Preparative HPLC method development

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... 

For a liquid or semi-solid pharmaceutical dosage form, it is crucial to include a preservative in the formulation. Commonly used preservatives in these systems include sodium benzoate, EDTA, sorbic acid, and parabens. A generic HPLC method is also recommended for the preservatives used in liquid formulations for routine monitoring to ensure the stability of the preservative itself and it must be validated specific to its use with the dosage form. (See chapters on Sample Preparation and Method Development.)... 

A typical example of HPLC method development and validation was provided by Boneschans et al. [9]. They developed an HPLC method for piroxicam benzoate and its major hydrolytic degradation products, piroxicam and benzoic acid. The authors utilised a robust stationary phase (Phenomenex Luna, Cig), with an optimised mobile phase comprising of acetonitrile/water/acetic acid (45/7/8 v/v), and a flow rate of 1.5 ml/min. The operating pH of the mobile phase (pH 2.45) was selected on the basis that it is ca. 2 pH units from the pKa of the drug, and hence reasonably insensitive to changes in mobile-phase preparation. The injection volume was 20 pi with a detection wavelength of 254 nm. They utihsed... 

HPLC method development has already been covered in Chapter 8. The focus of this chapter is to utilize HPLC and the data generated by this technique to help in developing a robust formulation for a drug product. However, the current section will discuss sample preparation solvent since it becomes an integral part of HPLC when we are discussing HPLC methods for a particular formulation. Any sample preparation solvent that is chosen for any HPLC method must be compatible with the HPLC solvents utilized for that particular test method. The current section will assume that a new molecular entity (NME) is utilized for a particular drug product. 

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... 

Interestingly, the increased selectivity of MS instruments has made it possible to circumvent the process of HPLC method development and inject minimally prepared samples directly into the MS... 

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]. 

A. Preparation for Analysis HPLC METHOD DEVELOPMENT IN-PROCESS CONTROL TESTS... 

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. 

The hydrophilic surface characteristics and the chemical nature of the polymer backbone in Toyopearl HW resins are the same as for packings in TSK-GEL PW HPLC columns. Consequently, Toyopearl HW packings are ideal scaleup resins for analytical separation methods developed with TSK-GEL HPLC columns. Eigure 4.44 shows a protein mixture first analyzed on TSK-GEL G3000 SWxl and TSK-GEL G3000 PWxl columns, then purified with the same mobile-phase conditions in a preparative Toyopearl HW-55 column. The elution profile and resolution remained similar from the analytical separation on the TSK-GEL G3000 PWxl column to the process-scale Toyopearl column. Scaleup from TSK-GEL PW columns can be direct and more predictable with Toyopearl HW resins. 

Assessing the resources available for method development should also be done before beginning a project. The resources available include not only HPLCs, detectors, and columns, but also tools for sample preparation, data capture and analysis software, trained analysts, and especially samples representative of the ultimate analyte matrix. Also, it should be considered whether a fast, secondary method of analysis can be used to optimize sample preparation steps. Often, a simple colorimetric or fluorimetric assay, without separation, can be used for this purpose. A preliminary estimate of the required assay throughput will help to guide selection of methods. 

Integration of sample preparation and chromatography by on-line coupling aims at reduction of analysis time. It is apparent from Section 7.1 that these hyphenated techniques are not yet contributing heavily to the overall efficiency of polymer/additive analysis in industry. On-line SFE-SFC requires considerable method development, and MAE-HPLC is off-line. Enhancement of sensitivity for trace analysis requires appropriate sample preparation and preconcentration schemes, as well as improved detection systems. 

For discovery PK assays, the most common sample preparation procedure is protein precipitation161720 24 because it is fast, easy to automate, and requires no method development. While protein precipitation typically will not provide as clean a sample as will alternative procedures, it is sufficient for most discovery PK samples that use HPLC/MS/MS for the analytical step.21101... 

A simple and rapid RP-HPLC method was developed for the determination of retinoid in galenicals. Commercial preparations were diluted, filered and used for separation. Measurements were carried out in an ODS column (150 X 4.6 mm i.d. particle size 3 /xm). Solvents A and B were methanol-10 mM ammonium acetate (75 25, v/v) and methanol-THF (84 16, v/v), respectively. The flow rate was 0.8ml/min. Gradient conditions were 0-25 min, 0 per cent B 35 min, 100 per cent B, isocratic for 10 min. Typical chromatograms are shown in Fig. 2.37. The repeatability of peak area ranged between 0.48 -3.2 per cent for UV-DAD and 0.57 - 3.1 per cent for fluorescence detection. The reproducibility varied between 0.26 - 4.6 per cent. It was found that the method is precise, selective, sensitive and linear, therefore, it can be employed for the routine quality control of this class of drags [85],... 

The aim of this work, to develop a RP-HPLC method for the detemtination of four components given above in a new symp preparation by optimizing the experimental conditions using two level fractional factorial design. 

The primary object of this book is to provide the HPLC practitioner with a handy guide to the use of HPLC for analyzing pharmaceutical compounds of interest. This means familiarizing the practitioner with the theory, instrumentation, regulations, and numerous applications of HPLC. This handbook provides practical guidelines using case studies on sample preparation, column or instrument selection, and summaries of best practices in method development and validation, as well as tricks... 

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