Suspensions and solutions

In suspensions and solutions, the dyes used are water soluble and include natural pigments such as chlorophylls, carotenoids and anthocyanins, and coal-tar-based dyes. More effort may be required to remove interference by these materials. Solid-phase extraction with ion exchange resins may be useful for removing anionic or cationic dyes, although simple extraction of the drug into organic solvent of moderate polarity may leave such dyes in the aqueous phase. Solutions tend to contain anti-microbial preservatives and anti-oxidants. These are usually either phenols or quaternary amines such as benzalkonium chloride and have strong enough chromophores to interfere in the analysis of a drug. These compounds have to be removed by extraction procedures prior to analysis. Suspensions also contain surfactant materials such as the polyethylene glycol-based detergents but these compounds do not have appreciable UV absorbance and thus have little potential for interference.

Creams and ointments

Sodium and potassium salts of fatty acids, cationic surfactants and non-ionic surfactants are used in creams and ointments. As discussed above, these compounds do not have strong chromophores but, particularly the fatty acids, may interfere in chromatography, for instance by contaminating reverse-phase HPLC columns, if they are not removed. Contamination of reverse-phase HPLC columns by lipophilic materials can often be observed through a loss of chromatographic peak shape. Creams and ointments contain large amounts of oily triglycerides, which have to be removed to avoid interference with the chromatographic process. Extraction of the cream with methanol can partly remove this type of interference. Partitioning of the extract between hexane and methanol or methanol/water mixtures may also be used; the highly lipophilic material is removed into the hexane layer.

Extraction of organic bases and acids utilising their ionised and un-ionised forms

Salts of organic bases such as sulphates and hydrochlorides are often highly water soluble and the free bases are usually quite organosoluble, particularly in relatively polar solvents such as chloroform or mixtures of chloroform and ethanol. Similarly the sodium or potassium salts of organic acids are freely water soluble while the un-ionised acids are usually quite organosoluble. These properties can be used to advantage in designing an extraction procedure. A flow diagram for the extraction steps which can be used for the separation of an organic base from a formulation is shown in Figure 15.1.

Fig. 15.1 Flow diagram for the extraction of an organic base. (1) Direct removal of neutral and acidic excipients. (2) Acidic excipients left behind in aqueous layer. (3) Neutral excipients left behind in organic layer.

This type of extraction is employed in the BP assay of Cyclizine Lactate Injection: the injection is diluted with dilute H₂SO₄ and then neutral and acidic excipients are extracted with ether. The solution is basified and the cyclizine is extracted into ether, leaving the lactate ion, which would not have extracted during the initial ether extraction step, behind in the aqueous layer. For convenience in measurement by UV spectrophotometry and in order to carry out volumetric dilution of the extract, cyclizine is then back extracted into dilute H₂SO₄ and subjected to further dilution.

The same principles apply to the extraction of an organic acid except that in this case high pH values are used to ensure that the acid remains in the aqueous layer and low pH values are used to ensure that it is extracted into the organic layer.

Partitioning between organic solvents

Partitioning between organic solvents is used in the extraction of analytes from oily excipients such as in the extraction of steroid creams prior to HPLC analysis. The most commonly used systems are methanol/hexane, aqueous ethanol/hexane or acetonitrile/hexane. In the case of analysis of corticosteroids in creams, the cream is usually dispersed by heating in hexane and then extracted with an equal volume of methanol. Methanol and hexane mix only very slightly and the oily excipients remain in the predominantly hexane layer, while the more polar corticosteroid partitions into the methanol layer. Use of an internal standard with a structure closely related to that of the analyte is essential in order to achieve good precision in this type of analysis through compensating for incomplete recovery of the analyte. Examples of pharmacopoeial methods using this type of partitioning include assays of: hydrocortisone acetate cream, fluocinolone cream and beclometasone cream.

Ion pair extraction

Ion pair extraction provides a standard method for estimating ionic surfactants either colorometrically or titrimetrically. For example, a cationic surfactant such as cetrimide can be estimated by pairing it with a lipophilic anionic dye such as bromocresol purple. The ion pairing creates a coloured lipophilic ion pair, which can be extracted into an organic solvent such as chloroform and a quantitative measurement of the colour extracted can be made spectrophotometrically. This type of assay is described in the BP for Clonidine Injection and Benzhexol Tablets.

Ion pair extraction has also been used to extract polar analytes in bioanalytical procedures. Figure 15.2 exemplifies the determination of the amino acid taurine by gas chromatography–mass spectrometry (GC–MS); this figure also illustrates a useful property of amines (and phenols), which is that they will react more rapidly than water with an acylating reagent in an aqueous environment, thus improving their organosolubility. After acylation and ion pair extraction with tetrabutyl ammonium sulphate, the taurine is converted to an amide prior to analysis by GC–MS.

Fig. 15.2 Extraction and derivatisation of taurine.