Physicochemical and Biomimetic Properties in Drug Discovery, Enhanced Edition by Valko Klara; Valko Klara ;

Author:Valko, Klara; Valko, Klara ;
Language: eng
Format: epub
Publisher: Wiley
Published: 2013-11-19T05:00:00+00:00

Thereafter, we need to make corrections to the pH change caused by the increasing acetonitrile concentration in the mobile phase. The CHI value of the compound indicates the volume percentage of acetonitrile when the compound elutes. Then the and pH shifts due to the presence of organic solvent have to be estimated. Instead of this, we have taken a pragmatic approach to measure the gradient retention times using only three different mobile phases with acidic, neutral, and basic pH values.

The acidic mobile phase was 0.01 M phosphoric acid or 0.01 M formic acid if volatile mobile phase has to be used because of the MS detection. This provided a starting mobile phase pH of approximately 2.6.

To assess the compound's lipophilicity at the blood pH (pH 7.4), a carefully adjusted 50 mM ammonium acetate buffer has been used, while for high mobile phase pH, the ammonium acetate buffer pH has been increased to pH 10.5 by adding significant amount of concentrated ammonia solution [42]. The buffers are compatible with both UV and MS detections.

It has already been discussed in Chapter 5 and demonstrated in Figure 5.17 that the mobile phase pH changes with increasing acetonitrile concentration during the short (2–3 min) gradient run. Fortunately, the neutral pH remains close to neutral, the acidic pH increases from pH 2.6 up to pH 5, while the basic pH decreases to 8.3 when the acetonitrile composition reaches 90%. Interestingly, the shift for acids and bases makes them weaker; so, practically, at higher pH values, due to the higher concentration of the acetonitrile, the acid shifts to higher, while the base shifts lower. It means that we can assume that, for example, a basic compound with a 9, then at the starting mobile phase pH 10.5, is practically unionized, and it stays so when the acetonitrile concentration increases, regardless of whether the pH drops to 8.5. From the CHI measurements that are carried out at three pH values (using parallel HPLC systems as is described in Reference [43]), we cannot derive the values of the compounds, but we can group them confidently into five different groups, such as acidic, basic, neutral, weak acid, and weak base. These groups are automatically calculated from the differences at the three pH CHI values. Supposing that the accuracy of the CHI determination is within 5 CHI units, a larger difference than 5 obtained in any of the three pH values refers to the change in ionization of the molecule. It means that when the CHI values are the same (more precisely, they are within 5 units) at all the three pH values, then the compound is said to be neutral. If the CHI values obtained at pH 7.4 and 10.5 are the same but lower at the acidic pH, then the compound is a weak base and it is not ionized at physiological pH. If the acidic pH CHI is higher, then the compound is an acid and it has significant negative charge at physiological pH values. If the pH 2 and pH 7.

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