In setting the specifications for a pharmaceutical substance, its unknown impurities must typically be less than 0.1% area/area with respect to the main signal peak using the standard detector for that particular method. The usual instrumentation is reverse-phase liquid chromatography. Even so, regulatory agencies prefer that impurities be identified. Impurities that might structurally resemble genotoxic substances should be absent. If an unequivocal structure has been assigned to a minor component, it is possible a higher concentration of that impurity level can be accepted by regulators providing an extra incentive to discover that structure.
HPLC-Mass Spectroscopy & HPLC-MS-MS
With the correct instrumentation and method development, a skilled analyst can greatly reduce the number of possible structures for an HPLC impurity peak. Nearly always this requires that the HPLC mobile phase consist of either a volatile salt buffer, such as ammonium acetate, or no buffer at all. When developing new analytical test methods the first choice for buffers should be volatile buffers. General analytical methods are well described and easily searchable so no more need be said here.
It is worth discussing what can be done when the above standard approaches fail or are inapplicable for some reason. For one thing, it is sometimes possible to narrow down a list of suspect structures and devise means to isolate or synthesize a sample of the hypothesized material.
It is worth discussing what can be done when the above standard approaches fail or are inapplicable for some reason. For one thing, it is sometimes possible to narrow down a list of suspect structures and devise means to isolate or synthesize a sample of the hypothesized material.
Clues Suggestive of Structure
- Is the impurity acidic, basic or neutral? This can be determined by aqueous acidic and basic extractions.
- Does the quantity of impurity remain fairly constant with respect to the main component even when different methods of purification are tried? If yes, this is suggestive, either of a homologous structure in which there is some slight difference in a side-chain between the impurity and the active drug, or of a positional isomer relationship between the impurity and the main constituent. Such impurities usually come from impurities in one of the starting material building blocks.
- HPLC is the most common present-day method of pharmaceutical analysis and diode array variable UV wavelength detectors are routinely available for such analyses. So how does the UV spectrum of the impurity measured by the diode array detector compare to the UV spectrum of the main product? Does this narrow down structural possibilities?
- Is the impurity’s increase most probably a function of the degree of scale-up?
- Have you performed a laboratory run in which the duration of the addition times are the same as in the plant? Even if obtaining very slow rates of addition on the laboratory scale is too technically difficult or requires unavailable equipment the same effect might be obtained by adding a quarter of the dropping funnel charge, then waiting for ¼ of the plant addition time; adding the next quarter of the charge and waiting another quarter of the plant addition time and so on. Impurities often arise from the wider variation in the stoichiometric ratios that are present during the lengthened addition period on scale.
- Is the impurity occurring only in the most recent runs and not occurring under somewhat different earlier conditions? This is a key question that arises out of Kepner-Tregoe problem analysis. The answer may trigger an insightful guess at the structure of the impurity connected with the possible change that caused it.
- Do you already know some means to obtain a sample free of this impurity? Even if this is expensive and impractical it provides information to fashion a separation/identification method.
- Is the main component (active API) reactive with some easily removed and quantitatively reacting material (such as hydrogen)?
- If one prepares the sample for analysis differently, does the impurity increase, decrease, or remain the same? That is to say- is it actually an artefact of the analytical method?
Swish Chromatography
Does “swish chromatography” increase the relative concentration of the impurity versus the main peak?
Could trituration (swish TLC) with an atypical liquid in which the main component is poorly soluble give an enriched composition? Among atypical solvent I would include methylamine, ethylamine, sulphur dioxide, dinitrogen-pentoxide, carbon disulfide, nitromethane, acetonitrile, perfluoromethylcyclohexane, tetrachloroethylene, trifluoroacetic acid, carbon tetrachloride, dicyclopentadiene and perylene sulfone. These solvents are quite volatile and can be readily removed. The perylene sulfone and dicyclopentadiene both decompose to volatiles upon heating under vacuum and methylamine, ethylamine, carbon disulfide, sulphur dioxide and dinitrogen pentoxide are gases under normal conditions or are very volatile.
Is extractive crystallization possible to selectively phase switch the impurity?
Switching from HPLC to TLC for Isolation
Is there some means to find the equivalent TLC, Rf for the impurity, which you are identifying in HPLC by a RRT?
On this question, I do not know what the literature provides but here is a possible method: Suppose one runs a preparative TLC using a solvent system that gives an Rf of 0.05-0.1 for the main component but one performs multiple elutions on the plate and then removes the adsorbent in horizontal strips from that plate and runs these according to one’s HPLC procedure. Multiple elutions with a poor solvent system provides the best chance for separation of impurities. If the impurity is not co-eluting on TLC, this HPLC analysis of bands from the prep plate will locate the TLC Rf range where the HPLC impurity of interest is located. Even when this Rf region has been located, the impurity is not necessarily one that can be readily visualized on the TLC plate. The HPLC impurity may be undetectable by conventional TLC at the concentration being spotted. Nevertheless, if it works, you have found a preparative scaleable method for separating the impurity even if you cannot detect the impurity by normal visualization. If the location of the HPLC impurity is well resolved from the main compound, you will be able to simplify your TLC method by increasing the polarity of the elution solvent trying to get a method that only requires a single development of the plate, but if the separation is difficult, the simplification may not be possible and you will have to live with a multiple elution method.
Run a preparative scale column, collecting multiple fractions in the Rf region where your qualitative study has shown the impurity to come and analyze these fractions by HPLC to pick out those with the highest concentration of the unknown impurity.
Hypothesizing the Structure of Potential Impurities
Perhaps you can hypothesize a possible identity for the troublesome impurity from the answers to series of questions.
What modifications of conditions increase the impurity? What modifications decrease it? Does the Rf provide a clue to the polarity and so make some structures more likely and others less likely? Is the Rf consistent with the proposed functional groups? Does the degree that it partitions between different liquid phases provide a clue?
What modifications of conditions increase the impurity? What modifications decrease it? Does the Rf provide a clue to the polarity and so make some structures more likely and others less likely? Is the Rf consistent with the proposed functional groups? Does the degree that it partitions between different liquid phases provide a clue?
If you have a potential mechanism for formation of the hypothesized impurity, is it going to be easier to simply synthesize this potential impurity and test it instead of trying to isolate and purify the impurity from the product mix?
Is a composition that results from intramolecular self-reaction possible? Think of possible side reactions that could yield such products. These materials, because they have a molecular weight about double the API itself and similar functional groups can be difficult to crystallize out. Size exclusion chromatography can be very powerful for distinguishing materials differing by 700 atomic units. Advantageously the high molecular weight impurity elutes first!
Is reaction with a solvent, solvent impurity, or reagent impurity possible?
Could the impurity be present in a very small amount but have a very large extinction coefficient compared to the main substance, so that the HPLC detector signal was exaggerated in a molar comparison? The diode array spectrum may be useful to assess the likelihood of such a situation. Absorptions with very high extinction coefficients usually have extensive conjugation and longer wavelength absorption.
Can color-forming TLC reagents be useful to identify the functionality in the impurity? If the impurity is separable by TLC one can often perform derivatizations on the TLC plate, which signal some functionality by a coloration of the spot or band. In contrast, this is not readily applicable to HPLC separation.
Could you obtain a KBr/IR by concentrating a preparative plate sample on a triangle piece of KBr. Could you obtain a mass spectrum or ms/ms from a TLC or HPLC sample?
Does steam distillation help to decrease the impurity level? If steam distillation reduces an impurity the most frequent conclusion is that the impurity was some solvent-like material.
Combining Yield Optimization with Impurity Identification
Performing simplex or other process step optimization can provide some conditions, which result in a dramatic increase in the impurity level. These conditions are unsuitable for optimizing but such a sample may be an easier mixture from which to purify the impurity.
Using Coloured Derivatizing Agents
Can colored derivatives be useful? Note you will not know whether the impurity of interest formed a derivative unless you know that it contained the prerequisite functional group. The main component should not form the derivative.
Can colored derivatives be useful? Note you will not know whether the impurity of interest formed a derivative unless you know that it contained the prerequisite functional group. The main component should not form the derivative.
Chromatography of colored derivatives is simpler because the experiment can be followed visually on the chromatographic plate or on a column whichever is used.
4-phenyl-azo-benzenesulfonic acid chloride has been used as a derivatizing agent for primary and secondary amines [R.D. Desai and C.V. Mehta, Indian J. Pharm. 13, 211 (1951).] Hydrolysis in conc. HCl-dioxane E.O. Woolfork, W.E. Reynods and J.L. Mason J. Org. Chem. 24, 1445 (1959).
4-phenyl-azo-benzoyl chloride is a derivatization for alcohols. [E.O. Woolfork, F-E. Beach and P. McPherson, J. Org. Chem. 20, 391 (1955).]
4-(4-nitrophenyl-azo)-benzoylchloride can be used to form derivatives from primary and secondary alcohols, amines and thiols. E. Hecker, Ber. 88, 1666(1955).
E.S. Amin and E. Hecker Ber. 89, 695 (1956).
A. Butenandt, T. Beckmann and E. Hecker, Z. Physiol. Chem.324, 71 (1961).
A. Butenandt, D. Stamm and E. Hecker, Ber. 94, 1931 (1961).
Because an impurity that moves closely with the main product probably has the same polar functionalities as the main compound, if one can make a colored derivative of the main composition, the unidentified impurity will also most likely form the same derivative. Now, however, a TLC chromatographic separation will much more easily show up the impurity and allow sensitive variation of the elution system to separate this small minor band. Swish TLC can also probably be usefully applied to the now colored, highly crystalline derivative of probably decreased solubility, and the impurity itself recovered by breaking apart the derivative.
Because an impurity that moves closely with the main product probably has the same polar functionalities as the main compound, if one can make a colored derivative of the main composition, the unidentified impurity will also most likely form the same derivative. Now, however, a TLC chromatographic separation will much more easily show up the impurity and allow sensitive variation of the elution system to separate this small minor band. Swish TLC can also probably be usefully applied to the now colored, highly crystalline derivative of probably decreased solubility, and the impurity itself recovered by breaking apart the derivative.
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