Preparation Of Sample For Analysis

Analytical chemistry: The branch of chemistry that deals with the separation, identification and determination of components in a sample. It also traditionally includes coverage of chemical equilibrium and statistical treatment of data. Analytical chemistry can be broken down into two general areas of analysis: - Qualitative analysis - Quantitative analysis

Quantitative analysis: Determining how much of a material present in a sample. Qualitative analysis: Attempting to identify what materials are present in a sample.

The most common pharmaceutical analysis is the quantitative measurement of the active ingredient and related compounds in the pharmaceutical product. These determinations require the highest accuracy, precision, and reliability because of the intended use of the data for manufacturing control, stability evaluation, and shelf-life prediction. Determination of drugs and their metabolites in biological samples like plasma or urine, is important in elucidation of drug metabolism pathways as well as comparing bioavailability of different formulas.

Basic steps in an analysis: Prior to starting an assay, one must concentrate to several aspects: Technique to be used Sampling and sample preparation Proper application of method Data analysis and reporting All of these together comprise an analytical method.

Sampling: Sampling is a process by which a sample population is reduced in a size to an amount of homogenous material that can be conveniently handled in the laboratory and whose composition is representative of the population. Regardless of the motives the analysts must take sample for analysis that will give the most accurate picture of the composition of the whole batch.

There are three main methods for obtaining samples: Continuous sampling: In continuous sampling, a certain portion of the material to be analyzed is continuously diverted to form the gross sample. For example, a water line may be tapped in such a way that small portions of water are continuously removed for analysis. Intermittent sampling: In intermittent sampling, every nth unit is set aside to form the gross sample. At the production level, this may mean that

In most instances, however, the contents (either liquid or tablets) have already been analyzed, and this serves only as a final check on the product. Grab sampling: Grab sampling is usually associated with homogenous materials (usually liquid). In small scale preparation, where through mixing is possible, grab sampling is quite successful. But in large scale preparation, where through mixing is not possible, it is preferable to take the sample from several depths of container.

Pharmaceutical samples: Table below lists types of samples that are typically found in the pharmaceutical analytical chemistry lab. Sample type

Explanation

Plasma sample

Metabolism or bioavailability

Tablet, capsule

Solid mixture

Packaging material, rubber gasket, bottle

Solid mixture

Transdermal patch, topical gel

Semisolid solution

Elixir

Solution

Aerosol

Solid in gas

Oral suspension, topical lotion

Solid suspended in liquid or

Oral suspension, topical cream

liquid–liquid suspension (emulsion)

Candy lozenge

Solid–Solid suspension

rimary concern for a sample preparation -

Range Selectivity Recovery Stability

Range: The instrumental method must be developed before the sample preparation method. The concentration of the prepared sample must be within the working concentration range of the instrumental method. If the sample is too diluteted or concentrated, then the sample concentration must be adjusted using an appropriate sample

Selectivity: The sample preparation method must not only deliver a measurable amount of sample but the compounds accompanying the analyte (undergo analysis) must not interfere with the analysis process. In gravimetric assay (methods based on a measured weight) the detection step has no discriminating power and the sample preparation provides all of the specificity. A common example of gravimetric analysis is the determination of the amount of water in a hydrate by heating the sample to remove the water such that the difference in weight is due to the water lost.

Recovery: The recovery of a sample preparation must be assessed, because the recovery determines the accuracy of the analysis. For drug substance and drug product analysis, recovery of 100% is generally required to maintain required levels of accuracy and precision. For biological samples, less than quantitative recovery is generally acceptable if the recovery is reproducible.

Stability: After the sample is prepared and is awaiting instrumental analysis, the analyte must be stable for a reasonable amount of time. If necessary, the sample may require one additional step, such as pH adjustment. ∗∗∗ In general, a pharmaceutical sample for analysis must be in solution form and should have following properties: - Concentration must be in the measurable rage of instrument - Sample should be compatible with the system - Quantitative recovery (desirable) - The analyte is stable until the sample is

Prevention of segregation of sample Various methods have been suggested to prevent physical separation of the components (segregation): Direct dissolution of tablet in a suitable solvent and assay aliquot of solution. The resieving and regrinding of the ground tablet and assay sievings. The grinding of a composite with a suitable organic solvent and the evaporation of the solvent and assay residue. The dissolution of the composite powdered tablet sample in a solvent and assay aliquot of solution.

Direct dissolution in a suitable solvent usually produces the most accurate and precise analytical result. For enteric coated tablets, manual grinding with a mortar and pestle can lead to erratic result which are overcome by repeated sieving and grinding of the particle to a uniformly sized powder. Alternatively, removing the tablet coating with an organic solvent prior to manual grinding facilitates more uniform grinding of the tablets. For example, ethinyl estradiol tablets are powdered and triturated with four 20ml portions of chloroform, decanted, filtered and analyzed by TLC.

Fundamental theories controlling sample preparation techniques Before designing sample preparation technique, it is necessary to review are some fundamental theories that control the separation process. A. Physicochemical interactions: Several types of intermolecular physicochemical interactions have to consider during development of sample preparation methods. The interactions can be divided into 4 types: ionic, dipole-dipole, hydrogen bonding and hydrophobic interactions.

Ionic interactions are interactions of two charged species of opposite charge. Examples of sample preparation techniques using ionic interactions are ion-exchange or ion-pairing chromatography. The strength of these interactions depends on the ionic concentration of the solutions. As the ionic strength increases, the charge-charge interaction decreases. The presence of competing charged species (high ionic strength) results in weaker attractions of the species of interest. The pH of the solution plays a critical role, since most charged pharmaceutical compounds are weak acids or bases.

Dipole-dipole interactions (when two opposite charge of separate dipole interact) take place when the adsorption of a solute occur onto the stationary phase in normalphase chromatography. Hydrogen bonding occurs when a hydrogen of one molecule interacts with an electronegative dipole of another molecule or functional group. Hydrogen-bonding interactions behave similarly toare dipole-dipole Hydrophobic interactions important in interactions. liquid-liquid extractions as well as in reversedphase chromatography.

B. Solubility: Many factors affect solubility and must be considered. For example, pH, ionic strength, and temperature can significantly affect solubility. For example, the aqueous solubility of a carboxylic acid can be higher at a higher pH. This is simply due to increase in the ionization of the molecule. For aqueous sample preparations, addition of a water-miscible solvent (organic) such as acetonitrile or alcohol can be used to enhance solubility. For example, the solubility of acetaminophen in water is approximately 11 mg/mL, but the solubility is doubled by adding

B. Phase equilibrium: Phase equilibrium theory is the fundamental basis for many of the separations techniques used for sample preparation, including liquid-liquid extraction, solid phase extraction, solid-phase microextraction, and HPLC. When a solute X is exposed to two immiscible solvents, the partition equilibrium can be described by the following equation: X1 : X2 and the partition coefficient Where,is defined as Kp = Kp = partition coefficient follows: X1 = concentration of X in X1 solvent 1

Kp for a liquid-liquid extraction is influenced by many other variables, such as pH, temperature, ionic strength of the aqueous phase and the volumes of the two solvents. Therefore, method development efforts must include identification and control of these critical variables that will influence the extraction.

Specific sample preparation techniques Liquid-solid extraction: A frequently encountered procedure is the extraction of a substance from a solid dosage form sample, such as in the analysis of tablets. This is relatively a simple procedure involving the selection of a solvent or solvent combination which ideally provides good solubility of the substance of analytical interest and minimal solubility of the components that interfere with the analysis. For the majority of procedures, the first step requires grinding of the solid matrix into a fine powder followed by solvent extraction, and

For semisolid formulations, solvent extraction is generally performed at elevated temperatures so as to melt the semisolid and increase the extraction efficiency. In developing a method that requires filtration, adsorption of the analyte onto filter must be taken into consideration. This is specially true for dilute solutions, in bulk formulation filter adsorption is not an important concern.

Soxhlet extraction A classical liquid-solid sample preparation technique is soxhlet extraction (hot extraction). A soxhlet extractor is a piece of laboratory apparatus. Typically, a soxhlet extraction is only required where* the desired compound has only a limited solubility in a solvent, and * the impurity is insoluble in that solvent. If the desired compound has a high solubility in a solvent then a simple filtration can be used to separate the compound from the insoluble substances (as in cold extraction).

Fig: A schematic representation of a soxhlet extractor 1: Stirrer bar 2: Solvent pot 3: Distillation path 4: Thimble 5: Solid 6: Siphon top 7: Siphon exit 8: Expansion adapter 9: Condenser 10: Cooling water in 11: Cooling water out

Normally a solid material containing some of the desired compound is placed inside a "thimble" made from thick filter paper, which is loaded into the main chamber of the soxhlet extractor. The soxhlet extractor is placed onto a flask/pot containing the extraction solvent. The soxhlet is then

The solvent is heated to reflux. The solvent vapor travels up a distillation arm, and floods into the chamber housing the thimble of solid. The condenser ensures that any solvent vapor cools, and drips back down into the chamber housing the solid material. The chamber containing the solid material slowly fills with warm solvent. Some of the desired compound will then dissolve in the warm solvent. When the soxhlet chamber is almost full, the chamber is automatically emptied by a siphon side arm, with the solvent running back down to the distillation flask. This cycle may be allowed to repeat many times, over hours or days.

During each cycle, a portion of the compound dissolves in the solvent. After many cycles the desired compound is concentrated in the distillation flask. The advantage of this system is that instead of many portions of warm solvent being passed through the sample, just one batch of solvent is recycled. After extraction the solvent is removed, typically by means of a rotary evaporator, yielding the extracted compound. The insoluble portion of the extracted solid remains in the thimble, and is usually discarded. Figure: Extraction in progress

The soxhlet technique is not often used in routine pharmaceutical sample preparations for two reasons: The high temperature can cause degradation which is unacceptable for stabilityindicating assays. The technique is difficult to perform on multiple samples because of time and space requirements.