Micellar Monolithic Liquid Chromatography for High-Throughput

Department of Pharmaceutical and Biomedical Analysis, Pharmaceutical Institute, Vrije Universiteit Brussel-VUB,. Laarbeeklaan 103, B-1090 Brussel, Bel...
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Anal. Chem. 2004, 76, 1903-1908

Evaluating “Fast” Micellar Monolithic Liquid Chromatography for High-Throughput Quantitative Structure-Retention Relationship Screening Ann Detroyer, Yvan Vander Heyden,* Katrien Reynaert, and Desire L. Massart

Department of Pharmaceutical and Biomedical Analysis, Pharmaceutical Institute, Vrije Universiteit Brussel-VUB, Laarbeeklaan 103, B-1090 Brussel, Belgium

The recently introduced monolithic silica columns were tested for their use in micellar liquid chromatography. Micellar methods are utilized in high-throughput quantitative structure-retention relationships to estimate an indicator of the membrane permeability of drugs, namely, the octanol-water partition coefficient, log P. The monolithic column’s ability to function at higher flow rates might be useful to speed up these chromatographic methods estimating the log P. Therefore, the elution behavior of diverse basic pharmaceutical substances was determined on a classical particle-based and a monolithic column, both with and without a micellar medium in the mobile phase. Utilizing among others principal component analysis, the extent to which these methods differ in retention characteristics was examined in the context of highthroughput determination of log P. Results indicate that combining monolithic columns with micellar media leads to faster log P and possibly even better permeability predictions. One of chromatography’s recent research developments is the monolithic column. Although trial preparations started in the late 1960s, the introduction of the so-called continuous beds by Hjerte´n et al.1 in 1989 and of the continuous macroporous polymer rods by Svec2 in 1992 was the onset for many papers dedicated to the monolith’s preparation, characterization, and application in diverse domains.3-5 Part of the enormous interest stems from the monolith’s ability to speed up chromatographic analysis. Due to its larger porosity, higher flow rates can be applied compared to particle-based columns while retention characteristics should be maintained and better efficiencies and performances are seen.3,6 In the pharmaceutical industry, any development to speed up methods is encouraged as it leads to larger throughput and lower costs. Here monoliths might be applied for the early and rapid estimation of the partitioning over a biological membrane and thus * To whom correspondence should be addressed. E-mail: [email protected]. Phone: (+32) 2 477 47 23. Fax: (+32) 2 477 47 35. (1) Hjerten, S.; Liao, J. L.; Zang, R. J. Chromatogr. 1989, 473, 273-275. (2) Svec, F.; Frechet, J. M. Anal. Chem. 1992, 64, 820-822. (3) Tanaka, N.; Kobayashi, H.; Nakanishi, K.; Minakuchi, H.; Ishizuka, N. Anal. Chem. 2001, 421A-429A. (4) Miyabe, K.; Guiochon, G. J. Phys. Chem. B 2002, 106, 8898-8909. (5) Que, A. H.; Novotny, M. V. Anal. Chem. 2002, 74, 5184-5191. (6) Ishizuka, N.; Kobayashi, H.; Minakushi, H.; Nakanishi, K.; Hirao, K.; Hosoya, K.; Ikegami, T.; Tanaka, N. J. Chromatogr., A 2002, 960, 85-96. 10.1021/ac030339e CCC: $27.50 Published on Web 02/24/2004

© 2004 American Chemical Society

the potential bioactivity of drug candidates. Because the partitioning of drugs is often attributed to their hydrophobic character, frequently the logarithms of the retention factors (log k) from classical RP-HPLC are correlated with hydrophobicity, represented by the octanol-water partition coefficient, log P.7,8 Utilizing monolithic instead of classic particle columns for quantitative structure-retention relationship (QSRR) delineation might result in faster screening methods. However, according to Dorsey and Khaledi,9 biological partitioning is generally entropy driven as opposed to the bulk-phase hydrocarbon-water partitioning (e.g., in classical RP-HPLC), which is, overall, enthalpy driven. Techniques with a micellar medium are proposed as better chromatographic screeners of biomembrane permeability.10 Consequently, combining monolithic columns with micellar media might result in faster and better permeability-predicting methods. For this paper, “fast” micellar liquid chromatography (MLC) methods for QSRR have been developed utilizing monolithic columns and they are evaluated against existing chromatographic methods. Therefore, the elution behavior of a set of diverse basic pharmaceutical substances was determined, on a particle and a monolithic column, both with and without a micellar medium in the mobile phase. When possible, analysis was accelerated by increasing the flow rate. Because MLC on monolithic columns is a new field, characteristics of these methods were investigated. By applying chemometric methods, the particle versus monolithic and the micellar versus aqueous-organic modifier methods are examined, and the influence of the acceleration of these methods on their QSRR with log P is evaluated. EXPERIMENTAL SECTION Reagents. Acebutolol hydrochloride, alprenolol hydrochloride, atenolol, carbamazepine, clonidine hydrochloride, desipramine hydrochloride, diphenhydramine hydrochloride, imipramine hydrochloride, ketotifen fumarate, metoprolol tartrate, nadolol, oxprenolol hydrochloride, pindolol, promazine hydrochloride, propranolol hydrochloride, ranitidine hydrochloride, timolol maleate, trifluoperazine hydrochloride, and sodium dodecyl sulfate (SDS, 99% purity) were from Sigma (St. Louis, MO, or Steinheim, (7) Biagi, G. L.; Recanatini, M.; Barbaro, A. M.; Borea, P. A. Process Control Qual. 1997, 10, 129-149. (8) Valko, K. Trends Anal. Chem. 1987, 6, 214-219. (9) Dorsey, J. G.; Khaledi, M. G. J. Chromatogr., A 1993, 656, 485-499. (10) Woodrow, B. N.; Dorsey, J. G. Environ. Sci. Technol. 1997, 31, 28122820.

Analytical Chemistry, Vol. 76, No. 7, April 1, 2004 1903

Table 1. Overview of the Methods, Their Abbreviations, and Their Grouping method name

stationary phase(s)

mobile phase(s)

flow rate (mL/min)

method group abbreviation

A1-9 A10 B1-9 C1-8 D1 D2

monolithic (M) monolithic (M) monolithic (M) particle-based (P) particle-based (P) particle-based (P)

aqueous-organic (a/o) aqueous-organic (a/o) micellar (mic) aqueous-organic (a/o) micellar (mic) micellar (mic)

1-9 1 1-9 1 1 1

Ma/o-LC Ma/o-LC Mmic-LC Pa/o-LC Pmic-LC Pmic-LC

Germany). Bisoprolol fumarate, sotalol, thioridazine, sodium dihydrogen phosphate, 1-propanol, NaOH, acetic acid, phosphoric acid, boric acid, and ethanol 96% were from Merck (Darmstadt, Germany). Chlorpromazine hydrochloride came from Fluka Chemie (Buchs, Switzerland) and carteolol hydrochloride from Madaus (Ko¨ln, Germany). Cimetidine was a gift from Smith-Kline Beecham (Herts, U.K.) and esmolol hydrochloride from Du PontDe Nemours (Le Grand Saconnex, Switzerland). The acetonitrile (ACN) and methanol (MeOH) for HPLC came from BDH laboratory supplies (Poole, U.K.). Chromatographic Studies. The chromatograph consisted of an isocratic L-6000 pump, an L-7400 UV detector, and a D-7500 integrator, all Merck-Hitachi (Tokyo, Japan). Monolithic Column Experiments. A Chromolith Performance RP-18e analytical column (100 × 4.6 mm i.d.) from Merck was used. The injection volume was 20 µL and the detection wavelength 254 nm. Monolithic aqueous-organic modifier RP-HPLC (Ma/o-LC) methods (methods A1-A10 in Table 1) were based on a protocol described by Nasal et al.11 The substances were dissolved and diluted in MeOH to 100 µg/mL. The flow rate increased with 1 mL/min from method A1 (1 mL/min) to method A9 (9 mL/min). Method A10 was conducted at 1 mL/min. The mobile phase (MP) consisted of an ACN/Britton-Robinson buffer pH 7.4 (methods A1-9, 10/90% v/v; method A10, 60/40% v/v). This universal buffer consisted of a solution of 0.04 M acetic acid, 0.04 M phosphoric acid, and 0.04 M boric acid in MilliQ water. The buffer was adjusted to pH 7.4 with NaOH and filtered through a Schleicher and Schuell membrane filter (Dassel, Germany) with a pore size of 0.2 µm. The dead time (t0) was determined by the injection of MeOH. The monolithic micellar LC (Mmic-LC) measurements (methods B1-B9 in Table 1) were based on a protocol described earlier.12 Stock solutions containing 100 µg/mL of the substances were prepared in an ethanol/0.05 M SDS aqueous solution (10/90% v/v). The flow rate increased with 1 mL/min from method B1 (1 mL/min) to method B9 (9 mL/min). The MP consisted of SDS (0.1125 M)/1-propanol (10% v/v) in a 0.01 M sodium dihydrogen phosphate buffer. Before adding 1-propanol, the pH was adjusted to 7.4 with NaOH. The MP was filtered through Super R-450 membranes of 0.45-µm pore size and 47-mm diameter from Gelman Sciences (Ann Arbor, MI). The t0 was measured by injecting the 0.05 M SDS solution. Particle Column Experiments. The results of the particle aqueous-organic modifier RP-HPLC (Pa/o-LC) methods (methods (11) Nasal, A.; Bucin ˜ski, A.; Bober, L.; Kaliszan, R. Int. J. Pharm. 1997, 159, 43-55. (12) Detroyer, A.; Vander Heyden, Y.; Carda-Broch, S.; Garcı´a-Alvarez-Coque, M. C.; Massart, D. L. J. Chromatogr., A 2001, 912, 211-221.

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C1-C8 in Table 1) were extracted from the literature.11 These methods included a chiral R1-acid glycoprotein (AGP) column at pH 6.5 in an 2-propanol/phosphate buffer (C1), an immobilized artificial membrane IAM column at pH 7.0 in an ACN/phosphate buffer (C2), a Suplex pKb-100 column at pH 2.5 in an ACN/ Britton-Robinson buffer (C3), a Suplex pKb-100 column at pH 7.4 in an ACN/Britton-Robinson buffer (C4), a RP-Spheri column at pH 2.5 in an ACN/Britton-Robinson buffer (C5), a RP-Spheri column at pH 7.0 in an ACN/Britton-Robinson buffer (C6), an Aluspher RP-select B column at pH 7.3 in a MeOH/BrittonRobinson buffer (C7), and a Unisphere PBD column at pH 11.7 in a MeOH/Britton-Robinson buffer (C8), all conducted at 1 mL/ min. Except for the results on the AGP and IAM columns (methods C1 and C2), retention factors k were obtained by extrapolation of retention results measured at several MP compositions to the 100% aqueous buffer MP. In the original reference,11 these retention factors were calculated to obtain a hypothetical RP-HPLC system setting for which the retention of all substances could be compared. The particle micellar LC (Pmic-LC) measurements (methods D1 and D2 in Table 1) were conducted similar to those on the monolithic column. However, for method D1, a Discover C-8 column (5 µm, 50 × 4.6 mm i.d.) from Supelco (Bellefonte, PA), and for method D2, a Kromasil C-18 analytical column (5 µm, 120 × 4.6 mm i.d.) from Scharlau (Barcelona, Spain) were used. The flow rate was 1 mL/min, the injection volume 20 µL, and the detection wavelength 225 nm. The MP consisted of SDS (0.15 M)/1-propanol (15% v/v) in a 0.01 M sodium dihydrogen phosphate buffer (adjusted to pH 7.4). The MP was filtered through Nylon membranes from Schleicher & Schuell of 0.45-µm pore size and 47-mm diameter. The t0 was measured as the time at which a potassium iodide (4.2 µg/mL aqueous solution) peak appears. Calculations. In Table 2 the log P values were calculated from the molecular structure by applying the freely available on-line interactive LOGKOW program of the Environmental Science Center of Syracuse Research Corp. (Syracuse, NY) (http:// esc.syrres.com/interkow/kowdemo.htm). These data have been shown to be correlated to experimental log P values.13 The acidbase dissociation constants, pKa, were obtained from the ACD/ pKa database 4.06 (1999) of the Advanced Chemistry Development Corp. (Toronto, Canada) (http://www.acdlabs.com/products/ phys_chem_lab/pka/). The other calculations including the PCA algorithms were executed with the Matlab 4.2c.1 software from MathWorks (Natick, MA). RESULTS AND DISCUSSION “Fast” MLC versus Aqueous-Organic LC: Monolithic Column Performances. For aqueous-organic modifier RP(13) http://esc.syrres.com/interkow/logkow.htm,

Table 2. The Substances and Their Characteristics a

a

substances

log P

pKa

substances

log P

pKa

atenolol ranitidine sotalol cimetidine nadolol acebutolol carteolol pindolol metoprolol timolol oxprenolol bisoprolol clonidine

0.03 0.29 0.37 0.57 1.17 1.19 1.42 1.48 1.69 1.75 1.83 1.84 1.89

9.17 8.40 9.19 6.72 9.17 9.11 9.13 9.21 9.18 8.86 9.13 9.16 8.01

esmolol carbamazepine propranolol alprenolol diphenhydramine ketotifen promazine desipramine imipramine trifluoperazine chlorpromazine thioridazine

2.00 2.25 2.60 2.81 3.11 3.64 4.56 4.80 5.01 5.11 5.20 6.45

9.17 9.17 9.15 9.17 8.76 8.75 9.43 10.40 9.49 7.82 9.43 9.66

a

In aqueous medium.

HPLC methods on silica particle columns, it was shown that they can be easily transferred to silica monolithic columns.5,14 Moreover, by increasing the flow rate, the analysis times with these methods might be accelerated without noticeable efficiency problems.15 We wanted to investigate the monolith’s abilities when applying MLC and selected an existing method as starting point for that purpose. Applying Ma/o-LC and Mmic-LC methods on the same monolithic column and increasing the flow rate (methods A1-A9 and B1-B9 in Table 1) led to the following results. Using the micellar MP on the monolithic column (methods B1-B9) posed no practical problems. When the flow rate was increased with 1 mL/min, pressure increased ∼30 kg/cm2. Because of the pressure drop in monolithic columns,16 experiments could still be carried out when the mobile phase was pumped at 9 mL/min (method B9). However, since a pressure limit of 200 kg/cm2 is recommended for this type of column, using the given Mmic-LC method below or equal to 7 mL/min (method B7) is advisable. When the flow rate is increased, it is known for RP-HPLC methods on particle columns that 1/t0 is proportional to the flow rate.17 It seems this is also the case on monolithic columns when the flow is varied between 1 and 9 mL/min. The linear correlation between flow rate and 1/t0 is high, r > 0.999, both for the Ma/oLC methods A1-A9 and for the Mmic-LC methods B1-B9. The linear regression line does however have a steeper slope for the Mmic-LC (slope 0.822) than for the Ma/o-LC (slope 0.634). Consequently, for a given flow rate, slightly shorter t0’s and thus a reduction in average dead volume, V h 0, for micellar MP is seen (V h 0 ) 1.16 mL compared to 1.40 mL for Ma/o-LC). This might indicate a narrowing of the mesopores and occasionally the macropores of the monolithic column due to adsorbed SDS monomers on the surface. The phenomenon of V h 0 reduction is also observed on particle-based columns.18 Due to covering of the stationary phase with surfactant monomers the method’s efficiency, N, is expected to be worse. (14) van Nederkassel, A. M.; Aerts, A.; Dierick, A.; Massart, D. L.; Vander Heyden, Y. J. Pharm. Biomed. Anal. 2003, 32, 233-249. (15) McCalley, D. V. J. Chromatogr., A 2002, 965, 51-64. (16) Vervoort, N.; Gzil, P.; Baron, G. V.; Desmet, G. Anal. Chem. 2003, 75, 843850. (17) Poole, C. F. The essence of chromatography; Elsevier Science: Amsterdam, 2003. (18) Borgerding, M. F.; Hinze, W. L.; Stafford, L. D.; Fulp, G. W.; Hamlin, W. C. Anal. Chem. 1989, 6, 1353-1358.

Table 3. Efficiency (N) Evolution for Timolol as a Function of Flow Rate flow rate (mL/min)

N with Ma/o-LC methods A1-9

N with Mmic-LC methods B1-9

1 2 3 4 5 6 7 8 9

720 638 471 480 387 355 436 353 418

2498 1418 2537 1459 936 724 554 432 346

Table 4. Various Substances’ Correlation for 1/tr versus Flow Rate as a Function of Flow Rate. Average k h and Standard Deviation s for the Measurements over the Nine Flow Rates (n ) 9) r

kh (s)

atenolol acebutolol esmolol timolol

Ma/o-LC Methods A1-9 0.994 0.975 0.978 0.975

0.76 (0.09) 17.78 (2.13) 38.62 (4.12) 17.63 (2.08)

atenolol acebutolol esmolol timolol

Mmic-LC Methods B1-9 0.999 0.998 0.999 0.999

1.31 (0.04) 2.55 (0.07) 4.41 (0.10) 4.74 (0.12)

From Table 3 it seems for both the Ma/o-LC and Mmic-LC methods N decreases with increasing flow rate. This is consistent with the Knox plot findings for micellar and aqueous-organic modifier methods on particle columns by Berthod and Garcia-AlvarezCoque.19 However, contrary to the same findings, at 1 mL/min, N is considerably higher for Mmic-LC and it is only at high flow rates that N values become comparable with Ma/o-LC. Possible explanations related to the way of calculating N, the high mass transfer of monoliths, the high aqueous content in the MP of the Ma/o-LC method, and differences in retention windows should be considered. Since for the QSRR modeling only retention times and not peak shapes are of importance, the above observation was not further taken into account. As the retention factor k is used in the QSRR with log P, the retention behavior of some substances as a function of the flow rate was studied (Table 4). The substances’ inverse retention times (1/tr) are found to be linearly proportional to the flow rate, having an r above 0.975 for the Ma/o-LC methods A1-A9 and 0.998 for the Mmic-LC methods B1-B9. The different k for a given substance measured at different flow rates fluctuate randomly around a mean kh (n ) 9); i.e., no flow effect on k is observed. For the Ma/o-LC, ∼10% relative standard deviation around kh is seen, while for MmicLC, the fluctuations are restrained to 2%. Thus, speeding up MmicLC apparently does not change the nature of the interaction mechanisms based on a more complex three-phase model.20,21 “Fast” MLC and Analysis Time Comparisons. Overall MLC methods have the advantage of being able to determine substances (19) Berthod, A.; Garcı´a-Alvarez-Coque, M. C. Micellar Liquid Chromatography; Marcel Dekker: New York, 2000. (20) Armstrong, D. W.; Nome, F. Anal. Chem. 1981, 53, 1662-1666. (21) Armstong, D. W.; Stine, G. Y. Anal. Chem. 1983, 55, 2317-2320.

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Table 5. Absolute Analysis Times (tr) and Retention Factors (k) of the Different Methods for the Least and Most Retained Substances least retained substance (atenolol)

most retained substance (trifluoperazine)

method

tr (min)

k

tr (min)

k

retention window (min)

A10 C1-8a B1 B9 D1 D2 B7

1.63

0.22

97.34

72.11

95.71

2.60 0.32 1.06 2.52 0.40

1.27 1.38 0.79 1.89 1.35

25.66 3.21 5.75 28.33 3.98

21.39 23.12 8.67 31.69 22.39

23.06 2.89 4.69 25.81 3.58

a Results not taken into account because unavailable or mainly obtained through extrapolations.

from a large log P range using only one mobile phase12 (e.g., PmicLC methods D1 and D2). With aqueous-organic modifier RPHPLC there is, in general, a need to use several mobile phases, resulting, compared to micellar media, in a more complicated and much longer analysis, involving inter- or extrapolations (e.g., Pa/oLC methods C3-C8).22,23 For the Ma/o-LC methods, only rarely can one MP be found that determines all 25 examined substances (method A10 in Table 1). In this case, the retention window was very large (between 1.6 and 97 min for the least and most retained substances, respectivelysmethod A10 in Table 5) while the peaks of the least retained substances were not completely separated from the solvent peak. Utilizing flow rates up to 9 mL/min with this method did shorten the analysis times, but differentiation from the solvent peak of the least retained substances became almost impossible, making the calculation of the corresponding k values difficult. Moreover, the differences in retention times between the least retained substances became very small (close to zero) resulting in a loss of information. As the k for the least retained substance at 1 mL/min was only 0.22 with this method (A10 in Table 5), there is no room left for the fast analysis of other, even less retained, substances or for a differentiation between such substances. With MLC (Mmic-LC methods B1 and B9; Pmic-LC methods D1 and D2), these problems did not occur. All 25 substances could be clearly distinguished and were retained even at high flow rate. Moreover, compared to Ma/o-LC method A10 at 1 mL/min, much shorter retention times for the most retained substances and in general longer retention times for the least retaining substances were observed (Ma/o-LC method A10 versus Mmic-LC methods B1 and B9 and Pmic-LC methods D1 and D2 in Table 5). Thus, for MLC, a smaller retention window was found with a remarkably lower maximal analysis time both on a particle-based and on a monolithic column. This is caused by the extra hydrophobic and electrostatic interactions that are possible between the micelles in the mobile phase and the substance.20,21 From Table 5, it seems the Mmic-LC at 9 mL/min (method B9) gave the fastest analysis (the longest retained substances trifluoperazinestook only 3.2 min to elute). Taking into consid(22) Valko´, K. J. Liq. Chromatogr. 1984, 7, 1405-1424. (23) Valko´, K.; Bevan, C.; Reynolds, D. Anal. Chem. 1997, 69, 2022-2029.

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eration the above-mentioned pressure limits, the Mmic-LC method at 7 mL/min (method B7) was still faster (just under 4 min for trifluoperazine) than all the others. Pmic-LC method D1, an MLC method making use of a classical short column, is a good alternative. It is known that the use of particle-based short columns is a way to speed up analysis.24 However, k values obtained with this method were lower compared to the Mmic-LC methods B1, B9, and B7 in Table 5. So less possibility remains for the analysis of even less retained substances or for a differentiation between such substances. Moreover, due to the smaller particle size used to compensate for the loss in efficiency, pressure build-up is higher and thus limiting the applicable flow rates. The use of short monolithic columns might overcome this problem and push the “fast” MLC methods to an even higher analysis speed. Of course, these methods will have to generate a retention window that allows the retention of and the discrimination between the different substances over a broad log P range. “Fast” MLC and Method Comparison with Principal Component Analyisis (PCA). To situate “fast” MLC relative to the other methods, the influence on the retention of a change in stationary phase, MP, and flow rate are evaluated. For this purpose, Ma/o-LC method A10, Mmic-LC methods B1 and B9, Pa/oLC methods C1-C8, and Pmic-LC methods D1 and D2 in Table 1 were selected. The retention results with the different methods on the particle-based column were compared with the monolithic column methods using PCA. This chemometric technique was successfully applied before on retention results from several chromatographic systems, revealing information about the behavior of both substances and systems.11,25,26 Here the table of retention results can be considered as an n × m matrix, where n represents the objects (the 25 substances) and m the variables (log k from 13 of the methods in Table 1). With PCA, the number of original variables is reduced to a few latent ones called principal components (PCs), which still contain the main information from the original data set. The first new latent variable (PC1) is chosen in the direction of the largest variance in the data and thus contains the main information. The second PC is defined in such a manner that it is orthogonal to the first and it represents a maximum of variance not explained by PC1, etc. Mathematically each PC can be described as a linear combination of the original variables where the importance of each original variable is given by its so-called loading.27 From this equation, for each object, values, called scores, can be calculated on each PC. The PC loadings, which represent information about the methods, can be easily visualized and assessed. On the obtained loading plots usually the outlying or clustered loadings for methods are of interest. Here autoscaling27 was applied on the logarithm of the retention results. This transformation (or scaling) gives rise to (24) Nguyen Minh Nguyet, A.; Tallieu, L.; Plaizier-Vercammen, J.; Massart, D. L.; Vander Heyden, Y. J. Pharm. Biomed. Anal. 2003, 32, 1-19. (25) Bober, L.; Nasal, A.; Kuchta, A.; Kaliszan R. Acta Chromatogr. 1998, 8, 48-69. (26) Detroyer, A.; Schoonjans, V.; Questier, F.; Vander Heyden, Y.; Borosy, A. P.; Guo, Q.; Massart, D. L. J. Chromatogr., A 2000, 897, 23-36. (27) Vandeginste, B. G. M.; Massart, D. L.; Buydens, L. M. C.; De Jong, S.; Lewi, P. J.; Smeyers-Verbeke, J. Data handling in science and technology 20B: Handbook of chemometrics and qualimetrics: part B; Elsevier Science: Amsterdam, 1998.

Figure 1. PC1-PC3 loading plot. Numbers refer to the methods in Table 1.

variables (the methods) that are independent of the measurement units, which have equal range and therefore equal importance. When PCA was applied on the autoscaled log k of the 13 methods, it is seen on the PC1-PC3 loading plot (Figure 1) that the PC1 loadings of all methods are very similar and positive. This PC also represents, by definition, most of the variance (47.8%) in the measurements, i.e., retention factors. The fact that loadings are similar for the first PC indicates that the main retention characteristic with all methods is the same. The PC1 scores of the substances, calculated from these loadings, are directly related to their log P (r ) 0.96), meaning that the main characteristic represented by this PC appears to be hydrophobicity. For PC2, the MLC methods (methods B1, B9, D1, and D2) have a loading close to zero and thus have no influence. Mostly a differentiation between the aqueous-organic modifier methods was seen (results not shown). The PC3 loadings (Figure 1) explaining 10.3% of the total variance essentially represent the contrast between the micellar (methods B1, B9, D1, and D2) and the aqueous-organic modifier MPs (methods A10 and C1-C8). Possibly the different thermodynamic signature of micelle-water as opposed to aqueous-organic modifier partitioning is expressed on this PC.10 On all loading plots, the MLC methods on both monolithic and particle-based columns (methods B1, B9, D1, and D2) are found in each other’s vicinity. Thus, they should give highly correlated information and their retention characteristics should be very comparable. From Table 6, it seems the correlation is indeed high between the MLC methods, independent from their flow rate (methods B1 versus B9) and their stationary phase (methods B1 versus D1 versus D2). As from previous studies it seemed a small difference in MP does not affect retention characteristics.12 When PCA is applied solely to the autoscaled log k of MLC methods B1, B9, D1, and D2, it is seen in Figure 2 that PC1 represents almost all variation (93.6%) and the PC1 loadings are very similar and positive. Again the corresponding PC1 scores of the substances are closely related to their log P (r ) 0.89). Thus, the methods are very similar and their main retention characteristic is again hydrophobicity. Since the “true” hydrophobicity (log P) depends on the pKa value of the analyte as well as on the pH of the chromatographic system,28 PC1 actually represents a combination of the “intrinsic” hydrophobicity and the acid/base properties of the drugs. However, since all substances are basic, the acid/base properties cannot be very influential on PC1. (28) Buchwald, P.; Bodor, N. Curr. Med. Chem. 1998, 5, 353-380.

Because the variation between the MLC methods is very small and correlation is high, it can be concluded that the used stationary phase, flow rate, or MP has little influence on the retention characteristics. Thus, MLC methods can easily be transferred from particle to monolithic columns and speeded up to “fast” MLC methods without changes in retention characteristics. This transfer should be even better than the one with aqueous-organic modifier RP-HPLC methods from particle to monolithic columns since with MLC the monomers of the SDS cover the stationary phase and thus reduce possible differences in selectivity. “Fast” MLC and QSRR with log P. In view of selecting not only the fastest but also the best method to estimate log P, the different methods’ abilities to predict this property are tested. For this purpose, methods A10, B1, B9, C1-C8, D1, and D2 in Table 1 were selected. It should be remarked that in this study basic substances are investigated at pH’s where they are ionized (except method C8). Originally the log P of a substance is defined for its uncharged form. As the partition characteristics change for the ionized form a corrected parameter, log D, is calculated. Each substance’s log P is corrected proportional to the amount of ionized form present during the experiment at a given pH.28 For basic molecules when pH is near pKa

log D ) log P - log(1 + 10pKa-pH)

(1)

Contrary to what is expected, the correlations of the log k from the different methods with log D are somewhat less good than with log P (rd versus rp in Table 7).29 As the substances are uncharged, the correlation coefficients rd and rp for method C8 are the same. For the methods using aqueous-organic modifier MPs (methods A10 and C1-C8), the correlation with log D tends to get worse with decreasing pH. Possibly the pH of the systems is too far from the pKa of the substances to obtain proper estimates when applying eq 1. The lower rd for the MLC (methods B1, B9, D1, and D2) might be explained by the fact that micellar media can shift the pKa’s obtained in aqueous surroundings due to the interactions between the cationic solutes and anionic micelles. The acid/base equilibrium and thus log D of each of the basic substances is influenced depending on their interaction strength.30,31 For calculations not the aqueous but new protonation constants for MLC on the column should be determined. However, for these basic substances, the chromatographic column’s pH range is too small. Thus, we have opted to compare the different methods for their predictability of hydrophobicity, expressed as log P. The correlations between log k and log P rather than between log k and log D were considered. From Table 7, we can conclude that the rp between log k of aqueous-organic modifier MPs (methods A10 and C1-C8) and log P might be slightly higher than for the log k of MLC (methods B1, B9, D1, and D2). The variation in rp observed between the aqueous-organic modifier methods depends on the stationary phase and the pH used. Compared to MLC on particle columns (29) Detroyer, A.; Vander Heyden, Y.; Cambre, I.; Massart, D. L. J. Chromatogr., A 2003, 986, 169-337. (30) Khaledi, M. G.; Rogers, A. H. Anal. Chim. Acta 1990, 239, 121-128. (31) Rogers, A. H.; Khaledi, M. G. Anal. Chem. 1994, 66, 327-334.

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Table 6. Correlation Matrix between the log k’s

method

method A10 Ma/o-LC 1 mL/min

methods C1-8 Pa/o-LC 1 mL/min

method B1 Mmic-LC 1 mL/min

method B9 Mmic-LC 9 mL/min

method D1 Pmic-LC 1 mL/min

method D2 Pmic-LC 1 mL/min

A10 C1-8 B1 B9 D1 D2

1 0.63-0.96 0.89 0.89 0.87 0.87

1 0.70-0.92a 0.70-0.92a 0.69-0.92a 0.72-0.92a

1 1.000 0.996 0.997

1 0.996 0.997

1 0.997

1

a High correlations found with micellar methods due to method C (IAM particle phase). It contains structures similar to the ones in micellar 2 MPs.

tives. In view of these results, the “fast” MLC system can be proposed as a faster method to predict log P and possibly even membrane permeability. As log P might not be the most ideal parameter to mimic membrane permeability,9 our findings should be further correlated with other permeability-describing parameters, e.g., Caco-2 in vitro membrane passage permeability coefficients.33 Our MLC methods were selected to investigate basic substances, as the vast majority of drugs are basic. However, results might change for acids and MLC systems with cationic micelleforming agents could be required. This hypothesis might be a topic for further investigation. CONCLUSIONS Figure 2. PC1-PC2 loading plot for the micellar methods. Numbers refer to the methods in Table 1. Table 7. Statistical Tablea method

rd

rp

QCb

RMSPE

A10 C1-8 B1 B9 D1 D2

0.95 0.53-0.92 0.84 0.84 0.82 0.81

0.96 0.69-0.94 0.90 0.90 0.88 0.88

>100 19.43-53.47 45.91 26.11 43.39 23.42

0.65 0.26-0.91 0.21 0.22 0.18 0.29

a With r the correlation coefficient between log k and log D, r the d p correlation coefficient between log k and log P, QC the quality coefficient, and RMSPE the root-mean-squared prediction error of model: log k ) a log P + b for each method. b Calculated according to ref 32.

It is shown that micellar media can easily be applied on monolithic columns even at very high flow rates (9 mL/min). Their retention mechanisms, even at 9 mL/min, are not different from their application on classic particle-based columns. However, analysis is much faster. Thus, the use of monolithic columns might be proposed to speed up any existing MLC procedure. The QSRRs between log P and the proposed “fast” monolithic micellar systems appear to be comparable, sometimes even better, than with classic organic modifier/buffer HPLC methods. As molecules from a broader log P range can be analyzed with a micellar system, “fast” MLC might be proposed as a faster and potentially better membrane permeability predicting system. ACKNOWLEDGMENT

(methods D1 and D2), MLC on monolithic columns gives slightly higher rp (methods B1 and B9). However, when prediction models are being validated, the use of the quality coefficient (QC) and root-mean-squared error of prediction (RMSEP) is preferred. Both based on the deviation of the estimated response for the measured one, they give a better idea of the spread of the data points around the model, in this case log k ) a log P + b, and give an indication of the error to be expected for estimations.32 For the MLC methods, the QC and RMSEP are lowest. Overall the monolithic, micellar, 9 mL/min method (method B9) seems to give the best results. The results from the Mmic-LC at 7 mL/min (method B7) are closely correlated to those at 9 mL/min (r ) 0.999) and thus comparable QSRR are expected. However, the MLC methods on particle-based columns (methods D1 and D2) seem good alterna1908 Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

Research financed with a Specialisation Grant from the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT). Y.V. is a postdoctoral fellow of the Fund for Scientific Research (FWO-Vlaanderen).

Received for review September 23, 2003. Accepted January 5, 2004. AC030339E

(32) Massart, D. L.; Vandeginste, B. G. M.; Buydens, L. M. C.; De Jong, S.; Lewi, P. J.; Smeyers-Verbeke, J. Data handling in science and technology 20A: Handbook of chemometrics and qualimetrics: part A; Elsevier Science: Amsterdam, 1997. (33) Hidalgo, IJ. Curr. Top. Med. Chem. 2001, 1, 385-401.