HPLC Analysis of Nonvolatile Analytes Using Charged Aerosol Detection

Feb 01, 2005
Volume 23, Issue 2, pg 150–161

There continues to be strong demand for improvements in sensitivity, selectivity, throughput, qualitative content, and many other performance characteristics of high performance liquid chromatography (HPLC). A major need is for methods that provide both universal detection and quantitative analysis. It is widely recognized that no single HPLC detector is capable of distinguishing all possible analytes from a given chromatographic eluent and the term "universal" often is used to describe detection of a diverse range of analytes.

A primary goal for most analyses that seek universal detection is to obtain a consistent relationship between the magnitude of response and quantity injected for a range of analytes. This "consistency of response factors" allows the use of global mathematical relationships to estimate quantity (for example, use of parent drug response factor to quantify metabolites and degradants). This characteristic is useful in many applications, where it is impractical or impossible to use individual standards to calibrate the response for each analyte such as drug library QC, pharmaceutical impurity testing, complex lipid analyses, and many applications requiring mass balance assessment. Currently, performing such an analysis is difficult with available technologies.

Several techniques are employed for universal detection that are based upon measuring bulk properties or generic attributes among diverse analytes. These techniques include refractive index (RI), low-wavelength UV, evaporative light-scattering detection (ELSD), and chemiluminescent nitrogen detection. These techniques are in contrast to those geared toward selective detection, which typically is based upon measurement of specific properties or reactivity of analytes (for example, fluorescence at a specific wavelength, oxidation at a specific potential, and mass-to-charge ratio [m/z]). It should be noted that for most detectors, selectivity and universality are mutually exclusive. Universal detectors often require very selective sample prepurification and analytical separation techniques for analysis of biological matrices.

Various characteristics of commercially available instruments employed for universal detection have been compared previously (1-3). RI, while widely used, has significant limitations in sensitivity and is not compatible with gradient elution. Low-wavelength UV provides higher sensitivity and improved gradient compatibility. However, detection is limited to chromophores, and response magnitude depends upon molar absorbtivity, which can vary by orders of magnitude even among analogous structures. Although UV detection remains the primary technique for many HPLC analyses, it is unable to see compounds that lack a sufficient UV chromophore such as many underivatized amino acids, carbohydrates, lipids, polymers, surfactants, drug substances, and natural products. ELSD has become widely used alone or to complement absorbance and mass spectrometry (MS) detectors (3). ELSD can see compounds lacking a chromophore provided they have low volatility. Also, ELSD response magnitude often is less dependent on analyte chemical properties than MS or UV. ELSD, however, has significant limitations in precision, sensitivity, dynamic range, and the nature of calibration curves (1,4,5). Chemiluminescent nitrogen detection is a relatively new technology for nitrogen-containing molecules and, while less universal, can detect a broad range of pharmaceutical compounds. It has been reported that this detection technique can provide a more linear response and lower limit of detection (LOD) than ELSD, but with poorer precision, higher maintenance, and no compatibility with nitrogen-containing mobile phases such as those with acetonitrile and triethylamine (1). The demand for improved methods continues to drive novel approaches toward universal HPLC detection such as those involving condensation nucleation light scattering (5) and inductively coupled plasma MS (6).

A new HPLC detection method has been developed based upon charged aerosol detection (CAD). This technique is fundamentally different from that of other detectors and is based upon the coupling of HPLC with widely used electrical aerosol analyzer technology (4,7-10). A similar HPLC detection method, termed aerosol charge detection, was described by Dixon and Peterson (4). The detection principle involves the charging of aerosol particles via corona discharge with subsequent electrometer-based measurement and thus has some commonality with atmospheric-pressure chemical ionization (APCI) MS. However, CAD operates by detecting charged particles that have a selected range of mobility rather than by measuring individual gas-phase ions that are differentiated based upon m/z. Furthermore, previous studies have shown that the signal obtained with electrical aerosol technology depends primarily upon particle size across a wide range and does not depend significantly upon individual analyte properties (9). This more generic principle can thus theoretically provide consistent interanalyte response factors and complement atmospheric pressure ionization MS techniques such as electrospray and APCI.

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