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Figure 5 Schematic of traditional sequential mode injection to injection cycle time (injection overhead) operation of an LC system.

Figure 6 Traditional sequential mode injection showing cycle to cycle inject time. Separation conditions and peak identification are identical to those reported in Figure 2B. Data system run time was extended to 20 minutes to capture LC instrument timing.

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Equilibrating the system separately from the column can also save additional time. Equilibration time is an essential part of gradient chromatography. Both the LC system and the column must be returned to initial mobile phase conditions prior to the next run to ensure repeatability. Traditionally, equilibration times of 5–10 column volumes have been used (9). However, with the use of smaller diameter and shorter LC columns, a 10-column volume is not always enough to equilibrate both the LC system and the column. To compensate for this situation, the reequilibration volume has been divided into two parts: the volume required for system equilibration and the volume required for column equilibration (10). Total equilibration time is then given by the formula:

Tr (3VT 5VC )/F

where Tr is the equilibration time (min), VT is the total system volume, VC is the column volume (mL), and F is the flow rate (mL/min). As the column gets smaller (with a correspondingly lower flow rate), more equilibration time is taken up by returning the system volume of the LC to initial conditions. In advanced LC systems with integrated fluidics and control, a purge step can be added post run at high flow rates (5–7 mL/min) with the column off-line to significantly reduce system equilibration times. By starting the gradient but holding off on the injection for the amount of time proportional to the system volume, the volume of the system can also be used to aid in column equilibra- tion—a technique referred to as a ‘‘just-in-time gradient.’’ Loading the sample loop, as illustrated in Fig. 8 (p. 123), during the equilibration saves additional time by further reducing injection overhead. Comparing Figs. 6 and 8, the parallel mode with rapid equilibration increases throughput by about 30% without sacrificing chromatographic information or integrity.

Rapid equilibration techniques are particularly helpful to save time as the flow rate decreases, in, for example, microbore applications. Figure 9 (p. 124) shows a separation on a 1 50 mm column, at 0.3 mL/min, with a cycle-to-cycle inject time of 5.5 min using the parallel mode with rapid equilibration. Run in the traditional sequential manner, the corresponding cy- cle-to-cycle inject time would be in excess of 7.5 min. At lower flows, the time saving during reequilibration would be even more significant.

Some general notes on operating LCs in a high-throughput mode are as follows:

With the trend toward smaller columns, extra care should be taken to minimize extra column band broadening by using reduced diameter tubing, smaller volume detector cells, and properly fitting connections (9).

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Figure 7 Schematic of parallel mode operation with rapid equilibration.

Photolabile or temperature-sensitive compounds may require special handling. LC systems are available that maintain samples in the dark and/or chilled if required.

The small peak volumes and resolution demands from high-throughput analyses require faster data collection rates for accurate results. Adequate detector time constants should also be used.

III.ADVANCED HIGH-THROUGHPUT ANALYSIS TECHNIQUES

A. Sample Pooling

Sample pooling can also be used to reduce analysis times. Pooling is commonly used in drug discovery and early development for pharmacokinetic screening in animals, intestinal permeability screening using Caco-2 cell– based assays, and combinatorial library analyses. Sample pooling is an injection technique that uses multiple sample aspirations and then coinjects the samples, decreasing analysis times by reducing the number of chromato-

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Figure 8 Parallel mode injection showing cycle to cycle inject time. Separation conditions and peak identification are identical to those reported in Figure 2B. Data system run time was extended to 20 minutes to capture LC instrument timing. Note differences in cycle to cycle timing relative to Figure 6.

graphic runs. It is performed as diagrammed in Fig. 10 (p. 126) either, for example, within a microtiter plate (intraplate) or between plate (interplate) pooling, with mixing or air or solvent segmentation. Figure 11 (p. 128) illustrates the results of an interplate pooling experiment using two compounds and two internal standards, run parallel with rapid equilibration mode conditions used in Fig. 6. The technique is quantitative (good accuracy and precision), as summarized by the data shown in Table 1. In this simple experiment, sample throughput is doubled without compromising inject-to-inject cycle time or injection overhead.

B. Two-Column Regeneration

A technique referred to as two-column regeneration has also been used to increase sample throughput. In this technique, while one LC column is running the gradient method, a second isocratic pump is used to equlibrate a second

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Figure 9 A bore separation cycle time comparison. Separation was performed on a Waters Alliance HT HPLC System (Waters Corporation, Milford, MA) using a 1.0 by 50mm 3.5 micron particle size Symmetry C18 column (Waters Corporation,

Milford, MA) at 60° C. The mobile phase consisted of 0.1% phosphoric acid as the A solvent, and acetonitrile as the B solvent, run as a linear gradient from 10–95% B over 2 minutes at 300 L/min. UV detection at 220nm, and a 2 L injection was used. Peaks 1–6 (0.1mg/mL each in 50/50 methanol/water) are uracil, caffeine, primidone, phenacetin, benzophenone and biphenyl, respectively.

Table 1 Quantitative Results of Interplate Pooling Experiment from Figure 9 (n 6)

			A/IS	B/IS
	A/IS	B/IS	Pooled	Pooled
Avg. ratio	1.129	2.632	1.123	2.618
%RSD	0.55	0.52	0.21	0.11
Avg. cycle	4.68	4.69		4.69
time (min.)
%RSD	0.30	0.08		0.10

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column to initial gradient conditions. After the analysis is complete, the columns are switched, and the first column is reequilibrated while the second column is run. Postrun equilibration time can be decreased using this technique on a traditional LC system; however, when modern LC systems are used that operate in the parallel rapid equilibration mode outlined above, any throughput advantages from the two-column regeneration technique may be minimal on the analytical scale. Throughput can, however, be increased by as much as 25% on the preparative scale (11).

C. Automated Multichannel Chromatography

Another way to save time is to use multiple separation and analysis channels on the same instrument. Recently, new technology was reported that allows multiple samples to be analyzed in parallel as illustrated in Fig. 12 (p. 129) (12). This technique allows for four LC columns running identical gradients in parallel on a single LC system to be multiplexed to one MS detector. In the LC/MS interface the traditional electrospray probe and outer source assembly are replaced by an array of four miniaturized, pneumatically assisted electrosprays. The interface has a sampling rotor that is monitored in real time enabling the four liquid inlets to be indexed. Four separate chromatograms are collected into four separate data files, allowing conventional data analysis, as presented in Fig. 13 (p. 130). Depending on LC conditions, analysis time for an entire 96-well microtiter plate can be reduced to as little as 1.1 h, resulting in sample throughput approaching 2000 samples per day.

D. Auxiliary Detection

As noted previously, fast, generic LC/MS methods are the techniques of choice for assessing the progress and final quality of large combinatorial arrays in drug discovery. However, ultraviolet (UV) or MS detection alone cannot always provide the type of quantitative data required when assessing compound purity. To obtain accurate quantitative data, UV detection requires the use of well-characterized reference standards due to the differences in molar absorptivity that may exist between members of a combinatorial library. Since reference standards may not be available for even a fraction of the members of any combinatorial library, detectors that respond to physical characteristics independent of compound-to-compound variations must be used, i.e., the ‘‘universal’’ detector. While no truly universal detectors exist, detectors do exist that provide similar responses to compounds in a particular class. These detectors include those used in evaporative light scattering detection (ELSD),

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Figure 10 Injection pooling schematic.

chemiluminescent nitrogen detection (CLND) and its cousin, chemiluminescent sulfur detection (CLSD). Used alone or in combination with UV and MS, use of these auxiliary techniques allows a more accurate and complete assessment of purity to be obtained.

The principles of ELSD date back a number of years (13–15). More recently, ELSD has been applied specifically to pharmaceutical analyses and combinatorial uses (2,16–18). Detection by light scattering is based on the available mass and not absorptivity (UV) or ionization efficiency (MS), making it more accurate in some applications. In ELSD, nebulized column effluent enters a heated drift tube where rapid evaporation of the LC mobile phase takes place. A stream of nitrogen gas sweeps any nonvolatile solutes toward a detection region. Detection is accomplished by a laser and photodiode at an angle of 90° , perpendicular to the central axis of the drift tube. As the solute particles pass through the laser beam, the source is scattered. The intensity of the scattered light measured by the photodiode is proportional to the amount of solute in the column effluent.

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Although most compounds respond well to ELSD, the volatility of some low molecular weight compounds may cause problems during the evaporation process. Varying response in different stages of the gradient can also cause problems. Use of ELSD in combination with UV detection minimizes these potential limitations.

CLND and, more recently, CLSD play a role similar to that of ELSD in quantitative assays of true unknowns. The CLND and CLSD detectors respond to the nitrogen and sulfur content of a compound, respectively. Both detectors operate under relatively the same principles and have been used with considerable success in drug discovery (19–21). In CLND, the analyte is oxidized at 1050° C, converting nitrogen-containing compounds to nitric oxide. The nitric oxide reacts with ozone to produce nitrogen dioxide in the excited state, which releases a photon when decaying to the ground state. The photons are measured by a photomultiplier tube and converted to an analog signal dependent only on the total mass injected.

Since a vast majority of drug compounds contain nitrogen, CLND is very useful for pharmaceutical analyses. The CLND response has been shown to be independent of gradient composition and, again, is often used in combination with other (UV and MS) detection systems (18). However, the presence of nitrogen-containing impurities in the sample or solvent will bias results. Mobile phases must of course be nitrogen-free, dictating the use of methanol or another alcohol rather than acetonitrile as mobile phase modifier.

IV. LC/MS INSTRUMENTS IN ROUTINE HIGH-THROUGHPUT USE

A. Open Access Instrument Operation

Traditionally, MS analyses have been performed in a centralized facility, often on highly specialized instruments that required constant operator intervention and maintenance. This situation is highly impractical when supporting a combinatorial program because it inhibits high throughput and general access to instrumentation and data. In response, instruments are now often operated in an ‘‘open access’’ environment. In such an environment, people not trained in LC or MS can submit samples on a continuous basis and get rapid turnaround. The use of MS and its wealth of information is promoted, and spectrometrists are freed from the mundane, tedious task of repetitive sample analysis of perhaps thousands of samples.

An open access setup usually consists of a workstation at the point of need. The walkup user has access to the workstation as well as to the sample

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Figure 11 Interplate pooling with internal standards. Separation conditions are identical to those reported in Figure 2B. Peaks A and B are acetylfuran and valerophenone. The internal standards for peaks A and B are acetanilide and benzophenone, respectively. Data system run time was extended to 15 minutes to capture LC instrument timing. Cycle to cycle injection time is 4.7 minutes including interplate pooling.

manager or autosampler used for injection. A system administrator is responsible for setting up the system and has access to and control of all components. The walkup user, perhaps a medicinal or synthetic organic chemist, logs in sample(s) (or an entire plate) at the workstation, places them into the directed location, and, depending on the queue and system setup, gets a postanalysis report at the point of use or e-mailed to his desk. Results are typically displayed in an integrated browser format, similar to that illustrated in Fig. 13. The browser provides a graphical display for review of the results, a confirmation of molecular weight, and displays corresponding chromatograms and spectra.

B. Mass-Directed Autopurification

As mentioned previously, during lead optimization and testing leading to candidate drug selection, compounds are often required in larger quantities. Analytical techniques used in lead discovery eventually give way to semipreparative or preparative mass-directed autopurification techniques, capable of isolating and purifying 10–20 mg or more of the compound of interest during a single chromatographic analysis. Short, wide-diameter columns operated at

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10–50 mL/min are the norm. Isolation and purification on the preparative scale involves fraction collection and reanalysis. Fractions are reanalyzed on the analytical scale to see how efficient the purification was. The analytical scale separation is also run on the system to check to develop the preparative method prior to actual use. Generic methods exactly like those outlined previously and scalable column chemistries are used.

Automated purification has evolved over the years from ‘‘collect everything’’ on a time basis to UV-based collection. In both instances, time-con- suming secondary analyses must be carried out to correctly identify the correct or desired fraction. More recently, techniques have evolved using mass-di- rected and intelligent automated purification. Mass-directed fraction collection is defined as collecting a fraction of a certain specified mass only. Intelligent automated fraction collection takes fraction collection one step further by allowing fraction collection based on masses, substructures/fragments, multiple masses, adducts, etc. This results in higher quality data, single fractions, less time, fewer steps, and less chance for errors. A typical high-throughput LC/MS mass-directed autopurification system is highlighted in Fig. 14.

The system as diagrammed in Fig. 14 used two-column regeneration to improve throughput. The system has both preparative and analytical capability so that samples can be run on the same system for rapid screening of original samples, or an initial purity assessment, as well as fraction collection capability. In addition to the two 6-port column-switching valves, two flow splitters

Figure 12 Schematic of an automated multichannel LC/MS system.