Completeness of Extraction

During formulation development, all experiments are considered GMP experiments. Therefore, all test methods that are utilized to determine any results must be developed for the intended purpose and they must be validated according to regulatory requirements. Method validation is covered in Chapter 9, so it will not be repeated here except for completeness of extraction that is specific to drug products. 

For drug products containing constituents that are insoluble in the extraction medium used in the analytical procedure, it may be deemed adequate to perform a separate test for completeness of extraction (in addition to recovery experiments as described above). The completeness of extraction can be evaluated two ways kinetically (over some elapsed time t) and thermodynamically (change in volume). 

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The cumulative sums of selected major and trace metals extracted by the two SSD procedures from representative arid-zone soils are shown in Fig. 4.6. As can be seen from the figure, the Rehovot procedure is stronger in attacking desired fractions, such as the carbonate bound, Mn oxide bound and organically bound fractions. Extraction of certain major elements, indicating selectivity, specificity and completeness of extraction of given soil components, was found to differ between the two procedures. Calcium and Mg were more completely extracted from the carbonate fraction in arid zone soils by the Rehovot procedure. Calcium and relevant trace elements bound in the carbonate fraction, which were not completely dissolved by the Bonn procedure at this step, were released at the following steps, such as the ERO, OM or RO fractions. 

PCBs in biological samples are usually extracted by a Soxhlet column and with a nonpolar solvent such as hexane. The sample is first mixed with sodium sulfate to remove moisture. The extraction of PCBs from sediments was tested with sonication, with two sonications interspersed at a 24-h quiescent interval, with steam distillation, or with Soxhlet extraction (Dunnivant and Elzerman 1988). Comparison of the recoveries of various PCB mixtures from dry and wet sediments by the four techniques and the extraction efficiency of four solvents showed that the best overall recoveries were obtained by Soxhlet extraction and the two sonication procedures. In comparisons of solvent systems of acetone, acetonitrile, acetone-hexane (1+1), and water-acetone-isooctane (5+1.5+1), recoveries of lower chlorinated congeners (dichloro- to tetrachloro-) were usually higher with acetonitrile and recoveries of higher chlorinated congeners (tetrachloro- to heptachloro-) extracted with acetone were superior (Dunnivant and Elzerman 1988). The completeness of extraction from a sample matrix does not seem to discriminate against specific isomers however, discrimination in the cleanup and fractionation process may occur and must be tested (Duinker et al. 1988b). 

Successive extractions, whilst increasing the efficiency of extraction of both solutes, may lead to a poorer separation. For example, if DA = 102 and I)v = 10 one extraction will remove 99.0% of A and 9.1% of B whereas two extractions will remove 99.99% of A but 17% of B. In practice, a compromise must frequently be sought between completeness of extraction and efficiency of separation. It is often possible to enhance or suppress the extraction of a particular solute by adjustment of pH or by complexation. This introduces the added complication of several interrelated chemical equilibria which makes a complete theoretical treatment more difficult. Complexation and pH control are discussed more fully in Chapter 3. 

In Soxhlet extractions (see Basic Protocols 1 and 3), the amount of solvent must be sufficient to cover the thimble and have at least 50 ml solvent in the flask and may require an additional amount of solvent. After completion of extraction, it is essential to let the system cool and let all the solvent transfer to the flask. In addition, accurate weighing of flask is essential. Use of gloves to avoid fingerprints on flasks which may affect the weights is critical. 

Normally, odd-numbered fatty acids are used as internal standards. While the use of internal standards ensures the correctness of the extraction procedure, it does not guarantee the completeness of extraction for different fatty acids. Due to this reason, a comparison between the methods is essential to truly determine the efficacy of extraction. Chavarri et al. (1997) compared two sample preparation procedures. The first method was the direct method developed by de Jong and Badings (1990), described above. The second method involved saponification with TMAH as described by Martin-Hemandez et al. (1988) and the formation of methyl esters in the injector prior to analysis. The authors found that separation of the FFAs from the triglycerides prior to derivatization improved the analysis. Another comparative study by Ardo and Polychroniadou (1999) reported that the saponification method described above (Martin-Hernandez et al., 1988) was found suitable for both low and high FFA levels in cheese. 

Extraction scientist who is responsible for sample preparation should be certified prior to extracting real study samples. The validated extraction method has to be followed exactly. The raw data entries have to be documented promptly such as lot numbers of STD and QC, IS, extraction reagents, matrix, the IDs of automation tool and pipette, the time for study sample removal and return to storage and the completion of extraction. Instrument operator who is responsible for analysis has to perform SST test and assess sensitivity and carryover prior to initialing batch. Instrument operator has to... 

To check for completeness of extraction, add 20 mL of dichloromethane to the column, and collect the eluate in a second tared flask. Evaporate the contents of the second flask to dryness, and examine it for residue. Determine the weight of the residue (IVR1), if present. If residue is present, repeat the procedure with an additional 20 mL of dichloromethane. 

The mass (Om) and concentration (oQ degrees of extraction are characteristics of the completeness of extraction of a substance... 

In the isolation of hemicelluloses from plant materials containing a relatively large amount of pectic substances, such as the cambium layer of a wood or the leaves and stems of plants, the water-soluble materials and insoluble pectic substances are first removed. The hemicelluloses are then extracted by an alkaline solution. Sodium hydroxide solutions at concentrations varying from 2 to 17% have been used for this purpose. Norman recommends a 2% solution of sodium carbonate when high purity is more desirable than completeness of extraction. Most investigators have used a cold sodium hydroxide solution of approximately 4% concentration. " ... 

The equipment and issues pertaining to this analysis are similar to those for soil chloride (see Method 5.5.1) using ion chromatography. The method is specific to sulfate, whereas ICPAES measures total S in the aqueous extracts. Accordingly, results will usually be lower for this analytical finish compared with Method 5.7.3. The only post-extraction preparation necessary, other than dilution, is the removal by filtration of particulate matter >0.20 pm. Since the concentration of SO -S can be influenced by biological transformations, aqueous soil extracts should be kept at 4°C if there is any delay (e.g. overnight) between completion of extraction and analysis. Under such conditions filtered aqueous samples should remain stable for at least 28 days (O Dell etal. 1984). The analysis can be undertaken in sequence with other anions (Dick Tabatabai 1979 O Dell etal. 1984). 

After completion of extraction, the cellulose thimble is placed in a drying-oven at 120°C. After drying, the solid remainder in the cellulose thimble is weighed. The extract contains the liquid product and solvent (THF). THF is distilled from the product and reused in a subsequent analysis. 

On completion of extraction, the mineral aggregate is removed with its container and dried to a constant mass. 

The column is inserted into the extractor and eluted with approximately 200 mL of refluxing solvent (for solvent composition see Section 18.5) to which a suitable internal standard has been added. At the beginning of the extraction, the solvent passing through the column from the bottom up, removes a considerable volume of water from the interior of the resin beads (approximately 30 mL for a 75 mL resin bed). Depending upon the saUnity of the sample water, dissolved sea salt may precipitate in the boiling flask. Four to six hours of continuous extraction are usually sufficient to remove all adsorbed materials. Completeness of extraction may be checked as described in Section 18.6. 

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