Evaluation of Charged Aerosol Detection (CAD) as a Complementary Technique for High-Throughput LC-MS-UV-ELSD Analysis of Drug Discovery Screening Libraries

The successful application of high-throughput screening (HTS) as a tool for drug discovery research has driven the development of both high-throughput organic synthesis (HTOS) for compound collection expansion and the need for high-throughput LC-MS analysis for compound library quality assessment. Providing rapid evaluation of both the purity and quantity of material present in large numbers of samples in the absence of individual, fully characterized reference standards (e.g., as required for quantitation using UV detection) represents an ongoing challenge for pharmaceutical industry research.

A preliminary evaluation of charged aerosol detection (CAD) as an addition to the LC-MS quantitation toolbox was performed at Berlex Biosciences (Richmond, CA). In the preliminary phase of this study, the detector response of CAD was compared to evaporative light scattering detection (ELSD) for a series of common LC-MS calibration standards. Based on the initial observations, a set of diverse standards with appropriate chromatographic characteristics were selected to generate a calibration curve for quantitation. Finally, 20 building blocks from the HTOS reagent collection were analyzed to test the general applicability of the calibration approach.

Background

The ultimate goal of HTS of large compound collections in drug discovery research is the identification of biologically active compounds of pharmacological interest as starting points for drug discovery programs. Improvements in both biological screening methodology and HTOS have made the overall hit-to-lead process substantially more effective. Clearly, the usefulness of the biological data obtained is enhanced by improving the quality of the samples screened. For this purpose, quality can be defined as both purity and quantity of active ingredient in the solution used for screening.

The purity of HTOS compounds is often measured using HPLC separation with UV detection coupled to mass spectrometry for identification of the desired compound. However, significant differences in UV molar absorptivity for typical HTS collection compounds mean that the method cannot be used for quantitation without well-characterized standards for each sample.

Alternative detection techniques, including ELSD and chemiluminescent nitrogen detection (CLND), have been investigated for quantitation of HTOS compounds.1,2 Both of these techniques have been successfully applied under a narrow range of conditions. However, ELSD has a limited sensitivity range, leading to overestimation of purity through underestimation of volatile impurities. In addition, ELSD response signals show significant dependence on the organic modifier content of the mobile phase, leading to nonuniform response factors during gradient HPLC analyses. By the nature of the technique, CLND is only applicable for nitrogen containing compounds. Thus, the method does not detect impurities that do not contain nitrogen, again leading to overestimation of sample purity. In addition, CLND cannot be used with typical HPLC mobile phases with nitrogen containing solvents or mobile phase additives.

The recent introduction of a commercially available CAD instrument (ESA, Chelmsford, MA) has led to considerable interest in evaluating this method as a “universal” detector for both purity detection and quantitation in high-throughput analytical laboratory applications.3,4 In common with ELSD, the CAD method vaporizes the HPLC mobile phase eluent in a nebulizer chamber. The vapor phase containing the analyte then flows into a high-voltage chamber in which a secondary carrier gas stream of positively charged nitrogen is introduced. A corona discharge produces positively charged analyte particles that are carried in the gas phase through an electrometer, where the signal response is measured. Thus, the technique offers the potential for higher sensitivity to a wider range of molecular weight species when compared to ELSD, which relies on particle size dependent light scattering detection of analytes.

Experimental

All studies at Berlex were performed using a single-quadrupole ZMD 2000 mass spectrometer (Waters Corp., Milford, MA) with electrospray ionization (ESI) interface, an LC-20AD HPLC pump (Shimadzu Scientific Instruments, Columbia, MD), an HTC-PAL Compact autosampler (LEAP Technologies, Carrboro, NC), a model 2487 UV detector (Waters Corp.), a Corona CAD (ESA, Chelmsford, MA), and a Sedex 75 ELSD (SEDERE, Lawrenceville, NJ). The CAD and ELSD data were collected in separate experiments, in which each detector was connected in series to the outlet of the UV detector in the LC-MS system. Standards and test compounds were prepared as dimethylsulfoxide (DMSO) solutions and diluted to target concentrations with a 1:1 methanol/water mixture. Flow injection experiments used acetonitrile/water mixtures (with 0.01% trifluoroacetic acid [TFA]) as the carrier phase with a 0.55 mL/min flow rate. For LC-MS experiments, a Synergi hydro-RP column, 2 × 50 mm, 4 μm (Phenomenex, Torrance, CA), was used with a 1–99% acetonitrile/water (±0.01% TFA) gradient over 5 min with a 0.55 mL/min flow rate.

Results and discussion

Comparison of CAD and ELSD

Figure 1 - Flow injection MS-ELSD signal response.

A series of commonly used LC-MS standards was analyzed to compare the detector response of CAD with ELSD. Flow injection analysis (FIA) was used at constant concentration (125 µM) across a range of solvent compositions to confirm the previously reported dependence of both ELSD and CAD signals on the organic modifier content of the mobile phase in LC-MS studies.5,6 The results of the comparison of the CAD and ELSD signal responses for propranolol, theophylline, and thymidine are summarized in Figure 1 for ELSD and in Figure 2 for CAD.

Figure 2 - Flow injection MS-CAD signal response.

The ELSD and CAD signals both show a molecular weight dependence on response factor. However, the ELSD signal exhibits a nonlinear response with respect to the organic modifier content of the carrier phase (Figure 1). In comparison, the CAD signal has an essentially linear response to carrier phase organic modifier content (Figure 2). This observation suggested the potential for developing a generalized calibration curve for quantitation of diverse compounds with similar elution times in an LC-MS-CAD analytical system. If the retention times of the analytes are sufficiently close to the standards chosen, then a reasonable estimate of the quantity of material present can potentially be obtained.

Calibration standard selection

Figure 3 - LC-MS-CAD calibration samples.

Figure 4 - LC-MS-CAD calibration curves.

The series of calibration standards (Figure 3) was tested to evaluate the potential for a generalized CAD calibration approach. The results for LC-MS-CAD analysis for these standards as a function of concentration are shown in Figure 4. Three of the samples elute with similar retention times (RT = 2.24–2.88 min, Line A). Therefore, the mobile phase composition at the point of detection for L-tryptophan-2-naphthylamide, propranolol, and diphenhydramine should be comparable across the concentration range. The exception is adenosine (RT = 0.89 min, Line B); therefore, this compound was not used for the calibration curve when the sample test set was analyzed.