Exploring the Importance of the Relative Solubility of Matrix and

Martin Engler , Kerstin Scheubert , Ulrich Schubert , Sebastian Böcker. Polymers ... Pieter C. Kooijman , Sander J. Kok , Jos J.A.M. Weusten , Maarte...
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Anal. Chem. 2004, 76, 5157-5164

Exploring the Importance of the Relative Solubility of Matrix and Analyte in MALDI Sample Preparation Using HPLC Andrew J. Hoteling,†,‡ William J. Erb,‡ Robert J. Tyson,‡ and Kevin G. Owens*,‡

Research & Development Laboratories, Eastman Kodak Company, Rochester, New York 14650-2132, and Department of Chemistry, Drexel University, Philadelphia, Pennsylvania 19104-2875

New insight into the role of solubility in the sample preparation process for MALDI MS is reported. Reversedphase gradient HPLC conditions were developed that enable the analysis of a broad range of analyte polarities with a single method. This HPLC method was used to establish a relative polarity scale for a series of 15 MALDI matrix materials, a set of example peptides, and a series of model polymer materials with a broad range of polarity. Examples of each polymer type within the range of 600010 000 were analyzed with six matrixes that cover a broad range of polarity using MALDI TOFMS. With regard to polymer signal-to-noise ratio, the matrix and polymer combinations that had a close match of HPLC retention time produced the best MALDI spectra. Conversely, the matrix and polymer combinations that have a large difference in HPLC retention time produced poor MALDI spectra. The results suggest that there is a relationship between polarity (solubility) and effective MALDI sample preparation. The relative HPLC retention time of an unknown polymer can serve as a starting point for predicting the matrix (or range of matrixes) that would be most effective. Since its introduction in the late 1980s,1,2 matrix-assisted laser desorption/ionization (MALDI) has become the ionization technique of choice for a broad range of biological materials and synthetic polymers. Sample preparation for MALDI is often simply described as the combination of a solution of an appropriate matrix with a solution of the analyte of interest, each in an appropriate solvent, at a high matrix-to-analyte mole ratio. The resulting solution is deposited on a target, the solvent is evaporated, and the matrix/analyte combination is analyzed as a solid. The challenging and often empirical steps in this process are finding the appropriate matrix and the appropriate solvent. The key to understanding this is in defining the term “appropriate” in both cases. To do this, it is important to understand the role of solubility in MALDI sample preparation. With regard to MALDI sample * To whom correspondence should be addressed. Tel: 215-895-2621. Fax: 215-895-1265. E-mail: [email protected]. † Eastman Kodak Co. ‡ Drexel University. (1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (2) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshido, Y.; Yoshido, T. Rapid Commun. Mass Spectrom. 1988, 2, 151. 10.1021/ac049566m CCC: $27.50 Published on Web 07/29/2004

© 2004 American Chemical Society

preparation, it is helpful to make a distinction between two types of solubility. The first is the solubility of the analyte and the matrix (and cationization reagent, if necessary) in a solvent, and the second is the homogeneous embedding of the analyte (and cationization reagent) within the matrix in the solid phase after solvent evaporation.3-7 We refer to these as “solution-phase solubility” and “solid-phase solubility” (or solid solution), respectively. Solution-phase solubility has been demonstrated to play an important role in MALDI sample preparation. The solvent used in sample preparation has been identified as a potential cause of discrimination effects. For synthetic polymer analysis, it is generally thought that it is best to use the same solvent to dissolve the matrix, the analyte (polymer), and the cationization agent (if needed)5 and that sample segregation can occur when using mixed solvents.8 The effects of using mixed solvents in the MALDI analysis of polystyrene (PS) and poly(methyl methacrylate) (PMMA) polymers was investigated by Li and co-workers.9 When a polymer “nonsolvent” was used as part of the sample preparation, a significant bias in the molecular weight (MW) distribution toward lower MW was observed when compared to the preparations where polymer “solvents” were used. The authors explained that with a nonsolvent the polymer tended to precipitate out as the sample was drying on the probe, causing it to be segregated from (not incorporated within) the matrix. When placed in the mass spectrometer for analysis, these nonhomogeneous sample surfaces lead to what is often referred to as “sweet spots”. This sample inhomogeneity is the basis of the large shot-to-shot and spectrum-to-spectrum variability that is generally characteristic of the normal dry-drop sample preparation method. Such sample segregation has been demonstrated to occur with biomolecules as well.10-14 As it is often difficult to solubilize the matrix and (3) Karas, M.; Bahr, U.; Deppe, A.; Stahl, B.; Hillenkamp, F. Makromol. Chem. Macromol. Symp. 1992, 61, 397-404. (4) Strupat, K.; Karas, M.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1991, 111, 89-102. (5) Danis, P. O.; Karr, D. E. Org. Mass Spectrom. 1993, 28, 923-925. (6) Juhasz, P.; Costello, C. E.; Biemann, K. J. Am. Soc. Mass Spectrom. 1993, 4, 399-409. (7) Axelson, J.; Scrivener, E.; Haddleton, D. M.; Derrick, P. J. Macromolecules 1996, 29, 8875-8882. (8) Chen, H.; Guo, B. Anal. Chem. 1997, 69, 4399-4404. (9) Yalcin, T.; Dai, Y.; Li, L. J. Am. Soc. Mass Spectrom. 1998, 9, 1303-1310. (10) Cohen, S. L.; Chait, B. T. Anal. Chem. 1996, 68, 31-37. (11) Figueroa, I. D.; Torres, O.; Russell, D. H. Anal. Chem. 1998, 70, 45274533. (12) Green-Church, K. B.; Limbach, P. A. Anal. Chem. 1998, 70, 5322-5325.

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biomolecules in a single common solvent, mixed-solvent systems are often used. Efforts to overcome these sample segregation problems have generally focused on the method of sample deposition. The segregation effects have been demonstrated using the dried droplet method, which is caused by the slow evaporation of the solvent.4,10,15 Since crystallization is a separation technique, the slower the solvent evaporates, the purer the crystals. Efforts to overcome segregation and discrimination effects in the dry-drop method for biomolecules have focused on the following: control of the pH of the matrix/analyte solutions,10 drying time,4,10,15 water content,11 and addition of detergents (surfactants).13,16,17 All of these approaches involve different methods of manipulating the solubility of the components of the sample preparation mixture during the drying process. Additional efforts have focused on accelerating the solvent evaporation process to minimize the amount of segregation that is allowed to occur, including the use of heat,14 vacuum deposition,18 and electrospray deposition.19-21 In addition to the solubility of the components of a MALDI sample in the solvent phase (solution solubility), there appears to be relationship between the polarity of the analyte and the polarity of the matrix in successful MALDI samples. It has been widely suggested that a successful MALDI sample preparation is characterized by the analyte being homogeneously embedded within the matrix, such that the analyte molecules are well separated from one another. We will describe this desirable sample preparation as a “solid solution”, where the matrix is analogous to a solvent, and the analyte molecules are “solvated” by the matrix molecules in the solid state. It has been suggested by Linton and co-workers22 that solubility plays a role in the miscibility of a polymer analyte and matrix when solvent is removed. For example, they suggested that the phenyl functionality allows PS to be miscible with the highly aromatic dithranol matrix but not with the more polar 2,5-dihydroxybenzoic acid matrix. It has been observed by Hanton and Owens, using ME-SIMS as a probe,21,23 that the solid solution is best achieved by matching the relative polarity of the matrix and analyte. Since ME-SIMS is a surfacesensitive technique, it was a good probe for investigating whether the analyte (polymer) was incorporated within the matrix or segregated (at the surface). In cases where there was a good match of polarity between matrix and analyte, no polymer peaks (13) Breaux, G. A.; Green-Church, K. B.; France, A.; Limbach, P. A. Anal. Chem. 2000, 72, 1169-1174. (14) Bird, G. H.; Lajmi, A. R.; Shin, J. A. Anal. Chem. 2002, 74, 219-225. (15) King, R. C.; Sepa, J.; Owens, K. G. Proc. 42nd ASMS Conf. Mass Spectrom. Allied Topics, 1994; p 978. (16) Amado, F. M. L.; Santana-Marques, M. G.; Ferrer-Coreia, A. J.; Tomer, K. B. Anal. Chem. 1997, 69, 1102-1106. (17) Bornsen, K. O.; Gass, M. A. S.; Bruin, G. J. M.; von Adrichem, J. H. M.; Biro, M. C.; Kresbach, G. M.; Ehrat, M. Rapid Commun. Mass Spectrom. 1997, 11, 603-609. (18) Preisler, J.; Foret, F.; Karger, B. L. Anal. Chem. 1998, 70, 5278-5287. (19) Axelson, J.; Hoberg, A.; Waterson, C.; Myatt, P.; Shield, G. L.; Varney, J.; M., H. D.; Derrick, P. J. Rapid Commun. Mass Spectrom. 1997, 11, 209213. (20) Hensel, R. R.; King, R. C.; Owens, K. G. Rapid Commun. Mass Spectrom. 1997, 11, 1785-1793. (21) Hanton, S. D.; Cornelio Clarl, P. A.; Owens, K. G. J. Am. Soc. Mass Spectrom. 1999, 10, 104-111. (22) Belu, A. M.; DeSimone, J. M.; Linton, R. W.; Lange, G. W.; Friiedman, R. M. J. Am. Soc. Mass Spectrom. 1996, 7, 11-24. (23) Hanton, S. D.; Owens, K. G. Proc. 46th ASMS Conf. Mass Spectrom. Allied Topics, 1998; p 1185.

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were observed in the ME-SIMS spectrum, while peaks were observed in the MALDI spectrum collected from the identical location. In cases where the analyte and matrix polarity was mismatched, analyte peaks were observed in the ME-SIMS spectrum, and the MALDI spectrum was generally poor. The description of an analyte as being homogeneously embedded within the matrix (i.e., a solid solution) in MALDI has a great deal of similarity to the characteristics of polymer miscibility in a successful polymer blend. Both Hildebrand and Hansen solubility parameters have been used extensively to help guide polymer blend research. Coleman and co-workers24 demonstrated that nonhydrogen-bonded solubility parameters and strength of potential intermolecular interactions can be used as a guide to polymer miscibility. It was pointed out that the closer the match of the values of solubility parameter, the greater the probability of miscibility of two polymers. In a similar manner, we believe that matching solubility characteristics of the matrix and analyte (e.g., polymer) will result in better homogeneity of the solid solution. In this paper, we will discuss molecules that are referred to as polar as having strong intermolecular interactions (strong dipoledipole or H-bonding) and those that are nonpolar as having weak dipole-dipole or dispersion intermolecular forces. Recent work in our laboratory has focused on developing tools to objectively choose sample preparation conditions for a particular analyte. In an effort to extend the understanding of the role of solubility and polarity in MALDI sample preparation, we have used high-performance liquid chromatography (HPLC) to establish a polarity scale for matrixes, as well as several reference analytes. Reversed-phase HPLC techniques achieve chromatographic retention, based on partitioning processes that have been demonstrated to be related to solubility characteristics.25-27 The relationship between the octanol/water partition coefficient (Log P) and HPLC capacity factor has been well established using isocratic HPLC conditions. Muller and co-workers28 used the isocratic HPLC method to measure the Log P of a range of compounds over a relatively narrow polarity range. The range of polarities for the different matrix materials is expected to be relatively broad. To measure the different matrixes using the same conditions, an isocratic HPLC method would be extremely time-consuming. Dean and co-workers26 reported on the use of gradient HPLC in order to keep the analysis time short. The work reported here uses the HPLC retention time to predict what matrix material(s) will form a homogeneous solid solution with a particular analyte of interest. Note that the solubility/polarity properties are not the only contributors to the selection of sample preparation conditions. For instance, the proton affinity or relative cation affinities29 will also play a role in the ionization of a particular analyte. It is the intent of this work to provide a piece of the puzzle of polymer sample preparation, to make the selection of MALDI sample preparation conditions a more predictable process. (24) Coleman, M. M.; Serman, C. J.; Bhagwager, D. E.; Painter, P. C. Polymer 1990, 31, 1187-1203. (25) Mirrlees, M. S.; Moulton, S. J.; Murphy, C. T.; Taylor, P. J. J. Med. Chem. 1976, 19, 615-619. (26) Makovskaya, V.; Dean, J. R.; Tomlinson, W. R.; Hitchen, S. M.; Comber, M. Anal. Chem. Acta 1995, 315, 183-192. (27) Helweg, C.; Nielson, T.; Hansen, P. E. Chemosphere 1997, 34, 1673-1684. (28) Muller, K.; Gurster, D.; Piwek, S.; Wiegrebe, W. J. Med. Chem. 1993, 36, 4099-4107. (29) Knochenmuss, R. Anal. Chem. 2003, 75, 2199-2207.

EXPERIMENTAL SECTION Materials. The matrix materials nicotinic acid, 2,5-dihydroxy-p-benzoquinone (DHBQ), 2,5-dihydroxybenzoic acid (DHB), R-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), norharmane, 2′,4′,6′-trihydroxyacetophenone (THAP), trans-indoleacrylic acid (IAA), 2-(4-hydroxyphenylazo)benzoic acid (HABA), 2,6-dihydroxyacetophenone, 2-mercaptobenzothiazole (MBT), 5-chloro-2-mercaptobenzothiazole (CMBT), 1,8,9-anthracentetriol (dithranol), 2-[(2E)-3-(4-tert-butylphenyl)-2-methylpropenylidene]malanonitrile (DCTB), and all-trans-retinoic acid (RA) were purchased from Aldrich Chemical Co. (Milwaukee, WI). The concentration of residual alkali metals (Na and K) in the matrix materials was measured using either inductively coupled plasma atomic emission spectroscopy or flame atomic absorption spectroscopy. Matrixes used for MALDI measurements that contained more than 100 ppm of Na or K were cleaned prior to use by recrystallization. Narrow distribution poly(ethylene glycol) (PEG) 600, 2000, 4600, and 10 000 materials were purchased from Aldrich. Narrow-distribution PMMA 620, 2900, 6300, and 10 000 materials were purchased from Polymer Laboratories, Inc. (Amherst, MA). Narrow-distribution PS 510, 2500, 5050, and 13 000 materials were purchased from Polysciences, Inc. (Warrington, PA). The peptides leucine enkephalin, angiotensin II, and renin substrate were purchased from Sigma Chemical Co. (St. Louis, MO). All polymer and peptide samples were used as received, without further cleanup. pNitrophenol, p-chlorophenol, benzophenone, naphthalene, and anthracene were obtained from Eastman Kodak Co. (Rochester, NY). The deionized water (DI H2O) was prepared using a Milli-Q filtration system (Millipore, Bedford, MA). HPLC Sample Preparation. All matrix materials and polymer samples were prepared at a concentration of 0.5-1 mg/mL in HPLC grade unstabilized tetrahydrofuran (THF). The peptides were prepared at a concentration of 0.5-1 mg/mL in DI H2O. The molecules p-nitrophenol, p-chlorophenol, benzophenone, naphthalene, and anthracene were prepared at a concentration of 0.5-1 mg/mL in THF. MALDI Sample Preparation. Polymer samples for MALDI analysis were prepared at a concentration of 0.5-0.6 mM in THF. The matrix solutions were prepared at a concentration of 60-70 mM in THF. The cationization reagent, sodium trifluoroacetate (NaTFA) for PEG and PMMA, was prepared at 0.7 mM in THF. The cationization reagent silver trifluoroacetate (AgTFA) for PS, was prepared at 0.9 mM in THF. The samples were prepared by mixing the polymer solution with matrix solution and cationization reagent solution at a volume ratio of 1:9:1, resulting in an effective matrix/analyte mole ratio in the range of 1000:1-1200:1. A 0.5µL aliquot of the mixture was deposited on a sample plate and allowed to air-dry. HPLC Conditions. Reversed-phase gradient HPLC analyses were performed using an HP1090 liquid chromatograph (Agilent Technologies, Palo Alto, CA), equipped with a diode array detector (DAD) and a Sedex model 55 (Sedere, Alfortville Cedex, France) evaporative light scattering detector (ELSD). A YMC ODS-AQ (Waters Corp., Milford, MA) column (3.0 × 100 mm; S-5 120-Å particles) was used for all analyses. A binary gradient was used with the A phase being 0.1 M ammonium acetate buffer (pH 4.65) and the B phase being THF. The gradient conditions started at 10% B, ramped to 100% B in 10 min, and were held at 100% B for

1 min. For the DAD detector, the data were collected at 254 nm (full diode array data collected) with a bandwidth of 4 nm. The ELSD was run at a temperature of 42 °C, with a nitrogen purge pressure of 2.3 bar and a gain (PMT) of 12. The injection volume was 2 µL, and the flow rate was 1.0 mL/min. MALDI Time-of-Flight (TOF)MS Instrumentation. MALDITOFMS experiments were carried out using a TofSpec2E laser TOF mass spectrometer (Micromass, Inc., Manchester, U.K.), equipped with dual microchannel plate detectors for both linear and reflectron modes and a nitrogen laser operating at 337 nm. Positive ion mode was utilized for all analyses, with an accelerating voltage of 25 kV for linear mode and 20 kV for reflectron mode. Spectra were acquired using delayed extraction mode with a 500ns delay time. The delayed extraction pulse voltage was optimized for resolution, depending on the mass range of the individual polymer distributions. RESULTS AND DISCUSSION HPLC Analysis. The first step toward understanding the relationship between matrix and analyte with regard to solubility and polarity in the MALDI sample preparation process was to establish a relative order of polarity among a range of matrix materials. In a fashion similar to that of Dean and co-workers,26 reversed-phase gradient HPLC conditions were developed to allow for the analysis of a broad polarity range using a single HPLC method. In this method, a mixed-mode HPLC column was chosen (YMC ODS-AQ) because it contains both a hydrophobic high carbon loading and a hydrophilic surface.30 For the highly polar compounds, this type of packing gives longer retention times than traditional octadecylsilane (ODS) packings. To elute the highly nonpolar compounds in a reasonably short time, the gradient conditions were developed using THF as the B-phase. Table 1 lists 15 MALDI matrix materials, along with their respective retention times (RTs), using these gradient HPLC conditions. To examine how the solubility and polarity properties of the analytes compare with the matrixes investigated, a few example peptides and synthetic polymers were analyzed using the same HPLC conditions. It was important to use the ELSD HPLC detector to make these comparisons because some of the analytes have little or no absorption in the UV/visible range of the diode array detector. Figure 1 displays the HPLC chromatograms of three peptides in the mass range of 500-1500 Da, leucine enkephalin, angiotensin II, and renin substrate. Under these HPLC conditions, the peptide RTs fall in the range of 3.8-5.3 min. It is interesting to note that the matrixes that are commonly used for these peptides, namely, CHCA and SA, have RTs of 3.11 and 4.12 min, respectively (Table 1), which match closely with the RTs of the peptides. Although hydrophobic peptides were not studied here, Green-Church and Limbach12 found that hydrophobic peptides were soluble in solvents such as chloroform and that matrixes such as IAA and dithranol were found to provide strong MALDI signals. Hydrophobic peptides would be expected to elute later than those studied here and would be more closely matched with the later eluting IAA and dithranol matrixes. A set of three different synthetic polymers, which cover a broad range of polarities, was used to compare with the polarities of the matrixes. Initially, the low molecular weight analytes PEG 600, (30) http://www.ymc.co.jp/.

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Table 1. HPLC Retention Times for the Matrix Materials Employed in This Study matrix trivial name

monoisotopic mass (Da)

HPlC RT (ELSD) (min)

nicotinic acid DHBQ DHB CHCA nor-harmane sinapinic acid ferulic acid IAA HABA THAP MBT CMBT dithranol DCTB

123.032 140.011 154.027 189.043 168.069 224.068 194.184 187.063 242.069 168.042 166.986 200.947 226.063 250.147

0.73 0.88 1.64 3.11 3.39 4.12 4.85 6.14 6.27 6.38 6.81 7.45 7.66 8.71

RA

300.209

8.73

Figure 1. Reversed-phase gradient HPLC chromatograms of three polar peptides: (a) angiotensin II, (b) leucine enkephalin, and (c) renin substrate.

PMMA 620, and PS 510 were analyzed using the gradient HPLC method. The HPLC chromatograms of these reference standards are displayed in Figure 2. As expected from their polarities, the PEG 600 has a short RT, and the PS 510 has a long RT. Note that, for PMMA and PS, the multiple peaks in the chromatograms correspond to partial separation of the individual oligomers. To investigate the effect of increasing molecular weight on polarity, additional polymer reference standards were analyzed for PEG, PMMA, and PS over the MW range of approximately 100-10 000. Figure 3 displays the plots of RT versus molecular weight for the three polymer systems. Table 2 lists the RTs for all of the polymers used in this study. Note that when individual oligomers are resolved, their individual RTs are listed in the table. There are two interesting features to note from Figure 3 and Table 2: (1) as expected, the polarity order of these polymers from most to 5160 Analytical Chemistry, Vol. 76, No. 17, September 1, 2004

compound name pyridine 3-carboxylic acid 2,5-dihydroxy-p-benzoquinone 2,5-dihydroxybenzoic acid R-cyano-4-hydroxycinnamic acid 9H-pyrido[3,4-b]indole 3,5-dimethoxy-4-hydroxycinnamic acid 3-methoxy-4-hydroxycinnamic acid trans-indoleacrylic acid 2-(4-hydroxyphenylazo)benzoic acid 2′,4′,6′-trihydroxyacetophenone 2-mercaptobenzothiazole 5-chloro-2-mercaptobenzothiazole 1,8,9-anthracentetriol 2-[(2E)-3-(4-tert-butylphenyl)-2 methylpropenylidene] malanonitrile all-trans-retinoic acid

Figure 2. Reversed-phase gradient HPLC chromatograms of three low-MW polymers covering a broad polarity range: (a) PEG 600, (b) PMMA 600, and (c) PS 510.

least polar is PEG > PMMA > PS, and (2) within a polymer class, the polarity decreases with increasing molecular weight to a point and levels off. Both of these features suggest that the separation is based on functionality. The dramatic increase in RT with MW, which eventually levels off, suggests a substantial influence of the end groups. At very low MW, the end groups constitute a significant percentage of the polymer. With increasing chain length, the polymer becomes more nonpolar to a point where further increase in chain length no longer affects the polarity. This plot also demonstrates that there is a large difference in the polarity of these three classes of polymer. MALDI TOFMS. With the three sets of polymers and the range of matrixes analyzed under the same HPLC conditions, it

Figure 3. Reversed-phase gradient HPLC retention time plotted versus MW for three different polymers: (a) PEG, (b) PMMA, and (c) PS. Table 2. HPLC Retention Times for the Polymer Materials Employed in This Study PS

PMMA

PEG

MW

RT (min)

MW

RT (min)

MW

RT (min)

266 370 474 578 682 2500 5050 13000

9.57 9.77 9.92 10.05 10.16 11.01 11.29 11.32

202 302 402 502 602 702 2900 6300 10000

7.19 7.41 7.60 7.78 7.94 8.07 9.26 9.68 9.89

600 2000 4600 10000

0.87 3.37 3.47 3.63

is possible to investigate the relationship between HPLC RT and successful a MALDI sample preparation. Based on the observations in the ME-SIMS work of Hanton and Owens,21,23 it is expected that a polymer will form a good “solid solution” with a matrix that has an RT that closely matches the retention time of that polymer. Further, a sample preparation that produces a good solid solution is expected to produce a MALDI spectrum of the polymer with good S/N. To test this hypothesis of matching polarities of matrix and polymer, we investigated polymers with matrixes over a range of polarities. The MALDI TOF mass spectra of PS 7000 obtained using the matrixes RA, DCTB, IAA, CHCA, DHB, and DHBQ are displayed in Figure 4. In all cases, AgTFA was used as the cationization agent. Because each of the matrixes has a different molar absorptivity at the wavelength used, the laser intensity was adjusted to achieve the maximum S/N for each matrix. As expected, based on the majority of the literature work with PS, the matrix RA produces a spectrum with good S/N. The matrix DCTB also produces a spectrum with good S/N. Note that the S/N values given on the spectra indicate the ratio of signal to background (height) determined for the most intense peak observed in the polymer distribution. The HPLC retention time for PS 7000 is 11.3 min, and the retention times of RA and DCTB are 8.73 and 8.71 min, respectively. The data in Figure 4 show that, as the RT of the matrixes get further away from that of PS 7000 (i.e., going from nonpolar to polar), the S/N of the polymer peaks in the MALDI spectra decreases.

As shown in Figure 3, the polymer PMMA 6300 has an HPLC RT of 9.68 min. The MALDI TOF mass spectra of this polymer, using the same set of matrixes as with PS, are displayed in Figure 5. Note that NaTFA was used as the cationization reagent for this analyte. Figure 5 shows that the spectrum with the highest S/N uses IAA as the matrix. Although reasonable spectra are achieved with most of the matrixes, as noted for PS above, it appears that the spectra with the best S/N are achieved with the matrixes that have a close match of HPLC RT to that of the polymer. Note that a poor spectrum is achieved using DHBQ, where the HPLC RTs are still quite far apart. PEG was used to examine a polymer that is on the polar end of the HPLC RT scale. The MALDI TOF mass spectra of PEG 10 000, with the same set of matrixes and using NaTFA as the cationization reagent, are displayed in Figure 6. Note that the matrixes that produced poor S/N spectra for PS and PMMA produce strong MALDI spectra for PEG. An unexpected result was that reasonable MALDI spectra are produced for all of the matrixes examined. It was expected that the more polar matrixes would be the best match for PEG 10 000 and that the spectra obtained with the more nonpolar matrixes would be of poor quality. Two, possibly inter-related, explanations are proposed below to rationalize the results observed for the PEG polymer in context with the other polymers studied. First, the molecular weight range of the polymers that were examined by MALDI fell within the 6000-10 000 range. This mass range was chosen because it was expected to be low enough to effectively study the HPLC RT correlation, yet high enough to study true MALDI effects. One possibility that could complicate the interpretation of the MALDI results is direct laser desorption. For low molecular weight polymers, it is possible to observe signal when a sample prepared without a matrix is illuminated.31,32 It was expected that the mass range that was chosen would be high enough that laser desorption would not be a possibility. For a polymer that can directly laser desorb and ionize, it would be possible to obtain a spectrum, even if there were severe segregation in the sample preparation. A laser desorption/ionization (LDI) spectrum of PEG 10 000 (i.e., without matrix), with NaTFA added for cationization, was obtained. The spectrum contained low S/N peaks, which were consistent with molecular ions for the polymer distribution, demonstrating that it is possible to ionize PEG without a matrix even in this higher molecular weight range. However, the spectrum was dominated by extensive fragmentation. Spectra obtained from LDI analysis of the PS 7000 and PMMA 6300 polymers, with cationization reagent added, did not contain analyte peaks (vide infra). Second, the observation that reasonable MALDI spectra can be obtained from PEG, seemingly independent of matrix, could be explained with reference to the polymer blend work discussed by Coleman.24 For polymers that have very weak or nonexistent favorable intermolecular interactions (e.g., only dispersive interactions), the probability of obtaining a miscible blend between two polymers is very low. To achieve this, an almost perfect match of solubility parameters was necessary. Conversely, polymers that exhibit relatively strong favorable intermolecular interactions (e.g., strong dipole-dipole and H-bonding), the probability of obtaining (31) Cotter, R. J.; Honovich, J. P.; Oltoff, J. K.; Lattimer, R. P. Macromolecules 1986, 19, 2996-3001. (32) Hoteling, A. J. Ph.D. Thesis, Drexel University, Philadelphia, PA, 2004.

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Figure 4. MALDI TOFMS spectra of PS 7000 using AgTFA as the cationization reagent with six matrixes that cover a broad polarity range: (a) RA, (b) DCTB, (c) IAA, (d) CHCA, (e) DHB, and (f) DHBQ. The spectra are displayed at the same y-axis scale.

Figure 5. MALDI TOFMS spectra of PMMA 6300 using NaTFA as the cationization reagent with six matrixes that cover a broad polarity range: (a) RA, (b) DCTB, (c) IAA, (d) CHCA, (e) DHB, and (f) DHBQ. The spectra are displayed at the same y-axis scale.

a miscible blend between two polymers dramatically increases. In this case, Coleman found that the match of solubility parameters was still important; however, a broader range was tolerable. In 5162 Analytical Chemistry, Vol. 76, No. 17, September 1, 2004

the case of MALDI, PS is a very nonpolar polymer, which would have relatively weak intermolecular interactions with the other component (i.e., the matrix). In this construct, it seems reasonable

Figure 6. MALDI TOFMS spectra of PEG 10 000 using NaTFA as the cationization reagent with six matrixes that cover a broad polarity range: (a) RA, (b) DCTB, (c) IAA, (d) CHCA, (e) DHB, and (f) DHBQ. The spectra are displayed at the same y-axis scale.

that there would be a narrow range of polarity of matrixes that would form a solid solution with PS. PEG, however, is a very polar polymer, which would have relatively strong intermolecular interactions with the matrix (e.g., H-bonding with the carboxylic acid group). It seems reasonable to expect that a broader matrix polarity range would form a solid solution with PEG. For example, even though retinoic acid is a very nonpolar matrix, it contains a carboxylic acid group, which could interact favorably with the highly oxygenated PEG polymer chains. Therefore, a wider range of matrixes can be used for successful MALDI analysis of the more polar polymer analytes. Log P Relationship. Matching the polarities of matrix and analyte is a good first step toward predicting useful MALDI sample preparation conditions. For real world samples that need to be analyzed by MALDI, it would be useful to obtain the HPLC RT in order to help guide the matrix selection process. However, because of constraints in time and sample availability, it may not be practical to screen all samples by HPLC prior to MALDI analysis. Moving toward a prediction-based approach would be more desirable. In an effort to make the estimation of relative polarities a simpler process, we have considered the use of Log P prediction. A set of compounds with previously reported Log P values28 were analyzed to examine the relationship between Log P and RT using the HPLC method conditions employed to measure the matrixes and analytes in this work. These compounds were 4-nitrophenol, 4-chlorophenol, benzophenone, naphthalene, and anthracene and their respective Log P values were 1.91, 2.39, 3.18, 3.44, and 4.49. A plot of the gradient HPLC RTs versus Log P for these compounds is displayed in Figure 7. Note that the relationship between RT and Log P is linear with an R2 value of 0.9931. The linear relationship suggests that the gradient HPLC method conditions used here are reasonable for establishing the relative polarities for the test compounds. Work is currently underway in

Figure 7. Reversed-phase gradient HPLC retention time plotted versus literature Log P values for five reference materials (labeled on the plot). Literature values obtained from ref 28.

our laboratory investigating the limitations of various Log P prediction methodologies for its extension to MALDI sample preparation. CONCLUSION The relationship between solubility and MALDI sample preparation was investigated using a reversed-phase gradient HPLC method. The method was used to establish a relative polarity scale for 15 MALDI matrix materials that cover a broad polarity range. Three peptides ranging in MW from 500 to 1500 were analyzed under the same HPLC conditions to assess their RTs relative to the matrixes. It was found that the RT of these relatively polar peptides matched closely with the matrixes that are most commonly used in MALDI to analyze them. A series of polymer reference standards, which covered a range of polarity and a range of molecular weight for each polymer type, were assessed by the HPLC method. It was found that, as Analytical Chemistry, Vol. 76, No. 17, September 1, 2004

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molecular weight increases for a particular polymer, the RT increases and levels off. MALDI TOFMS analysis of this range of polymers with a broad range of matrixes was also performed. The MALDI analysis showed that the best MALDI spectra, with respect to S/N ratio, were obtained when the matrix and polymer had RTs that were closely matched. For the polymer systems examined here, matching the RTs of the matrix and analyte consistently produced the best MALDI spectra with regard to S/N. Consistent with previously published results from ME-SIMS, the sample preparation conditions that had a large difference in HPLC RT resulted in the poorest MALDI signal. It is suspected that sample segregation occurs when there is a mismatch of HPLC RT, which further results in poor MALDI spectra. The results presented here suggest that a good starting point for choosing the appropriate matrix for an “unknown” sample would be to match its relative polarity with that of the matrix. The HPLC method conditions reported here have also been shown to have a good relationship to measured Log P values. For

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a limited RT range, the known Log P values from a series of reference materials were demonstrated to have a linear relationship with the measured HPLC RT. This suggests that, with further work, it may be possible to establish a modeling process for predicting the correct matrix or range of matrixes that would work best for a particular “unknown” analyte. ACKNOWLEDGMENT We thank Mr. William Nichols (Eastman Kodak Co.) and Dr. Scott Hanton (Air Products and Chemicals, Inc.) for helpful discussions. We thank Mr. James Hauenstein (Eastman Kodak Co.) for assistance with the ELSD detector. A.J.H. gratefully acknowledges financial support from Eastman Kodak Co.

Received for review March 20, 2004. Accepted June 22, 2004. AC049566M