Method development, levels phases

The identification and quantification of potentially cytotoxic carbonyl compounds (e.g. aldehydes such as pentanal, hexanal, traw-2-octenal and 4-hydroxy-/mAW-2-nonenal, and ketones such as propan- and hexan-2-ones) also serves as a useful marker of the oxidative deterioration of PUFAs in isolated biological samples and chemical model systems. One method developed utilizes HPLC coupled with spectrophotometric detection and involves precolumn derivatization of peroxidized PUFA-derived aldehydes and alternative carbonyl compounds with 2,4-DNPH followed by separation of the resulting chromophoric 2,4-dinitrophenylhydrazones on a reversed-phase column and spectrophotometric detection at a wavelength of378 nm. This method has a relatively high level of sensitivity, and has been successfully applied to the analysis of such products in rat hepatocytes and rat liver microsomal suspensions stimulated with carbon tetrachloride or ADP-iron complexes (Poli etui., 1985). 

In the approach described here method development is broken down into discrete stages as shown in Figure 1. The levels described are correlated with clinical and regulatory phases in Table 1 to provide rough guidance as to when in the pharmaceutical development process each level is applicable. 

SuperChems Expert 161 is a code developed by Arthur D Little Inc. for risk assessment consequence analysis, which also has a relief system sizing option. The code has a physical properties package that can handle highly non-ideal properties. It can also consider the effect of chemical reaction in the relief system piping. The code uses the DIERS drift flux methods for level swell and has the option of a rigorous two-phase slip model for the. relief system capacity. 

Cosolvent effects on SCF solution behavior allow the tailoring of solvents for extractions and separations. The strong interactions in these systems currently defy prediction by popular computational methods. Only by understanding these interactions at a molecular level will we be able to guide the development of phase equilibria models successfully. One way of exploring the molecular level interactions is with spectroscopy of various kinds and we have demonstrated here an attempt to look at the cosolvent/solute interaction. 

To run a patient sample, you will need to go through exactly the same deproteination, SFE cartridge extraction, IS addition, mobile phases dilution, and injection steps (Fig. 12.4f). From the peak heights relative to the IS height, we can now quantitate the amount of each drug in the patient s blood. To insure linearity, you may need to dilute our windowed plasma blank and spike it with different levels of each standard and plot calibration curves for each compound, but basically, our methods development is done. 

The inclusion of basic additives in the run buffer leads to a reduction in the EOF. This is due to the reduction in the number of free silanol sites on the silica surface. However, above 50 mM the continued reduction in the EOF is less pronounced [63]. In practice, sufficient EOF is generated, even in the presence of mobile phase additives, to elute neutral species in acceptable times. The upper limit on the additive concentration is most frequently due to excessive baseline noise arising from high background absorbance. The inclusion of mobile phase additives leads to a further level of complexity in method development and prohibits coupling to mass spectrometry. However, this approach is a practical solution until better stationary phases are developed. 

Counter-ions, usually small polar or ionic compounds, are routinely used to enhance the aqueous solubility and/or stability of the API. Because of their polarity, counter-ions are rarely resolved from the chromatographic solvent front in reversed-phase HPLC and have characteristically poor chromophores which makes detection difficult. The counter-ion can be omitted from the achiral method development sample set with minimal risk when this holds true. Analysis of counter-ions is normally performed using ion chromatography.9,10 This assay is separate from the reversed-phase assay performed to measure organic impurity levels. 

A fourth experiment would be an evaluation of precision and recovery at one to four concentration levels (n — 6 to 24 plus a recovery standard), plus recovery of an internal standard (n — 3 plus recovery standard). Therefore, as a minimal method development exercise, 60—70 spiked samples would be prepared and extracted within 1 day. The experiments need to be performed sequentially because the results from each will impact how subsequent experiments are designed. Selectivity is assessed through the course of the method development. The analytical chemist with access to API LC/MS/MS will spend less time on solid-phase extraction selectivity development. 

In the screening phase of method development and in robustness tests, the factors usually are examined at two levels (-1, +1). On the other hand, in the response surface designs, applied in method optimization, the factors are examined at three or more levels, depending on the applied design (see further). 

The factors and their levels examined during a screening phase in method development (27), an optimization phase in method development (28), and a robustness test (29) are presented in Tables 2.2,2.3, and 2.4, respectively. 

A TLC- and HPLC-based method was developed to quantitatively detect the cytokinins and auxins produced in the acid and alkaline extracts of culture filtrates from different P. amygdali strains. The IAA and cytokinin (t-Z and t-ZR) quantification was performed by an HPLC method developed using a C-18 reverse phase column and a linear gradient of MeCN-H20 (7 3, v/v) for the analysis of IAA, and 100 pM TEAB (triethylammonium bicarbonate adjusted at pH=7 with COf) and MeCN-H20 (7 3, v/v) in steps 1 and 2 for the analysis of cytokinins. The presence and level of phytohormones produced by some strains were analysed with respect to their virulence on almond plants [41]. The same analytical methods were used to estimate the production of cytokinins and/or auxins by three wild strains of P. savastanoi and three phytohormone-deficient mutants. The pathogeneticity on olive and oleander plants of three wild-type strains of P. savastanoi (two from olive and one from oleander) was compared to those of three phytohormone-deficient mutants of oleander strains Iaa+/cytokinin, Iaa-... 

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