Characterization and Performance of MALDI on a Triple Quadrupole

Aug 5, 2005 - We report instrument characterization and small-molecule quantification performance data from a high repetition rate laser MALDI ion sou...
0 downloads 10 Views 519KB Size
Download PDF
Anal. Chem. 2005, 77, 5643-5654

Characterization and Performance of MALDI on a Triple Quadrupole Mass Spectrometer for Analysis and Quantification of Small Molecules Jason Gobey,*,† Mark Cole,† John Janiszewski,† Thomas Covey,‡ Tung Chau,‡ Peter Kovarik,‡ and Jay Corr‡

Groton/New London Laboratories, Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06340, and MDS Sciex, 71 Four Valley Drive, Concord, Ontario, L4K 4V8 Canada

The usefulness of MALDI for small-molecule work has been limited by matrix chemical interference in the mass range of interest, tedious sample preparation, and various crystallization and sample deposition issues. We report instrument characterization and small-molecule quantification performance data from a high repetition rate laser MALDI ion source coupled to a triple quadrupole mass spectrometer. The high repetition rate laser improves sensitivity and precision and allows a proportional increase in sample throughput. Tandem mass spectrometry is used to discriminate the signal from the high chemical background caused by the MALDI matrix. Successful quantification requires use of an internal standard and a means of sample cleanup for typical in vitro sample compositions. This instrument combination and analysis technique is relatively insensitive to sample crystal quality and spot homogeneity. Quantitative performance results are characterized for 53 small-molecule pharmaceutical compounds and compared to those obtained by ESI-MS/ MS. Further comparison between MALDI and ESI is examined, and the potential for high-throughput MALDIMS/MS quantification is demonstrated. Since its introduction, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has been applied to the analysis of a wide variety of molecules.1-4 Being a soft ionization technique with the ability to desorb and ionize large intact molecules, MALDI, commonly coupled with a time-of-flight (TOF) detector, has most often been applied to peptides and protein analysis. There are limited reports of the successful application of MALDI to small-molecule quantification.5-12 That MALDI has not been * Corresponding author. E-mail: [email protected]. † Pfizer Global Research and Development. ‡ MDS Sciex. (1) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int J. Mass Spectrom. Ion Processes 1987, 78, 53-68. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (3) Bahr, U.; Deppe, A.; Karas, M.; Hillenkamp, F. Anal. Chem. 1992, 64, 28662869. (4) Beavis, R.; Chait, B. Anal. Chem. 1990, 62, 1836-1840. (5) Cohen, L.; Gusev, A. Anal. Bioanal. Chem. 2002, 373, 571-586. (6) Ling, Y.; Lin, L.; Chen, Y. Rapid Commun. Mass Spectrom. 1998, 12, 317327. (7) Kang, M.; Tholey, A.; Heinzle, E. Rapid Commun. Mass Spectrom. 2000, 14, 1972-1978. 10.1021/ac0506130 CCC: $30.25 Published on Web 08/05/2005

© 2005 American Chemical Society

utilized effectively for small-molecule quantification is largely because of the high degree of chemical interference below molecular mass 500 Da, caused by the cocrystallized MALDI matrixes. Further, most commercially available instruments utilize a TOF detector, which is arguably less suited for quantification, since the dynamic range for a quantitative assay is generally limited to ∼2 orders of magnitude. To improve quantitative performance, previous reports have described a variety of approaches to improve the cocrystallization of the analyte with the matrix and to compensate for differences between spots. These include a fast-evaporation sample preparation procedure,8 use of isotopically labeled analogues or close structural analogues as internal standards,9,12 and electrospray sample deposition.10 Recently a charge derivatization approach was used to tag small amine molecules13 and take them out of the troublesome mass range by substantially increasing the weight of the protonated species that was detected. Recently high repetition rate lasers have been incorporated to improve throughput.14-18 These lasers allow a larger number of measurements (laser firings) to be gathered in a shorter amount of time, allowing rapid signal averaging, and increasing the (8) Horak, J.; Werther, W.; Schmid, E. Rapid Commun. Mass Spectrom. 2001, 15, 241-248. (9) Duncan, M.; Matanovic, G.; Cerpa-Poljak, A. Rapid Commun. Mass Spectrom. 1993, 7, 1090-1094. (10) Hensel, R.; King, R.; Owens, K. Rapid Commun. Mass Spectrom. 1997, 11, 1785-1793. (11) Gusev, A.; Wilkinson, W.; Proctor, A.; Hercules, D. Anal. Chem. 1995, 67, 1034-1041. (12) Kang, M.; Tholey, A.; Heinzle, E. Rapid Commun. Mass Spectrom. 2001, 15, 1327-1333. (13) Lee, P.; Chen, W.; Gebler, J. Anal. Chem. 2004, 76, 4888-4893. (14) McLean, J.; Russell, W.; Russell, D. Anal. Chem. 2003, 75, 648-654. (15) Hatsis, P.; Brombacher, S.; Corr, J.; Kovarik, P.; Volmer, D. Rapid Commun. Mass Spectrom. 2003, 17, 2303-2309. (16) Brombacher, S.; Hatsis, P.; Corr, J.; Kovarik, P.; Volmer, D. Comparison of Two Novel Prototype MALDI Mass Spectrometers for Quantitative Analysis of Small Pharmaceutical Drugs. Proceedings of the 51st ASMS Conference on Mass Spectrometry and Allied Topics; Montreal, Canada, June 2003. (17) Kovarik, P.; Corr, J. J.; Covey, T. R. Application of Orthogonal MALDI for Quantitation of Small Molecules using a Triple Quadrupole Mass Spectrometer. Proceedings of the 51st ASMS Conference on Mass Spectrometry and Allied Topics; Montreal, Canada, June 2003. (18) Corr, J. J.; Covey, T. R.; Chau, T. K.; Kovarik, P.; Fisher, W. MALDI MS/ MS on a Triple Quadrupole Mass Spectrometer: A New Technology for High Throughput Small Molecule Quantitation. Proceedings of the 51st ASMS Conference on Mass Spectrometry and Allied Topics; Montreal, Canada, June 2003.

Analytical Chemistry, Vol. 77, No. 17, September 1, 2005 5643

measurable sensitivity. A high repetition rate laser is necessary to derive quantitative data with sample acquisition times appropriate for normal throughput (e.g., three or more samples per minute).19,20 In this report, we characterize a high repetition rate laserMALDI ionization source coupled to a triple quadrupole mass spectrometer for fast, quantitative analysis of small molecules. Linearity and dynamic range were evaluated using typical smallmolecule pharmaceuticals. Additionally, 53 compounds were run through standard microsomal incubation assays. The resulting samples were analyzed using the MALDI-MS/MS instrument and compared to analysis by traditional LC/ESI-MS/MS techniques. Performance of small-molecule analysis by MALDI is compared to ESI in terms of coverage of small-molecule chemical space, sensitivity, and speed. EXPERIMENTAL SECTION ESI-Triple Quadrupole Mass Spectrometer. Data were obtained using a standard Sciex API3000 with a Turboionspray interface. The HPLC system has been previously described.21 Briefly, it consists of a dual-column/dual-injection system. Each injection port utilizes a six-port, two-position switching valve. Another 6-port valve directs the aqueous mobile-phase flow, while a 10-port valve directs the organic mobile phase and controls column switching. The system allows rapid LC/MS/MS data acquisition wherein all data points for a typical in vitro assay are collected into one data file at the rate of 3 samples/min. Prior to assaying samples, generic analytical conditions were obtained for each analyte of interest. The process has previously been described.22 Briefly, all analytes were arranged in 96-well plates. They were injected in rows, first in Q1 full-scan mode, to determine the parent ion. Then all analytes were injected a second time to determine the most intense product ion and the better of two standard collision energies, 28 or 48 eV. No other parameters were optimized, and this generic method/selective reaction monitoring (SRM) combination was used for subsequent assays. MALDI-Triple Quadrupole Mass Spectrometer. Data were obtained using a prototype MALDI source coupled to a modified API3000 triple quadrupole mass spectrometer (MDS Sciex). The API interface was removed and modified to accept the prototype orthogonal MALDI source. The MALDI target plate was under low vacuum (10 mTorr) and directly exposed to within 3 mm from the entrance quadrupoles. The ionizing region was pressurized by a nitrogen bleed to assist in optimizing ion kinetic energy and focusing. Samples were deposited onto the stainless steel target plate. The instrument was equipped with a solid-state diode pumped laser at 355-nm wavelength, 500-ps pulse width, with an output of ∼16 µJ/pulse (Nanolase, JDSUniphase). The laser is a (19) Gobey, J. S.; Janiszewski, J. J.; Cole, M. J.; Corr, J. J.; Kovarik, P.; Chau, T. K.; Covey, T. R. Performance of MALDI/MS/MS for Small Molecule Quantitation. Proceedings of the 51st ASMS Conference on Mass Spectrometry and Allied Topics; Montreal, Canada, June 2003. (20) Cole, M. J.; Gobey, J. S.; Janiszewski, J. J.; Corr, J. J.; Chau, T. K.; Kovarik, P.; Covey, T. R. Characterization of MALDI on a Triple Quadrupole Mass Spectrometer for Analysis and Quantitation of Small Molecules. Proceedings of the 51st ASMS Conference on Mass Spectrometry and Allied Topics; Montreal, Canada, June 2003. (21) Janiszewski, J.; Rogers, K.; Whalen, K.; Cole, M.; Liston, T.; Duchoslav, E.; Fouda, H. Anal. Chem. 2001, 73, 1495-1501. (22) Whalen, K.; Rogers, K.; Cole, M.; Janiszewski, J. Rapid Commun. Mass Spectrom. 2000, 14, 2074-2079.

5644 Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

Table 1. Quantification Performance of MALDI for Neat Calibration Curves compound

% precision (0.03 µM)

% accuracy (0.03 µM)

% precision (30 µM)

% accuracy (30 µM)

zolpidem verapamil buspirone quinidine olanzapine loperamide midazolam carbamazapine haloperidol

2.6 4.7 15 15 10 8.0 14 5.2 17

120 101 111 118 85 101 95 94 106

12 13 1.3 3.0 3.0 7.9

85 93 108 106 85 89

high repetition rate laser that delivers pulses at frequencies up to 1400 Hz, and the light is transferred to the MALDI source focusing optics through an attenuator and fiber optic. The MALDI target plate is held within an X-Y stage, and data acquisition is performed while moving the target plate in the laser focal plane. The mass spectrometer acquired data using the same generic method/MRM combination that had previously been determined using ESI-MS/MS as described above. Measurements from each sample spot were averaged together, and data for each compound were collected into a single data file for faster data collection. Materials. The MALDI matrix used for all sample analyses was R-cyano-4-hydroxycinnamic acid (R-CHC matrix solution; Agilent Technologies). The internal standard used for all analyses was prazosin (Sigma). Other commercially available compounds were obtained from Sigma. Sample Cleanup/Preparation. Three types of samples were examined, which necessitated different approaches to sample cleanup. Neat calibration curves were prepared for the compounds shown in Table 1. The actual MALDI-MS/MS samples were prepared using the dried droplet method, where the samples were thoroughly mixed with matrix solution (R-CHC containing a 1 µg/ mL concentration of prazosin) in equal volumes. A 0.25-0.35-µL spot was deposited on to the MALDI target and allowed to evaporate at room temperature. Microsomal incubation assays were performed, and the reactions were stopped by acetonitrile precipitation. The solid-phase extraction protocol typically consisted of the addition of 250 µL of water to the 50 µL of precipitate supernatant. The samples were transferred to a previously conditioned Oasis µElution HLB plate (Waters, Milford, MA) under ∼10 mmHg vacuum. Prior to the addition of sample, the extraction plate was conditioned with methanol, followed by water. After the sample was loaded, the sorbent was washed with 200 µL of 5% methanol solution. Samples were eluted with 25 µL of elution solvent. The elution solvent was prepared by mixing 3 mL of R-CHC MALDI matrix with 7 mL of 40/60 acetonitrile/2-propanol. A total of 100 µL of prazosin (100 µg/mL in acetonitrile) was added as internal standard. The eluate was collected into a new deep-well plate. Addition of the MALDI matrix directly to the elution solvent provided the advantage of homogeneous sample/matrix mixing and ease of sample handling. A 0.25-µL aliquot of sample is simply placed directly onto the MALDI plate for analysis. Sample cleanup was further optimized for the analysis of in vivo samples. Calibration curves were prepared in human serum. The extraction method was similar to that used for the in vitro

Figure 1. Full scan of R-cyano-4-hydroxycinnamic acid below m/z 1300, obtained by directly analyzing the matrix solution with the prototype MALDI triple quadrupole mass spectrometer.

samples. However, an Oasis µElution MCX plate (Waters) was used. A two-dimensional mixed-mode mechanism allowed a more aggressive wash protocol. The samples were loaded, washed with 2% formic acid solution, and washed with methanol. The samples were eluted with 50% methanol solution containing 5% ammonium hydroxide. The eluate was evaporated to dryness and reconstituted in 25 µL of R-CHC solution containing internal standard. A 0.25µL aliquot of this sample/matrix mixture was placed directly onto the MALDI plate for analysis. RESULTS AND DISCUSSION Successful quantification of small molecules from biological samples by MALDI mass spectrometry has four requirements. Tandem mass spectrometry is needed for selectivity, a high repetition rate laser provides sensitivity and speed, sample cleanup is required for proper cocrystallization and reduction of ion suppression, and an internal standard is necessary for precision. Tandem Mass Spectrometry. Matrixes used in MALDI to promote and assist desorption and ionization are typically small polar acids and bases. The mass spectra produced in MALDI have significant contributions below m/z 600 due to these matrixes, as shown in Figure 1. For the large-molecule analyses that typically employ MALDI, this chemical interference is irrelevant. However, small-molecule analyses directly compete in this high chemical background. There is significant intensity at every m/z below 600 that must be addressed for useful quantitative analysis. Figure 2a illustrates this challenge. A full-scan MALDI-MS spectrum from a sample spot containing 4 ng/µL carbamazepine shows a minor contribution from this relatively high concentration of analyte. Tandem MS provides an effective means of overcoming the interferences observed at low molecular weights. Similar to all tandem mass spectrometry measurements, however, discrimination is dependent on interfering components not having precursor/

product ion transitions in common with the analyte of interest. The abundant nature of the MALDI chemical background increases the probability of MS/MS interference. In practice, we observed a slight increase in chemical interference over ESI for particular ion transitions. Figure 2b shows a MALDI product ion spectrum of carbamazepine. The MS/MS spectra are similar to those obtained by ESI-MS/MS with the increased potential for additional ion peaks due to fragmentation of isobaric matrix components. Selected reaction monitoring experiments effectively discriminate analyte from background. Figure 2c shows the MALDI-MS/MS profile of five pairs of SRM (m/z 237-194) measurements from replicate sample spots containing 25 pg/µL carbamazepine each, with each pair separated by a single measurement of matrix blank. As expected, the high signal-tonoise ratio obtained by using tandem MS allows for limits of detection comparable to traditional ESI-MS/MS. High Repetition Rate Lasers. Traditionally, MALDI sources are coupled with TOF analyzers, where the TOF measurement is timed off of the laser pulse. Timing constraints limit the pulse repetition rate to 5-40 Hz. Recent developments in solid-state lasers have resulted in commercial products that obtain kilohertz pulse rates. Since the triple quadrupole mass spectrometer is a continuous beam analyzer and does not require the timing event of the laser pulse, it is ideally suited for taking advantage of higher repetition rate lasers. In this configuration, the MALDI ionization process occurs independently of the measurement process. The trace shown in Figure 3 is an expansion around a MALDI measurement such as those in Figure 2c. It illustrates the profile collected when the laser drills through the sample spot to complete ablation. At 1400 Hz, this depth profile of the spot is generated in ∼200 ms. During these 200 ms, ∼280 laser shots were fired. The power curve of this laser is shown in Figure 4. Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

5645

Figure 2. (a) Full-scan spectrum of 4 ng/µL carbamazepine neat standard by MALDI-MS. (b) MALDI -MS/MS scan of carbamazepine (products of m/z 237). (c) 25 pg/µL replicates of carbamazepine separated by blanks SRM m/z 237 f 194.

This curve is essentially flat across the operating range. The power measured is the unattenuated output as taken directly from the 5646 Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

laser. In practice, losses through transfer and focusing optics leave approximately 3-8 µJ incident on the sample. Note that the power

Figure 3. Hole drilled into sample spot by laser generating a “peak”.

Figure 4. Energy per pulse vs laser firing rate at 355 nm.

output is identical at 10 and 1400 Hz. These two points can be used to illustrate the advantage gained from operating at higher pulse rates. Figure 5 shows two profiles from the same sample. The first profile was obtained at 10 Hz and the second at 1400 Hz. Both profiles used similar numbers of laser shots, were generated from similar numbers of ions, and have similar absolute areas. All signal responses in the profiles are attributable to the analyte and not due to background noise. The ragged appearance of the 10-Hz profile is an artifact of maintaining a constant 20-ms dwell time for both measurements, which results in a ratio of 0.2 laser shots/dwell in the 10-Hz profile and 28 shots/dwell in the 1400-Hz profile. Since there is no measurable background signal, signal/noise is not meaningful, and the sensitivity is a function of

the detected signal rate. Adjusting the integration times to be similar on a relative basis smoothes the 10-Hz profile but does not result in increased sensitivity. At 10 Hz, 24 s was required to collect the profile, while only 180 ms was needed at 1400 Hz. Hence, the detected signal rate is proportional to laser pulse repetition frequency. This sensitivity gain is analogous to band compression achieved in chromatography. In addition to limiting sensitivity, the slower acquisition times associated with the lower pulse rate laser also inhibit the speed potential of the technique. Further comparison with a conventional 300-µJ N2 laser operating at 40 Hz shows the lower power, higher pulse rate laser also produces less in-source fragmentation. This characteristic contributes to greater quantitative sensitivity. Greater ion fragAnalytical Chemistry, Vol. 77, No. 17, September 1, 2005

5647

Figure 5. Comparison of data acquired for the same sample at different laser firing frequencies, 10 and 1400 Hz. The data acquisition time is much more rapid, and sensitivity is improved.

mentation is not inherent to the conventional N2 laser, and it is possible to operate this laser to produce primarily the MH+ species. However, the use of lower power with the N2 laser exacerbates loss of sensitivity by spreading the available signal over even longer time frames. The integration of the high repetition rate laser into the MALDI ion source allows quantitative data to be acquired rapidly, and with adequate sensitivity and statistical reproducibility. Crystal Quality and Sample Cleanup. It has been suggested that the MALDI ionization process is less susceptible to ion suppression than ESI,15 allowing greater latitude for direct sampling without prior cleanup. In our experience, however, a considerable degree of ion suppression is still evident in MALDIMS/MS. For in vitro samples, all initial attempts to analyze the assay samples directly by mixing with the MALDI matrix failed. This was due to poor crystallization and ionization suppression. Different ratios of sample to matrix solution were evaluated to overcome these problems, but this was also unsuccessful. Figure 6 shows the difference in crystal quality obtained from raw samples in contrast to those obtained from samples subjected to an SPE cleanup step. The top spot is representative of a clean sample. The crystals are small, dense, and evenly distributed. More notable is the complete ablation of the sample when rastered across the laser beam. The spectra obtained are intense and reproducible. The bottom spot in Figure 6 is typical of direct sampling. The crystals are large, sparse, and clumped. The major distinction is their resistance to ablation by the laser. Little ablation is physically apparent, and any resulting spectra are weak. Fortunately, simple established sample cleanup procedures are adequate for restoring analytical success. We chose SPE and used microelution plates to provide a small degree of sample concentration and avoid any further dry down/reconstitution steps. We also took advantage of the nonretentive behavior of the MALDI matrix 5648 Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

Figure 6. Crystal formation is critical for good MALDI-MS/MS performance. Samples were desalted using SPE cleanup (top). Raw samples did not yield good crystals, and as a result, data (below).

by using it as a component of the elution solvent. Doing this ensures a homogeneous sample/matrix mixture, allows preparation of 96 samples simultaneously, and avoids the tedious sample/ matrix mixing step. Furthermore, this method is amenable to automated sample spotting using common liquid handlers. This SPE-type sample cleanup is analogous to contemporary methodology in high-throughput analysis.21,23 Data Collection, Integration, and the Need for an Internal Standard. Data are collected by moving the sample spot under the laser focal point, creating a “raster” profile of the sample. A typical profile is shown in Figure 7a. Because the time necessary to ablate through the depth of the sample (∼200 ms) is shorter than the time required to move the x-y plate stage on this prototype source, a series of discrete profile “peaks” result as the sample is rastered across the laser beam. Area measurements for quantification are calculated from the integration of the entire packet of “peaks” collected across a sample. A typical sample area measurement integrates 2000-3000 laser shots. For analysis (23) Olech, R.; Pranis, R.; Perman, C.; Speziale, R.; Cole, M.; Janiszewski, J. Proceedings of the 50th ASMS Conference on Mass Spectrometry and Allied Topics; Orlando, FL, June 2-6, 2002.

Figure 7. Depiction of variability of spot shape. (a) A good profile consists of ∼10 separate measurements; each measurement is an average of ∼280 laser shots. (b) A spot with an irregular shape due to random variances in evaporation mechanism, or spot deposition will yield a smaller number of measurements. Both profiles yield useful data when analyte to internal standard ratios are used.

speed, related samples are spotted contiguously on the target plate, the entire sample set is rastered past the laser beam, and the data set is collected into a single data file. An example of a microsomal incubate time course collected in this manner is shown in Figure 8. The two aspects of MALDI sampling that are difficult to control are the uniformity of the cocrystallization of the sample with the matrix and the physical conformity of the sample spot (spot shape). Variations in these are responsible for most of the poor precision normally associated with MALDI measurements and make quantification difficult. Addition of an internal standard to all samples compensates for these variations and measuring the analyte to internal standard ratio returns high precision. Extreme measures to enhance spotting consistency (e.g., regulating sample volume, spot size, solvent composition, temperature, and deposition technique) are not practical, since despite careful practice, nonuniform sample spots occur. (See Figure 7b.) Despite the appearance of nonuniform spots, the analyte to IS ratios are identical for these samples as they are for similar samples forming more uniform spots, such as those in Figure 7a. Figure 9 illustrates the necessity of using an internal standard to compensate for measurement nonuniformity. Large variances in absolute analyte areas are corrected by the internal standard, and the resulting incubation time course profile is accurate and reproducible. Several approaches for choosing an internal standard were tested, and the choice of internal standard was not found to be particularly critical. We use prazosin because it responds well and has characteristics similar to the common analytes of interest (small-molecule pharmaceutics). For simplicity, the internal

standard is added to the bulk MALDI matrix solution. This pervasive use has the added advantage of providing ongoing instrument and source performance data and allows quantitative characterization of changes in experimental parameters. Interestingly, ions formed from the MALDI matrix itself can be successfully used as an internal standard, compensating for many of the analysis variables leading to imprecision. However, we found an externally added internal standard provided better performance in quantification. Quantitative Results. Satisfying the requirements of MS/ MS, high pulse rate laser, sample cleanup, and use of an internal standard allows for successful quantitative analysis by MALDI. Table 1 summarizes the initial data obtained from neat calibration curves. Individual curve points were spotted in duplicate (n ) 2). The LLOQ and ULOQ points were spotted five separate times (n ) 5) to allow for proper determination of precision and accuracy. Acceptable data were obtained, although for three compounds it was not possible to confirm linearity over 3 orders of magnitude. It should be noted that for in vitro ADME quantification, 2 orders of magnitude is sufficient, and it is of primary importance to be able to obtain good quantification at the LLOQ. A total of 53 diverse compounds were selected and assayed through a standard human microsome incubation time course study. The resulting samples were split and one set was analyzed through our standard LC/ESI-MS/MS system. The remaining set was processed through the SPE cleanup protocol, and analyzed using the MALDI-MS/MS instrument. Half-lives were calculated for all compounds, and the correlation of the two data sets is shown in Figure 10. Good correlation was obtained between the Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

5649

Figure 8. Example of microsome incubation time course. All time points were acquired to a single data file for clarity and speed.

Figure 9. Microsomal timecourse for a typical analyte, internal standard, and the analyte/IS ratio.

two techniques, and the unity slope ensures MALDI does not bias the analysis. Other in vitro samples from human hepatocytes, caco2, and MDR Pgp transport assays were also analyzed successfully. It is worth noting that the cocrystallized samples are very stable, and since only a portion of the spot is consumed during routine sample analysis, the MALDI target plate can be stored and the samples reanalyzed later. The results obtained for calibration curves in human serum were less successful. The curve for verapamil, for example, was linear from 5 to 5000 ng/mL. Verapamil has better than average 5650

Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

response in MALDI, but lower detection limits were not possible due to severe ion suppression. Further work will be required to develop this application, with particular attention to sample cleanup. Comparison of MALDI to ESI for Small-Molecule Analysis. Consideration and study of MALDI for small-molecule analysis inevitably leads to its comparison to LC-ESI. The specific qualities for routine quantification important to an ionization technique are its universality for ionization, coverage of chemical space, overall sensitivity, and potential for speed.

Figure 10. Human microsome half-lives for 53 compounds: ESI vs MALDI.

Small-molecule quantification by MALDI has its greatest utility when applied to higher throughput analysis of large numbers of samples generated from assays involving several hundred chemical entities. Current ESI practices in such an environment have several practical boundary conditions, such as the use of standardized mobile phases and chromatography conditions, the use of only a few template MS/MS condition settings, and the general inability to specifically optimize analytical conditions for any single compound. Similarly, these MALDI experiments were established with practical boundaries. Template MS/MS conditions were taken from the comparative ESI studies, R-CHC matrix was used exclusively, and no attempt was made to optimize matrix-to-analyte ratios for individual compounds. Since all MALDI experiments were performed in the positive ionization mode, only compounds known to produce positive ions were studied. The following comparisons of the two techniques were done within these practical boundaries, and the results represent those expected in a general higher throughput environment using semioptimized analytical conditions, but do not reflect what may be possible from a more customized and individual approach to analysis. The measure of universality is ionization success with sufficient sensitivity across a broad range of chemical space. For the comparison, 208 compounds forming positive ions under ESI conditions were chosen from our compound collection that were representative of the total chemical space available in the collection. Successful ionization was defined as obtaining a response from a 50 nM sample with a S/N greater or equal to 5. This criterion was chosen as representative of a measurement requirement for a typical microsomal incubation assay, where 5% of a 1 µM substrate remains at the end of the experiment. While determining sensitivity in this manner is not as rigorous as generating calibration curves for all 208 compounds, the criteria were chosen to be conservative but sufficient for determining relative performance between the two ionization techniques. Thirty-three of the 208 compounds (15.9%) failed the ionization criteria by MALDI and 14 of 208 (6.7%) failed by ESI. These data suggest that while MALDI is not as universal an ionization

technique as ESI, it may provide broadly successful ionization appropriate for routine quantitative analysis. Further evidence that the described ionization success experiment is valid for measuring relative performance is provided by comparing its failure rate with the weekly percent compound failure by ESI for routine ADME screening in our laboratories during the first 27 weeks of 2002 (the time period over which these experiments took place). This data set encompasses 15 000 compounds from diverse chemical series and has an average ionization failure rate of 6%. This failure rate correlates closely to the 6.7% of compounds that failed by ESI in the ESI/MALDI comparison experiment, and establishes a rationale for extending the MALDI results as broadly representative of the technique. While MALDI has an opportunity similar to LC-ESI for successful ionization, the factors controlling sensitivity in the two methods are very different. Sensitivity in a quantitative measurement is directly proportional to sample concentration and sample consumption. Here, LC-ESI can consume a larger proportion of the overall sample and deliver it to the ionization source in a concentrated band compared with MALDI. Chromatography serves to provide a local increase in concentration over the original diluted sample, which is complementary to the concentration dependence of ESI response. MALDI sample consumption and delivery concentration is small and relatively fixed. Since the samples are delivered to the MALDI measurements in a preset spatial array, the sample concentration available for measurement is limited to that of the original sample and the drying characteristic of its solvent makeup. Further, the sample consumption of the measurement is limited by the diminutive dimensions of the incident laser spot. Also contributing to overall sensitivity of a mass spectrometry measurement are the ionization and ion transfer efficiencies. Figure 11 shows MALDI and LC-ESI measurements of the same original sample. In MALDI, the sample consumed in a single laser ablation spot is equivalent to just over 2 nL of the original sample. A typical desorption envelope measurement consumes ∼25 nL of the original sample, as represented in Figure 11a. This MALDI Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

5651

Figure 11. Measurements for the same original sample by (a) MALDI MS and (b) ESI MS. MALDI is 100 times more efficient than ESI.

measurement provided an integrated area count of 26 970. In comparison, 25 µL of the original sample injected for an LC-ESI measurement (Figure 11b) provided an integrated area count of 217 100. These data suggest that MALDI provides a 100-fold increase in efficiency over ESI for the mass spectrometer used in these experiments. To date, the experiments to differentiate ionization efficiency from ion-transfer efficiency have not been performed. This efficiency gain is sufficient to bring the raw sensitivity of MALDI to within 10-fold of LC-ESI for typical quantitative measurements. MALDI’s retention of high S/N at limits of detection helps to provide ample precision. The final comparison between MALDI and LC-ESI involves their potential for analysis speed. LC-ESI analysis speed is mainly limited by the autosampler response and chromatographic elution times. Much work has been reported on minimizing the time necessary for LC-ESI analyses,21,24 and this technique has been pushed to ∼10 samples/min,25 with 4-6 samples/min reported in routine use. Speed is limited in MALDI by the time necessary to obtain sufficient quantitative precision through averaging measurements. A single laser shot is accomplished in less than 1 ms, but the precision is inadequate for quantification. The experimental measurements reported in this work consist of an envelope response obtained by moving the sample spot under the laser focal point. These measurements typically include 20003000 laser shots/sample. The speed of these measurements was limited by the relatively slow sample stage control motors, but still allowed for 8 samples/min analyses, as shown in Figure 12a. A practical minimum limit for the number of laser shots averaged is ∼250, which is the number of shots necessary to completely drill through a sample spot and provide a response profile that is easily integrated. This process occurs in 200-300 ms. The (24) Xu, R.; Nemes, C.; Jenkins, K.; Rourick, R.; Kassel, D. J. Am. Soc. Mass Spectrom. 2002, 13 (2), 155-165. (25) Whalen, K.; Courtney, J. C.; Smith, D.; Jaxheimer, B.; Tschopp, M.; Schelhorn, J.; Rogers, K.; Olech, R.; Janiszewski, J.; Cole, M. A Specialized High-Throughput Sample Delivery System Utilizing a Dual-Spray LC/MS Interface for the Bioanalysis of 500 Samples/Hour. Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics; Chicago, IL, May 2001.

5652

Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

response profile is the practical aspect of this limit. Producing a signal profile that has defined start and stopping points, along with a chromatographic peaklike shape, allows the use of common integration routines and data processing. Figure 12b is an expansion of a single sample measurement showing the envelope of individual drill-through responses. To determine the preservation of sufficient precision at the practical speed limit, these individual responses were integrated and plotted for a microsomal incubation time course and the results are shown in Figure 13. The larger diamonds are the overall average measurement of the entire response envelope for each sample, while the smaller circles are the individual measurements within these envelopes. Eight individual measurements were plotted for each sample, and the CVs ranged from 4 to 10%. The half-life calculation was 30.7 min with an overall CV of 7%. This half-life was identical to that obtained from the same sample set by LC-ESI, and the precision is comparable. These data suggest that MALDI analyses have the potential for speeds approaching 200-300 ms/samplesequivalent to ∼200 samples/min. CONCLUSIONS Liquid chromatography coupled to ESI-MS/MS is a wellestablished technique for quantitative analysis of small molecules. It provides universality and high sensitivity, and in recent years, its capabilities have been extended into higher throughput analyses. For a high-throughput screening analytical technique, however, it is encumbered by complicated solvent handling and plumbing and is slow relative to plate readers. Its main distinguishing strengths are selectivity, universality, and, to a lesser extent, sensitivity. The increasing demand for higher bioanalytical throughput and productivity in early Discovery ADME screening drives experimentation to develop mass spectrometry instrumentation and methodology into plate reader-style throughput and simplicity while preserving the present selectivity and universality. The high repetition rate MALDI triple quadrupole mass spectrometer has features addressing these issues. The potential for sample analysis times approaches plate reader speed and the lack of pumps, plumbing, and solvent provides simplicity. The capacity

Figure 12. (a) shows the acquisition of seven samples within 0.9 min. (b) depicts the expansion of a single sample measurement.

Figure 13. Example of a microsomal timecourse comparing the averaged data versus eight single measurements.

of such an instrument could be enormous, with small sample sizes and no real format or density constraints. This work was undertaken with a prototype ionization source. The laser transfer optics were crude and utilitarian, and the poor incident angle of the beam hampered its focus and energy transfer. No attempt was made to characterize or optimize the ionization event beyond what has been presented, and only a single matrix was studied. The pressure was adjusted in the ion-transfer region,

but little or no attempt was made to improve ion-transfer efficiency. Despite the crude nature of this setup, successful bioanalysis at subnanomole-level sensitivity across a broad range of molecular entities with impressive analysis speed was achieved. Good results were obtained on routine biological assay samples with a high correlation to those obtained from the LC/ESI-MS/MS analysis. Several challenges exist to the routine deployment of MALDI instrumentation in high-throughput quantification. Significant ion Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

5653

suppression was encountered with all biological matrixes tested. Relatively simple samples consisting of a buffer solution would need, at a minimum, to be desalted prior to MALDI-MS/MS analysis. More complicated samples need greater cleanup and possibly concentration. The SPE procedures employed in these experiments are successful, quick, and uncomplicated, but the present cost of SPE plates is prohibitive in a high-throughput environment. Development of either more tolerant MALDI methodology or less expensive sample preparation is necessary. The potential speed of MALDI analysis presents an engineering challenge in designing a system that does not require continuous operator intervention for sample loading. In addition, software analysis packages will need to address the challenges of capturing, integrating, and analyzing the types and quantities of data MALDI produces. Routine high-throughput analysis involves operating under calculated boundaries, some of which were previously mentioned. When possible, however, all attempts should be made to incorporate orthogonal or complementary secondary analysis options to improve success rates. Orthogonality between different ionization techniques is a particularly sought after quality. The ability to move failed analyses to an alternate successful method allows more flexibility and removes “blind spots” in the analytical toolbox. Only 4 of the 14 compounds failed by ESI were successful by MALDI, suggesting that MALDI does not provide an orthogonal ionization complement to ESI. A true determination of orthogonal-

5654

Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

ity, however, requires further studies. The total number of failed compounds studied was small, and no attempt was made to optimize the ionization conditions of either technique. The seemingly lower success rate of MALDI compared to ESI suggests that ESI will continue to play a role in high-throughput analysis. Strategies will be needed for optimally matching the analysis speed of MALDI with the complimentary use of ESI for picking up failed analyses. For example, one might use MALDI analysis to rapidly assay the large number of sample sets from all compounds, quickly screen the data for failed sets, and run those failed sets on the relatively slower ESI instrument. The entire process could be automated with the result of a significant increase in laboratory capacity and speed while maintaining analysis success. Finally, negative ionization by MALDI and the comparison to that in ESI was not studied. If MALDI is determined to be much less successful than ESI in negative mode, then ESI’s complementary role in a high-throughput strategy is more important. ACKNOWLEDGMENT The authors thank Nora Wallace for generating the microsomal incubation studies and Sabrina Zhao for the ESI-LC/MS analyses.

Received for review April 11, 2005. Accepted July 6, 2005. AC0506130