Polymorphism analysis, limitations

The /3-polymorphic form of anhydrous carbamazepine is official in the USP [3], The USP stipulates that, The X-ray diffraction pattern conforms to that of USP Carbamazepine Reference Standard, similarly determined. No limits have been set in the USP for the other polymorphs of anhydrous carbamazepine. Although several polymorphic forms of anhydrous carbamazepine have been reported, only the a- and /3-forms have been extensively studied and characterized [49]. A comparison of the powder x-ray diffraction patterns of these two forms revealed that the 10.1 A line (peak at 8.80° 26) was unique to a-carbamazepine, and so this line was used for the analysis (Fig. 5). It was possible to detect a-carbamazepine in a mixture where the weight fraction of a-carbamazepine was 0.02 at a signal-to-noise ratio of 2. Much greater sensitivity of this technique has been achieved in other systems. While studying the polymorphism of l,2-dihydro-6-neopentyl-2-oxonicotinic acid, Chao and Vail [50] used x-ray diffractometry to quantify form I in mixtures of forms I and II. They estimated that form I levels as low as 0.5% w/w can be determined by this technique. Similarly the a-inosine content in a mixture consisting of a- and /3-inosine was achieved with a detection limit of 0.4% w/w for a-inosine [51]. 

Another example of a well-designed method for the quantitative XRPD determination of polymorphs was developed for the phase analysis of prazosin hydrochloride [49]. As an example of an XRPD method for the determination of solvatomorphs, during the quantitation of cefepine dihydrochloride dihydrate in bulk samples of cefepine dihydrochloride monohydrate, a limit of detection of 0.75% w/w and a limit of quantitation of 2.5% w/w were associated with a working range of 2.5-15% w/w [50],... 

Haplotype analysis may be necessary if the candidate genes do not have known functional polymorphisms. Prior knowledge of haplotype tag single-nucleotide polymorphisms (SNPs) for each gene is essential to limit the number of assays required to avoid wasting finite patient samples. 

There has been much discussion about the potential utility of flow cytometry of chromosomes for clinical diagnosis. As regards its sensitivity, this technique appears to stand somewhere between the technique of flow analysis of whole cells for DNA content and that of microscope analysis of banded chromosomes. It may be a useful intellectual exercise for readers to ask themselves which technique or techniques would be most appropriate for detecting the following types of chromosome abnormalities (1) tetraploidy, where the normal chromosome content of cells is exactly doubled because of failure of cytokinesis after mitosis (2) an inversion in an arm of one particular chromosome and (3) trisomy (the existence of cells with three instead of two) of one of the small chromosomes. In addition to these limitations, the use of flow cytometry to look for abnormal chromosomes has been confounded by the fact that several human chromosomes are highly polymorphic, and flow karyotypes, therefore, vary considerably among normal individuals. 

Markers such as protein and blood group loci were initially used in the analysis of genetic traits however they were limited in utility due to low variation. Early in the 1980s these markers began to be replaced with DNA polymorphisms, initially RFLPs which are detected by the ability of a segment of DNA to be cut, or to not be cut, by a specific restriction enzyme. 

In essence, the test battery should include XRPD to characterize crystallinity of excipients, moisture analysis to confirm crystallinity and hydration state of excipients, bulk density to ensure reproducibility in the blending process, and particle size distribution to ensure consistent mixing and compaction of powder blends. Often three-point PSD limits are needed for excipients. Also, morphic forms of excipients should be clearly specified and controlled as changes may impact powder flow and compactibility of blends. XRPD, DSC, SEM, and FTIR spectroscopy techniques may often be applied to characterize and control polymorphic and hydrate composition critical to the function of the excipients. Additionally, moisture sorption studies, Raman mapping, surface area analysis, particle size analysis, and KF analysis may show whether excipients possess the desired polymorphic state and whether significant amounts of amorphous components are present. Together, these studies will ensure lotto-lot consistency in the physical properties that assure flow, compaction, minimal segregation, and compunction ability of excipients used in low-dose formulations. 

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