TYPES OF ANALYTICAL METHODS

Chromatographic methodologies have proved very useful for drug analysis.

From the mid-1970s to the early 1990s, the most widely used analytical methodologies in drug development were gas-liquid (GC) and high performance liquid chromatography (HPLC).

Gas-Liquid Chromatography

In GC, samples are vaporized in the injection port, and sample constituents are then separated as they are moved along the length of the column by the carrier gas. Separation of the constituents is achieved because each compound possesses a characteristic rate of dissolution into the stationary phase and revolatilization into the mobile phase that is dependent upon the characteristics of the compound, and the stationary phase used in the method (see Fig. 1) [8]. The extent of separation can be increased or decreased to some extent by altering the temperature of the oven in which the chromatographic column is housed. Some advanced GC systems also incorporates hardware that allows for variable injection port temperatures to increase analyte separation. However, the main means of increasing the separation of the analyte from other sample constituents is the choice of the stationary phase/column used in the method. As each analyte exits the column, it is detected and quantified by a detector (e.g., mass spectrometer, electron capture, flame ionization detectors, etc.). Gas chromatography is generally characterized by great analytical sensitivity, often as low pg/ml, but it is limited by the need to volatilize the compounds of interest. Compounds with high boiling points are difficult to vaporize and cannot be quantified by GC very readily [8]. For this reason, HPLC has been more widely used.

HPLC

In HPLC, the samples are dissolved in a solvent and injected into the system.

The analytes are then separated from other sample constituents by the differential rates of dissolution into the mobile phase and the stationary phase. The rate of this process is a characteristic of the analyte, mobile, and stationary phases used in the system. Increased or decreased separation can be obtained by altering the composition of the mobile phase solvent (i.e., changing the solvent polarity). Analytes are detected upon exiting the column by several types of detectors (i.e., UV-VIS, fluorescence, electro-chemical, mass spectrometers, Fourier Transformed Infrared (FTIR) detectors). The main limitation with HPLC is the ability to dissolve the

FIGURE 1 Chromatographic Separation. In GC, compounds are acted on by two forces: the carrier gas (mobile phase) which sweeps the molecules along the column (but does nothing to separate molecules), and dissolution of the compounds into the stationary phase. Separation is accomplished by the differences in the rate of dissolution of the molecules into and out of the stationary phase. The circles represent molecules with lower vapor pressures, which spend more time dissolved in the stationary phase. The circles are held up by the stationary phase, whereas the molecules represented by the squares have a higher vapor pressure (lower boiling point), and spend more time in the mobile phase, which sweeps these molecules out of the column faster than the circles.

Therefore, the squares are swept through the column to the detector faster than the circles. (The squares have a shorter retention time.) In HPLC, these interactions are similar. The difference is that a solvent is used in the mobile phase, and it contributes to the forces that separate the molecules.

sample in a solvent. This difficulty, however, is much less of a problem in HPLC than sample vaporization is in GC. The limit of detectability is usually lower with GC than HPLC (10 to 100 times), depending on the type of detector used. Generally, UV-VIS and fluorescence detectors in HPLC provide less sensitivity than GC detectors, but electrochemical and mass spectrometric detectors could provide equivalent sensitivity to GC systems.

LC/tandem Mass Spectrometry

Currently, the most widely used analytical technology is LC/MS/MS.

Traditionally, these systems were cumbersome and difficult to use, but recent advances in technology and automation have made LC/MS/MS systems the stalwart of current analytical methodologies. LC/MS/MS depends on HPLC to separate the analyte from other matrix constituents as described in the preceding section, but the use of tandem mass spectrometry allows for the detection of very small quantities of drug, in addition to generating information about the chemical structure of the analyte which allows for analyte identification.

Ligand-Binding Assays

In addition to LC/MS/MS, greater use is currently made of nonchromatographic techniques. The two most prevalent techniques, radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs) are ligandbinding techniques. These assays are based on specific or relatively specific antibodies that are developed for the analyte of interest (see Fig. 2).

RIAs

In a RIA, the analyte is incubated in a buffer with the antibody and a known quantity of radiolabeled analyte. After incubating these reactants for a period, the samples are centrifuged and the radioactivity in the bound, pellet fraction is counted (in some cases, the unbound tracer in the supernatant is counted instead). As the amount of analyte increases, more radioactive analyte is displaced and the amount of radioactivity in the pellet decreases.

Therefore, low radioactivity corresponds to higher amounts of actual analyte in the sample (see Fig. 3).

ELISAs

In an ELISA, the antibody is usually bound to a surface, and linked to some type of enzymatic reporter system (for instance, horseradish peroxidase).

Typically, the enzymatic reporter systems are linked to the surface of 96-well

plates. Samples are added along with the necessary reactants, and gently mixed. After a defined period of incubation, the reaction in each well is

“stopped” and the amount of analyte is quantified (often using a spectrophotmetric plate reader). One of the major drawbacks with ligandbased assays is antibody binding to nonanalyte entities. This type of binding will produce overestimates of the analyte quantity. It can be difficult to determine whether this process has occurred because unlike chromatography, there is no visual output to assess. Therefore, greater care has to be taken to ensure that no interference occurs in these types of assays.