Pharmaceutical profiling method for lipophilicity and integrity using liquid chromatography–mass spectrometry

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Abstract

A method is described for the simultaneous profiling of sample lipophilicity, integrity, and purity. The method is rapid and is applicable to high throughput profiling of pharmaceutical properties in drug discovery. A short Polaris C18 column is used with a rapid, wide-polarity mobile phase gradient, UV detection, and MS analysis. The lipophilicity of each component is estimated from a calibration curve using six drug or organic compounds and plotting their respective measured retention time versus Log D7.4 (literature). The correlation of Log D7.4 (literature) to Log D7.4 (HPLC) for 60 structurally diverse drugs has a correlation coefficient r2 of 0.89. The method is applicable to compounds with MW>200 and retention time>1.5 min for rapid, initial pharmaceutical profiling in drug discovery.

Introduction

Pharmaceutical profiling is the assessment of compound properties during drug discovery [1], [2]. This function is valuable for reducing attrition and delays in development, resulting from poor biopharmaceutical properties, as well as providing property information that can be used by research teams to make informed decisions on discovery experiments. Properties such as lipophilicity, solubility, permeability, stability, and pKa have a major effect on the concentration of drug delivered to the therapeutic target after dosing. Thus, properties affect the observed biological activity (pharmacology) in high throughput screening, enzyme/receptor assays, cell-based assays, animal models, and humans.

Lipophilicity is a physicochemical property of interest in drug discovery. This property provides discovery scientists with insights on the tendency of the compound to partition into lipid versus aqueous environments. Such insights are useful for correlation to important pharmaceutical processes such as gastrointestinal absorption, membrane permeation, solubility, volume of distribution, and protein binding.

Lipophilicity is commonly assessed via measurement of octanol–water partitioning [3], [4], [5]. The result is a value of Log P (the partition coefficient taken at a pH where, all of the compound is in the neutral state), or Log D (the distribution coefficient taken at a pH, where a significant portion of the compound exists in the charged state). The method has been scaled-down to 96-well plates, by Zaslavsky and co-workers, in order to increase throughput [6]. Lipophilicity has also been measured using microemulsion electrokinetic chromatography [7], pH-metric method [8], and HPLC [9], [10], [11], [12]. Lombardo et al. [12], [13] and Donovan and Pescatore [14] have described elegant methods for measuring Log D by HPLC. Valko and co-workers have described LC–MS methods for rapidly measuring hydrophobicity index [15], [16].

LC–MS is commonly used in drug discovery to rapidly profile the integrity and purity of compounds [17]. Extending this methodology to also profile lipophilicity in the same analysis was investigated. A combined method would provide additional pharmaceutical profiling data for drug discovery without expending additional time or precious material. Retention time was calibrated to lipophilicity by using compounds for which Log D at pH 7.4 had been measured using octanol–water partitioning [12], [13], [18]. Performance of the method for integrity/purity profiling was also assessed. A rapid LC–UV-MS method that provides estimates of Log D at pH 7.4 and integrity/purity at the rate of 5.5 min/compound for application in pharmaceutical profiling is described.

Section snippets

Experimental

DMSO was from Aldrich (Milwaukee, WI, USA). Acetonitrile and water were HPLC grade from EM Sciences (Darmstadt, Germany). Ammonium acetate, acetic acid and ammonium hydroxide were from J.T. Baker (Phillipsburg, NJ, USA). Test compounds were from Sigma (St. Louis, MO), Fluka (Steinheim, Switzerland), and Aldrich.

LC–MS analysis was performed using an Agilent 1100 HPLC and a Waters ZQ mass spectrometer system with electrospray ionization and alternating positive and negative ion quadrupole mass

Results and discussion

A set of six compounds, having Log D7.4 literature values from −2.00 to +5.50 were selected and used to calibrate tR versus Log D7.4. These compounds are listed in Table 1, along with the average literature Log D7.4 value [18] and tR, as determined using the method. The calibration line is shown in Fig. 1. A correlation coefficient of 0.994 was obtained.

Initial work with the method indicated that the volume of injection was critical for obtaining proper Log D predictions, especially for more polar

Conclusion

Pharmaceutical profiling assays provide a rapid assessment of the properties of compounds in drug discovery. They are not intended to provide the precision and accuracy of established methods, typically used for in-depth analysis in late discovery or development. Their utility is to rapidly provide property information to increase medicinal chemist’s early knowledge and assist informed decisions in drug discovery. Research teams typically assess libraries of thousands of compounds in a rapid

Acknowledgments

The authors thank Melissa Moyer for in-house log D determinations and Nelson Kung for assistance in sample preparation. Portions of this material were presented at the ASMS Annual Conference, June 2000, Long Beach, CA.

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