Method development mobile-phase considerations

In supercritical fluid chromatography, fluids above their critical point are used as mobile phases. This chapter discusses the principles of operation, mobile phase considerations, parameters that can be adjusted in method development as well as an overview of instrumentation required and a few pertinent examples from current literature. Not everything can be illustrated, but the advantages of this diverse technology will be highlighted. 

The following physico-chemical properties of the analyte(s) are important in method development considerations vapor pressure, ultraviolet (UV) absorption spectrum, solubility in water and in solvents, dissociation constant(s), n-octanol/water partition coefficient, stability vs hydrolysis and possible thermal, photo- or chemical degradation. These valuable data enable the analytical chemist to develop the most promising analytical approach, drawing from the literature and from his or her experience with related analytical problems, as exemplified below. Gas chromatography (GC) methods, for example, require a measurable vapor pressure and a certain thermal stability as the analytes move as vaporized molecules within the mobile phase. On the other hand, compounds that have a high vapor pressure will require careful extract concentration by evaporation of volatile solvents. 

Another equally important consideration before development of a determinative or confirmatory method is an understanding of the chemical properties of the analyte. Such an understanding becomes the cornerstone of a successful method since the unique chemical properties of each analyte provide the basis for isolation and detection schemes. Table 1 lists some of the important chemical properties that could be considered. For example, knowing the or p/fb of an analyte could influence the choice of a liquid-liquid extraction scheme, solid-phase extraction (SPE) cartridge, mobile phase pH, or mass spectrometric ionization. Knowing the overall polarity of the analyte can be very helpful in the evaluation of an extraction or separation. Currently, computational methods are available to obtain an estimate of the logP... 

The efficacy of CE separation depends considerably on the type of capillary. Fused-silica capillaries without pretreatment are used most frequently. Its outside is coated with a polymer layer to make it flexible and to lessen the occurrence of breakage. The polymer coating has to be dissolved with acid or burned away at the detection point. Capillaries with an optically transparent outer coating have also found application in CE. The objectives of the development of chemically modified capillary walls were the elimination of electro-osmotic flow and the prevention of adsorption on the inner wall of the capillary. Another method to prevent the adsorption of cationic analyses and proteins is the use of mobile phase additives. The modification of the pH of the buffer, the addition of salts, amines and polymers have all been successfully employed for the improvement of separation. 

This case study illustrates the SP method development of an assay method for a controlled-release analgesic tablet with a single API. Certain considerations were taken into account. First, the analyte within the tablet matrice core had to be extracted quantitatively. Second, the analyte was diluted into a final solution that was compatible with the HPLC mobile phase. Third, short SP time was required (i.e., 30 min) to maximize productivity of the work scheme for processing a large number of samples. 

Despite these considerations, the first approach in method development for ESl-MS is the formation of preformed ions in solution, i.e., protonation of basic analytes or deprotonation of acidic analytes. Thus, for basic analytes, mixtures of ammonium salts and volatile acids like formic and acetic acid are applied. Alternatively, formic or acetic acid may be added to the mobile phase, just to set a low pH for the generation of preformed ions in solution. The latter approach is successful if sufficient hydrophobic interaction between preformed aiialyte ions and the reversed-phase material remains. The concentration of buffer is kept as low as possible, i.e., at or below 10 nunol/1 in ESl-MS. The buffer concentration is obviously determined by the buffer capacity needed to achieve stable pH conditions upon repetitive injection of the samples. Constantopoulos et al. [99] derived an equilibrium partitioning model to predict the effect of the salt concentration on the analyte response in ESI. If the salt concentration is below 10 moFl, the analyte response is proportional to its concentration. The response is found to decrease with increasing salt concentration. 

Initially, MPH was extracted from plasma by LLE with cyclohexane. The reconstituted analytes were analysed using LC-MS [57]. A [ CDg]-analogue was used as ILIS. LC was performed using a 150x4.6-nun-ID Chirobiotic V column (5 pm) and methanol with 0.05% ammonium trifluoroacetate as mobile phase at a flow-rate of 1.0 ml/min. The first 3 min of the run were diverted to waste. The total ran time was only 7.5 min. The plasma-concentration-time profile for a child with ADHD after an oral administration of 17.5 mg of racemic MPH showed considerably higher plasma levels of d-threo-MPYi, as expected. The LOQ of this method was 87 pg/ml. This method was also applied in toxicokinetic studies in rat, rabbit, and dog [59]. In a further study [58], a semi-automated LLE in 96-well plate format was developed and validated. No chiral column was used in this case. 

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