a elaborated procedure. It has do do more than just the manual injection error. I recommend to use both internal and external standard.
The external standard is used when no matrix effects are present. The standards can be prepared in water or in the matrix IF it can be found without the analyte (e.g. plasma). The internal standards are used to correct for matrix effects (standard addition method with the same analyte added to the sample) or an internal standard different of the analyte, BUT , the standard must be a similar or labelled compound if corrections for loss sample are needed, in this case matrix effects are corrected also since the effect is the same for both analytes. However, this is an hypothesis and must be corroborated. In polarography for example, if you want to quantify Na+, any other atom is equal to sodium cation, so it is possible to use another metal with similar standard potential but similar matrix effects must be assumed. Corrections for loss of sample is not the unique aplication of the standard internal. Anyway, equal behaviour between the standard and the analyte it is expected related to the form those compounds are present in the matrix (bounded, free, complexed, etc...)
If it is quantitaive HPLC then external standard with a precise injection system is best. Adding an internal standard for quantitative analysis can add extra errors. Not many people realsie that UV detectors change wavelength with temperature as the spectrometers grating moves. So if you are measuring the analyte of choice at its maximum absorption wavelegthe errors are generally small if the wavelength shifts a little. However if you have an internal standard in the mixture and it is being measured not at it's ideal wavelength then the error due to wavelength change can be up to 10%. There are also extra errors associated with integrating the peaks when an internal standard is used.
Oh sorry, I tried to explain the quantification methods in general, when sample preparation is involved, but the question is "internal standard in HPLC", and I agree with the answer of Robert Taylor. Injection valves are very precise and no internal standard are necessary UNLESS you have very small amount of sample and injection is with the loop partially filled (e.g. 5 microliters of sample and the loop you have is 20 microliters). In this case the presition is given by the syringe and the internal standard is very convenient.
If you have a small amount of sample you can use internal loops of 0.5microlitre or less and they are very precise (valco make them).
External standard is depended upon ur sample u can use actone , acetonitrile etc
the question is not confusing (Can anyone explain when to use an external standard and when to use an internal standard method in HPLC? ) ....the solvent you use to dissolve the standards does not matter (remember the solvent elutes with the dead volume), unless it affect peak shape as occurs with not very well retained peaks (about k= 0.5 to 2) when you use strong solvents to dissolve the standards (acetonitrile, acetone) in RP-HPLC
Mario, the solvent used to dissolve the standard does matter. Ideally you want the compounds of interest to be in a solvent that is the same or weaker strength than the mobile phase. If the solvent is stronger than the mobile phase you can get peak broadening (If you use a very small injection volume a stronger solvent has a minimal effect). It is also not optimum to use a solvent that absorbs strongly in the UV region if that is the detector you are using.
Robert, I completelly agree....but as I said peak distortion or, as you said, peak broadening is more dramatic for less retained peaks. Of course, what is ideal is to inject in the same mobile phase.....and of course if you inject a solvent and it absorbs in the same UV region that the solute AND ALSO the peak solvent and the solute peak elutes AT THE SAME TIME it is imposible to quantify.....
According to my experience, both external and internal standard methods may be used with quantitative purposes. In external standard, calibration is carried out with pure standards which are the same compounds one wish to quantify in samples and a linear (ideal) relationship should be found between analyte concentration and detector response. Samples assays should then be done under the same analytical circumstances than standard and then the response obtained, which must fall within the linear range or the calibration curve, can be converted into concentration values by using the corresponding linear regression equation.
This is an universal calibration method suitable for most experimental situations. However, in many cases during the extraction of the samples some unexpected losses may affect to the compound of interest (adsorption to surfaces of the glassware used, co-precipitation along with other cellular components, uncontrolled evaporation, etc. In this cases the calibration by internal standard is strongly recommended because it may assume and correct these problems.
The point is the choice of the compound used as internal standard because it must be chemically similar to the compounds of interest in our samples but it should not elute at the same time than any of them. The bibliography at this point should help us to find the proper internal standard.
The construction of our calibration curve with the internal standard method requires employing both the "external" (our compounds of interest commercially available) and the "internal" standard. Mixtures with increasing concentrations of the external plus a given, an always the same, concentration of the internal standard are then prepared and chromatographied. Finally, the ratio of the concentration of the external/internal standard is plotted against the area external/area internal standard ratio, and the linear regression calculated.
To analyse samples, one must not forget to add a known concentration of the internal standard to the solvent where extraction will be carried out and analyse in the same conditions as standards.
I hope this serve you (sorry for the length)
Which method to use is totally up to target compound chemical and physical propertieas , the ways of extraction, clean up and related instruments for this specific test.
If the sample preparation is quite simple and the equipment, especially the autosampler, detector are working very well, an IS may not add any benefit.
An internal standard will benefit the method most commonly when there are multiple sample preparation steps, especially when volumetric recovery at each step may vary.
If it is determined that an IS is likely to improve the data quality, a suitable compound needs to be chosen for the IS.
Internal standard (IS ) characteristics are : sample doesn's have the internal standard; IS chemical property should be similar to target compound; IS should be eluted in same instrument condition; IS should be stable; IS should have similar responce to detector and available in pure form for making proper internal standard solution; IS concentration should be exatly same in both standard solution and in the sample extract.
Here's an article in LCGC that explains it pretty well: http://www.chromatographyonline.com/lcgc/Column%3A+LC+Troubleshooting/When-Should-an-Internal-Standard-be-Used/ArticleStandard/Article/detail/778040
There is a HUGE difference between the use of a surrogate compound (the procedure that Mr. D'Amico describes) and the use of an internal standard. An internal standard is NOT carried through the preparation procedure. It is used to control for variations in the chromatographic system (injection, mass transfer, detector, derivatization, etc).
In general, if you are using HPLC with a spectrometric detector (UV, FLD, etc) then you will see little to no difference between external standard calibration and internal standard calibration. If you are using a mass spectrometer then you may see significant differences between external and internal standard calibration.
If, as your questions suggests, you are actually using gas chromatography with an EC detector, then the use of an appropriate internal standard will potentially make a large difference. If you have excellent control of your chromatographic system, from injection through detection, then the calibrations will be similar in quality. If; however, you are uncertain as to the difference between HPLC and GC methods then I would strongly recommend the use of an internal standard.
I completely agree with Mark. There is a difference between internal std. and surrogate std. Surrogate std. is added before sample prepration and internal std. is added to final sample just to accomodate the variations in the injection volume, gas flow etc. Since you are using GC, I suggest you use an internal std.because unlike HPLC, the variations in GC injections are considerable. I observed one more thing in your procedure. It involves multiple extractions with low volumes and therefore chances of losses are very high. It would be advisable to validate your procedure and also run spiked standards along with each batch of samples.
when you are going to quantify the pure compound with using GC / GCMS/ HPLC by injecting fixed volume (using fixed loop or auto sampler) -- u can use external standard method. If u are extracting something from base material to quantify, u may required internal standard method to measure exact concentration. Because u dont know the efficiency of extraction method. It is also useful in manual injection in GC / GCMS, to avoid injection error due to temperature or vision problem.
At least as the terms are commonly used in the United States, there is no meaningful difference between internal standards and surrogate standards. Instrumentation for the past 20 years has been good enough that differences between injections due to the system are rarely significant. Internal (surrogate) standards should always be used when extracting analyte from complicated matrixes to account for losses in each step. Particulary in liquid-liquid extraction, losses will not be reproducible as different amounts are transferred each time. External standards may work well when doing dilute and shoot type of analyses.
I agree with Mark Krause. However, it is very common to find in the scientific literature the use of the term "internal standard" for both situations. As the method proposed uses a GC-ECD, the use of an IS will help to improve the calibration due to variations in the injection step in a GC system. Moreover, blood samples are very complex and always present a considerable matrix effect together with a large variability in the ionic strenght of each sample. My experience has showed me that the use of a surrogate is always needed to obtain accurate results in this type of samples.
The internal standard (IS) method is better than external method as general and especially when you determine your analyte in plasma. IS method will help you to compensate any expermental error by using the response factor (the ratio between the concentration of the analyte to the internal standard).
the use of internal standard is preferred in the detection of any drug in biological sample but you have to make a calibration curve using the suitable IS (deuterated amphitamine is better if available or any similar compound) using the same method of extraction with unchanged conc. of IS along calibration curve and application step.
External standard method is used when your unknown solution is prepared by dissolving an amount of the analyte in pure solvent(s), but internal standard is used when interference on the signal measured occurred due to matrix composition. So In this case you apply internal standard. By definition Internal standard is compound which has similar physical and chemical properties as your analyte.
The answers above are good. I like using internal and external standards--an external standard injected at the start of a sequence in order to evaluate day to day performance of the instrument, an internal standard to evaluate any loss during sample prep or problems with that particular sample. Of course an internal standard can tell you about the instrument day to day as well, but injecting the standard externally is easier to monitor and would usually be a faster to run, easier to interpret sample.
It is always good to use internal standards if there are interactions between the analyte of interest and your matrix (esp if it is a complex matrix). External standards will not be able to give you the actual concentration of the analyte of interest
I agree with Mark Krause. Internal standard is used along with the sample preparation and external standard solution individually but the concentration and the area are used for calculations.
I found the following resource quite helpful. I had similar questions as yours.
http://www.laserchrom.com/documents/CalibrationPart1ILMarticle.pdf
If using MS as a detector, it would be helpful to have a heavy labeled IS. The Analyte and IS should elute at the same time and resolution of IS and Analyte is by mass. Heavily deuterated IS's can have different elution times, and thus different matrix effects, and may have uncorelated intensity in MS. Other heavy labels (13C, 18O, 15N) can be used without affecting the chromatography.
I am in agreement with previous answers. However, simply conclusion for easily understanding are as follows:
we use internal standard by adding it with known amount in a sample or external standard to compensate or correct the change of the concentration of the target compound (s) in the sample or external standard compound. That means whenever the volume of sample or external standard solution is changed by solvent evaporation after long time storage, usage or accidentally losing the solution volume, the concentration of the target compounds or external standard compound will not change under the same analytical condition.
The internal standard compound must have similar properties with the target compound or internal standard.
Conclusion:
1. External standard is used for calibrating the instrument for specific analysis. The calibration curve with mostly linear equation, obtained by plotting the response (peak height or Area) versus a series of known concentrations of the external standard compound, can then be used for quantitatively estimating the concentration of target compound (s) in the sample solution.
2. Internal standard, by adding in the sample or external standard solution, is used for compensating or correcting the concentration of the target compound (s) or external standard whenever the sample solution volume is changed by any means.
Good luck
I agree with the answer however the assumption for using external standard is that there is a linear relationship over a given concentration range. In internal standard we have catered for any deviations as we run the process.
Hello everyone I want to ask the same thing should I add the internal standard in the reaction mixture after completion of reaction and then starting material, product and the internal standard with ether or
I first extract everything with ether and then add internal standard ????
which method can give me good results and which is standard method to use internal standard
I suggest first one if your internal standard have no reactivity with the crude mass of reaction mixture. If you follow second method there a chance of handling loss and also you not know the efficiency extraction method. This will misguide you. First thing will help you in better quantification of your product.
Theoretically, when the composition of the analyte is totally unknown, the sample is injected to get the peaks and assuming that the detector response ration of all the components is equal, the sum of areas of all the peakes is taken as 100%. Based on this every peak is aestimated for its purity. When tghe composition is known, the purity is calculated by comparison with that of known standard. This is aclled external standard method. In order to take care of manual error of injection volume, the internal stanadrd method is used so that even though there is an error in the injection volume, the ratio of peak area of sample and that of internal standard does not change and the answer is more accurate and reliable. Hope this explains the basics.
In HPLC the risk of volume variation in the injection is low. The main reason to use IST in HPLC is to avoid errors due to analyte loss in sample preparation. If your recovery is low, IST is advisable. One can also compare IST and EST in the same analysis since there is enough information for both calculations. Are results for IST and EST statistically the same? If the results are the same, IST is useless. If results are statistically different and IST is higher than EST, proceed with your recovery evaluation, but this is a strong indication that analyte loss in sample preparation has indeed occurred. In GC, however, the risk of volume variation in injection is too high and IST is always advisable.
Principles Underlying the Analytical Procedure
I will explain along your text
To 1 mL of the plasma sample, 3 mL of diethyl ether and 100 µL of 5 M sodium hydroxide were added.This mixture was shaken in tubes for 5 minutes and then centrifuged. The diethyl ether layer was transferred to a clean tube containing 1 mL of 0.1 M sulphuric acid. This tube was then shaken for 2 minutes and then centrifuged. The diethyl ether layer was removed and discarded.
It seems that most constituents - or interferents - in your sample are neutral in basic pH and can be transferred to the diethyl ether layer. However, when you add sulphuric acid and shake your mixture, at least your analyte is transferred back to the aqueous layer. In this way, you get rid of your interferents, but preserve your analyte.
To the acidic layer was added 3 mL of clean diethyl ether and 200 µL of 5 M NaOH. The tube was shaken for 5 minutes and centrifuged.
Your analyte is finally transferred to the diethyl ether layer.
The diethyl ether layer was transferred to a clean tube and evaporated to dryness under nitrogen.
You remove your solvent and concentrate your analyte.
The dried extract was derivatized using heptafluorobutyric anhydride and the derivative was injected onto a gas chromatograph using a CP-Sil 5 CB (fused silica) capillary (10m x 0.53mm), oven temperature 125OC, and electron capture detection.
You derivatize your analyte to turn it into a volatile molecule that can be traced and identified in your GC. Your derivative is halogenated due to your derivatization technique and the best detector is electron capture.
This is a very old thread, but since it was at the top of my search results when looking for something else, I figured I would chime in. As to WHEN to use an internal standard vs. external, that wasn't really answered. You want to use an internal standard when your instrumental response may change during your analysis. For example, we perform a lot of LC-MS/MS analyses, and over several hours, the instrument response tends to change. If I use an external calibration curve on a batch that is several hours long, I cannot be sure that an external calibration curve constructed at the beginning of the batch accurately represents samples the end of the run, and vice versa. Since the internal standard calibrates using a ratio of internal standard to analyte, instrumental drift becomes irrelevant.
Yes. And the earlier in the sample prep an isotope labelled IS is added the more it can also protect against errors/variance in the sample prep steps. But nothing is perfect.
This article is an adequate answer to that question:
https://www.scielo.br/pdf/qn/v33n4/41.pdf
Good reference. To add to the confusion, a "standard addition" can be another useful "classical" concept and can be used with the other approaches to build confidence, although it takes more time.
To my knowledge, we use external standard of targeted analyte for calibrating analytical instrument so that we can calculate the concentration of targeted analyte in a sample solution. If manual injection of HPLC is used, external standard cannot help to confirm the identification (retention time) of the targeted analyte since retention time may not the same except MS detector is used. To solve the problem, internal standard (isotopic standard of the analyte or any reference compound) can be used. By this means, this internal standard is added in the standard and sample before being subjected to injection. Then relative retention time (difference in retention times of the internal standard and the targeted analyte) will mostly the same even though the retention time of analyte peak is shifted.
Hope my explaination help you understand this matter.
Most people focus these "standard" terms of art on calibrating for a quantitative result and not confirming the identification of an analyte. With modern instruments retention times are quite good/reproducible. Identification is best confirmed with a signature spectrum, when available.
Continued from my first explaination, internal stardard will also help to correct your analysis results. By this means, it help any loss (further dilution without known volume but enough for analysis) or gain (unavoidable solvent evaporation during storage). In this case, internal standard will help correct the analysis result due to constant relarive response factor ( ratio of peak area/height of external standard/analyte and internal standard) to the external standard/analyte concentration will be the same. Remember, this internal stardard is added to external standard/sample befor injection.
If you use internal standard in the sample before extraction procedure for testing recovery, internal standard in this case is called surogate standard.
Uhm, one thing in different place may be known/called by different name. So, Understanding is very important.
With modern instrumentation of HPLC, autosampler is used and in this case internal standard is not needed for identification (rention time).
Also it is depended on the detector to be used. For MS detector, internal stardard is not need. Identification is helped by targeted fragment ion.
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