Preparative HPLC methods

Enantiomers of racemic cis-3H,7H- and frans-3H,7H-3-substituted 7-amino-l,2,3,5,6,7-hexahydropyrido[3,2,l-zy]quinazolin-l-ones were separated by using chiral preparative HPLC method (06USA2006/0004028, 07JP2007131577). 

Analytical chromatographic options, based on linear and nonlinear elution optimization approaches, have a number of features in common with the preparative methods of biopolymer purification. In particular, both analytical and preparative HPLC methods involve an interplay of secondary equilibrium and within the time scale of the separation nonequilibrium processes. The consequences of this plural behavior are that retention and band-broadening phenomena rarely (if ever) exhibit ideal linear elution behavior over a wide range of experimental conditions. First-order dependencies, as predicted from chromatographic theory based on near-equilibrium assumptions with low molecular weight compounds, are observed only within a relatively narrow range of conditions for polypeptides and proteins. 

This chapter briefly describes the various types of packing materials and equipment that can be utilized to isolate impurities from drug substances. It then goes into how to screen for a preparative HPLC method that could be used for impurity isolation and how to move from the screening phase of the project to develop an analytical-scale preparative method. The chapter concludes by giving rules on how to scale that preparative method up to any size column that will be used in the impurity isolation. 

It was decided that the most efficient method of identification was to couple succinic anhydride with the drug substance and isolate the peak of interest by preparative-scale HPLC. A solution of the drug substance was treated with dimethylaminopyridine and succinic anhydride. The resultant solution was stirred at room temperature for 48 hours. The reaction mixture was partitioned between ethyl acetate and water, and the aqueous layer was then treated with IN HC1. The two layers were shaken well, and the aqueous layer was removed. The organic layer was then washed with water, saturated sodium chloride solution, dried with magnesium sulfate, and concentrated by evaporation to afford a clear colorless oil (1.81 g). A suitable preparative HPLC method using a volatile mobile phase of 0.1% formic acid in water/ methanol was developed, and the crude reaction mixture was purified by preparative-scale HPLC. The solution was concentrated by evaporation and the water was removed from this solution by freeze-drying to afford a white lyophilate (40 mg). 

An enriched bulk lot containing 16% of the impurity of interest was identified and used to reduce the time required for isolation by a factor of 40. Isolation was required for further NMR analysis. The enriched bulk lot containing 16% impurity was used for isolation by preparative HPLC using 45 500-pL injections. A suitable reversed-phase preparative HPLC method using a volatile mobile phase (0.1% formic acid in water and acetonitrile) was developed based on the analytical purity and potency assay. The fractions containing the impurity were combined and concentrated by evaporation. A final analytical cleanup was performed to remove salts and... 

Hunter et al. developed a straight-phase preparative HPLC method for some steroid alkaloids... 

Preparative chromatography is the process of using liquid chromatography to isolate a sufficient amount of material for other experimental or functional purposes. This section describes the use of preparative HPLC to isolate tens of milligrams of pure unknown compound(s) for the purpose of structure elucidation by spectroscopic techniques, which is often referred to as semipreparative HPLC. This section will focus primarily on preparative HPLC methods with the following parameters ... 

Table 2 lists analytical and preparative HPLC methods developed for the isolation and quantification of benzophenones. Most methods have utilized reversed-phase C columns with mixtures of MeCN or MeOH and H20, with or without an acid modifier. Exceptions include... 

Matusheski NV, Wallig MA, Juvik JA, Klein BP, Kushad MM, Jeffery EH. Preparative HPLC method for the purification of sulforaphane and sulforaphane nitrile from Brassica oleracea. J Agric Food Chem 2001 49 1867-1872. 

For the determination of cichoric acid and other caffeoylics present in Echinacea preparations, HPLC methods have been described (see Fig. 2) [82, 104]. Cichoric acid can also be determined by a recently-developed capillary zone electrophoresis (MEKC) method (see Fig. 5) [44]. 

Reverse phase methods were also used to assay dimethylaminoetoposide (2) together with a stereoisomer and their AT-demethylated metabolites in the urine of cancer patients. A related paper discusses an analytical method for etoposide and isomers in plasma. Other work describes the isolation of glucuronides of metronidazole (3) and its hydroxy metabolite (4) by preparative HPLC methods, and the antipyrene metabolite derivatives 5-7 were identified by thermospray LC-MS methods. In the area of the metabolites of narcotics, two papers have described the reverse phase-HPLC analysis of morphine and its 3-and 6-glucuronides in biological samples. In the first case, fluorescence detection was used, in the latter electrospray MS procedures. Codeine and seven glycosidic metabolites have also been analysed by reverse phase-HPLC procedures with UV or electrochemical detection. ... 

Monobasic acids are determined by gas chromatographic analysis of the free acids dibasic acids usually are derivatized by one of several methods prior to chromatographing (176,177). Methyl esters are prepared by treatment of the sample with BF.—methanol, H2SO4—methanol, or tetramethylammonium hydroxide. Gas chromatographic analysis of silylation products also has been used extensively. Liquid chromatographic analysis of free acids or of derivatives also has been used (178). More sophisticated hplc methods have been developed recentiy to meet the needs for trace analyses ia the environment, ia biological fluids, and other sources (179,180). Mass spectral identification of both dibasic and monobasic acids usually is done on gas chromatographicaHy resolved derivatives. 

HPLC method with amperometric detection was applied for detenuination of phenols in sea sediment and some dmg preparation. Peaks of phenol, guaiacol, cresols, hydroquinon and resorcinol were identified on chromatogram of birch tai. The HPLC method with electrochemical detectors was used for detenuination of some drug prepai ation of aminophenol derivate. So p-acetaminophenol (paracetamol) was determined in some drug. 

Enantiomeric separations have become increasingly important, especially in the pharmaceutical and agricultural industries as optical isomers often possess different biological properties. The analysis and preparation of a pure enantiomer usually involves its resolution from the antipode. Among all the chiral separation techniques, HPLC has proven to be the most convenient, reproducible and widely applicable method. Most of the HPLC methods employ a chiral selector as the chiral stationary phase (CSP). 

The work presented here describes a RP-HPLC method for characterizing PB-DPE preparations. Components were purified by preparative RP-HPLC and identified by NMR. Components observed by GC under conditions similar to a published analysis (ref. 4), are identified by relation to the HPLC. A clear description of the bromination path can be made. 

The HPLC method for which data are given had previously been shown to be linear over a wide range of concentrations what was of interest here was whether acceptable linearity and accuracy would be obtained over a relatively narrow concentration range around the nominal concentration in the product the specification limits were 90-110% of nominal. Three concentrations were chosen and three repeat determinations were carried out at each. Two different samples were prepared at each concentration, namely an aqueous calibration solution and a spiked placebo. All samples were worked up according to the method and appropriate aliquots were injected. The area counts are given in the second, respectively the fifth column of Table 4.42. 

Next, reductive amination (step 4 in scheme 1) was exchanged with copper catalyzed palladium coupling (step 2 in scheme 1). Atomic absorption analysis for palladium in RWJ-26240 samples prepared by scheme 2 indicated that the level of palladium was reduced to an acceptable level. This improvement may be due to the two reduction steps subsequent to the use of palladium in scheme 2.177 The final major modification to the reaction scheme was the substitution of NaBH4 for NaBH3CN. The yield of product (60%) was determined by HPLC (Method 2). Reductive alkylation with formalin/NaBH4 afforded a pharmaceutically acceptable drug substance. 

HPLC methods have been widely used for the analysis of OTC in different samples. As described above in the Section 2.3, the HPLC method is described in most of compendia [1,2,4,7] for determination of OTC in bulk drug substances and in some pharmaceutical preparations. The application of HPLC methods for the analysis of antibiotics including oxytetracycline has been recently reviewed by Diaz-Cruz et al. [37] and Lunn [38], A summary of HPLC method for the analysis of OTC is presented in Table 3. 

In contrast to the metabolism of BA and BaP, the 5,6-dihydrodiols formed in the metabolism of DMBA by liver microsomes from untreated, phenobarbital-treated, and 3-methylcholanthrene-treated rats are found to have 5R,6R/5S,6S enantiomer ratios of 11 89, 6 94, and 5 95, respectively (7.49 and Table II). The enantiomeric contents of the dihydrodiols were determined by a CSP-HPLC method (7.43). The 5,6-epoxide formed in the metabolism of DMBA by liver microsomes from 3MC-treated rats was found to contain predominantly (>97%) the 5R,6S-enantiomer which is converted by microsomal epoxide hydrolase-catalyzed hydration predominantly (>95%) at the R-center (C-5 position, see Figure 3) to yield the 5S,6S-dihydrodiol (49). In the metabolism of 12-methyl-BA, the 5S,6S-dihydrodiol was also found to be the major enantiomer formed (50) and this stereoselective reaction is similar to the reactions catalyzed by rat liver microsomes prepared with different enzyme inducers (unpublished results). Labeling studies using molecular oxygen-18 indicate that 5R,68-epoxide is the precursor of the 5S,6S-dihydrodiol formed in the metabolism of 12-methyl-BA (51). 

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