Sample Preparation and Methods Development

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.)... 

Different approaches may be used to validate the sample preparation component of the dissolution test. However, it is important to understand that the objective of validation is to demonstrate that the procedure is suitable for its intended purpose. For example, one of the strategies will demonstrate the validity of different aspects of sample preparation during method development (prior to the formal method validation exercise). As a result, the final validation experiments will confirm the work done during method development. The strategy that will be followed for the method development and validation process will depend on the culture, expertise, and strategy of the analytical laboratory. 

Many advances have been made in recent years in chromatographic instrumentation and data handling including important advances in automation. However, for many methods, equally important is the sample preparation procedure. These are sometimes complex, and are often the key step in a method, but perhaps receive less attention than is warranted. The optimisation of sample preparation and the development of... 

The great advantage of NIR reflectance methods is their speed and the simplicity of sample preparation. Once method development has been completed, analysis of solid. samples tor several species can be completed in a few minutes. Accuracies and precisions of 1% U) 2% relative are regularly reported. 

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. 

It is generally difficult to identify developments with high potential where interferences do not preclude general application. To ensure the relevance of a method, its application to real sample analysis must be demonstrated. The accuracy of an analytical method should be confirmed by an independent method, or by the analysis of certified reference materials. Detailed comparative studies of the method developed with other well-established methods for polymer/additive analysis are not frequent in the analytical literature. Nevertheless, some examples may be found in Section 3.6. Improvements in analytical techniques are reasonably sought in sample preparation and in hyphenated chromatographic techniques. However, greatest efficiency is often gained from the use of databases rather than accelerated extraction or hyphenation. 

Direct injections using RAM or TFC have simplified sample preparation and increased throughput. Matrix ion suppression was greatly reduced or eliminated in several cases compared with traditional off-line sample cleanup procedures such as PPT, SPE, and LLE. Method development time was minimized with generic methods15 that suit most applications. Detailed applications can be found in a recent review.8... 

With the advent of API sources, LC/MS/MS allows the facile development of quantitative methods that are sensitive, selective, robust, and amenable to the rapid analysis of a majority of small molecules. In order to achieve high-throughput bioanalysis in support of pharmacokinetic studies, many approaches have been reported utilizing automated sample preparation and reducing analysis time by pooling samples, parallel analysis, and fast chromatography. 25,26,152,153... 

CE instrumentation is quite simple (see Chapter 3). A core instrument utilizes a high-voltage power supply (capable of voltages in excess of 30,000 V), capillaries (approximately 25—lOOpm I.D.), buffers to complete the circuit (e.g., citrate, phosphate, or acetate), and a detector (e.g., UV-visible). CE provides simplicity of method development, reliability, speed, and versatility. It is a valuable technique because it can separate compounds that have traditionally been difficult to handle by HPLC. Furthermore, it can be automated for quantitative analysis. CE can play an important role in process analytical technology (PAT). For example, an on-line CE system can completely automate the sampling, sample preparation, and analysis of proteins or other species that can be separated by CE. 

Specificity may be achieved through sample preparation, chromatographic selectivity, the selectivity of the detection method or combinations of these. It is often tempting to utilise the most selective detection method available (such as MS or MS/MS), since this can reduce the effort required in optimising the sample preparation and chromatography. However, whilst this is often the most expedient approach in early development, it may not always be suitable to transfer expensive, highly technical methods and instrumentation into a manufacturing environment if this is required. 

As well as typical sample preparation methods such as filtration and liquid-liquid extraction, newer developments are now extensively used. The first of these is solid-phase extraction (SPE). This is a rapid, economical, and sensitive technique that uses several different types of cartridges and disks, with a variety of sorbents. Sample preparation and concentration can be achieved in a single step. Interfering sugars can be eluted with aqueous methanol on reversed-phase columns prior to elution of flavonoids with methanol. 

The study of the precision of a method is often the most time and resource consuming part of a method validation program, particularly for methods that are developed for multiple users. The precision is a measure of the random bias of the method. It has contributions fi om the repeatability of various steps in the analytical method, such as sample preparation and sample injection for HPLC [5-9], and from reproducibility of the whole analytical method fiom analyst to analyst, fiom instrument to instrument and fiom laboratory to laboratory. As a reproducibility study requires a large commitment of time and resources it is reasonable to ensure the overall ruggedness of the method before it is embarked upon. 

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