Article pubs.acs.org/jced
Robustness of an Immobilized Artificial Membrane HighPerformance Liquid Chromatography Method for the Determination of Lipophilicity Moira R. Ledbetter,*,† Steve Gutsell,‡ Geoff Hodges,‡ Judith C. Madden,† Philip H. Rowe,† and Mark T. D. Cronin† †
School of Pharmacy and Chemistry, Liverpool John Moores University, Liverpool, L3 3AF, England Safety and Environmental Assurance Centre, Unilever Science Park, Sharnbrook, Bedfordshire, MK44 1LQ, England
‡
S Supporting Information *
ABSTRACT: Hydrophobicity is an intrinsic property that relates to a chemical’s tendency to partition between a polar and a nonpolar phase. Traditionally, hydrophobicity has been described by the logarithm of the octanol−water partition coefficient (log KOW or log P). Immobilized artificial membrane high-performance liquid chromatography (IAM-HPLC) is an alternative method to determine hydrophobicity that may be more biologically relevant. This paper examines the robustness of an IAM-HPLC assay optimized using the IAM.PC.DD2 column to determine the retention factor (log kIAM (pH 7.4)). The method has been shown to be robust following robustness testing of five compounds (log KOW ranging from 0.29 to 6.03) assessed across five columns (which included three batches of stationary phase) and two HPLC systems.
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INTRODUCTION
alternative for determining lipophilicity, an understanding of the robustness of the IAM-HPLC method is required. There are three IAM HPLC stationary phases available commercially. These are available in columns of various lengths (10 to 150 mm). All of these stationary phases have a phosphatidylcholine (PC) backbone bonded to aminopropyl silica; phosphatidylcholine being the major phospholipid in cell membranes. The IAM.PC.DD stationary phase has a single PC chain, and the residual aminopropyl groups are end-capped with C3 and C10 alkyl chains to improve column stability. The IAM.PC.MG and IAM.PC.DD2 stationary phases both contain double chains of PC. The IAM.PC.MG stationary phase is endcapped with methylglycolate, and the IAM.PC.DD2 stationary phase is end-capped with C3 and C10 acyl chains.10,11 This study has considered the optimization of an assay using columns that contain the IAM.PC.DD2 stationary phase (the IAM.PC.DD2 stationary phase is depicted schematically in Figure 1). The assay was optimized following investigations by this research group into the effect of experimental variability of log kIAM and the requirement for a standardized method.12 The use of the IAM.PC.DD2 stationary phase has a number of advantages over other commercially available IAM stationary phases including (1) there are a greater number of existing results obtained using this column in the literature; (2) it is available in a shorter
Hydrophobicity is an intrinsic chemical property that relates to a chemical’s ability to partition between a polar (aqueous) and a nonpolar phase. Traditionally, hydrophobicity has been described by the logarithm of the octanol−water partition coefficient (log P or log KOW).1,2 Log KOW has numerous applications, including being a common descriptor in quantitative structure−activity relationships (QSARs) to model and estimate biological activity of chemicals from their physicochemical properties.3−5 However, octanol−water partitioning models a single partitioning process. In contrast, within biological systems, partitioning occurs across multiple membranes and across multiple matrices. Thus, for experimental measurements of hydrophobicity to be used as a surrogate for membrane partitioning in modeling biological systems, more sophisticated techniques may be advantageous. Immobilized artificial membrane high-performance liquid chromatography (IAM-HPLC) is an alternative method to determine and estimate membrane partitioning that could be more biologically relevant than log KOW.6,7 IAM-HPLC attempts to simulate the hydrophobic and hydrophilic contributions involved in the partitioning across biological membrane(s). The IAM-HPLC column mimics both of these contributions through the compound’s interaction with the mobile and stationary phases. The IAM column combines hydrophobic, ion pairing, and hydrogen bonding interactions in the partitioning process.8,9 For IAM-HPLC to be a reliable © 2012 American Chemical Society
Received: August 16, 2012 Accepted: October 25, 2012 Published: November 5, 2012 3696
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Ledbetter et al.12 optimized an IAM-HPLC method to determine the logarithm of the IAM-HPLC retention index (log kIAM (pH 7.4)); the method was used to determine log kIAM (pH 7.4) for 66 compounds covering highly hydrophilic and highly hydrophobic compounds (log KOW = −1.35 to 6.03), as well as compounds ionized under the conditions of analysis. A good correlation was obtained between log KOW and log kIAM (pH 7.4) (n = 66, r2adj = 0.865, s = 0.561, F = 418)13 (full data is available as Supporting Information, SI, Tables 1 and 2). A clear distinction was reported between the response for unionized and ionized compounds. As discussed above, log KOW and log kIAM (pH 7.4) describe different partitioning behaviors. Therefore, a 1:1 relationship between these parameters is not expected. For ionized compounds the IAM partitioning system has additional electrostatic interactions, which are not present in octanol/water partitioning. It has been suggested that to bring ionized and unionized compounds together a hydrogen bond acidity term is required.14,15 However, this is outside the scope of the study, which is to determine the robustness or otherwise of the optimized IAMHPLC method for the analysis of both ionized and neutral compounds. Log kIAM (pH 7.4) values were determined for five compounds (covering a range of hydrophobicities (log KOW 0.29 to 6.03)). The robustness of the method was assessed using five different IAM.PC.DD2 columns, including three batches of stationary phase. The analysis was performed using two HPLC systems. The five compounds, analyzed are detailed in Table 1. These were chosen in part cover a wide range of log KOW values (from 0.3 to 6.03) consistent with many materials of interest to industry. In addition benzoic acid, butanone and bibenzyl were selected because they are reference materials for the determination of log KOW by reverse phase-HPLC (RP-
Figure 1. Structure of the IAM.PC.DD2 stationary phase.
length than comparable columns, leading to shorter analysis times (it is noted that for highly hydrophobic compounds this is not ideal; however, longer column lengths are available); (3) the column stationary phase is end-capped with C3 and C10 alkyl chains improving stationary phase stability.
Table 1. CAS Number, Structure, and Both Experimental and Predicted KOW Values for Samples Assessed for Robustness Testing
a
From KOWWin.17 bFrom the OECD Guideline for determining KOW by HPLC.16 cPreviously corrected for ionization through the following relationship: log P(corrected) = log P(pH) + log(1 + 10(pKa−pH)). 3697
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were prepared in 10 mM PBS, and bibenzyl and p-terphenyl were prepared in methanol. Samples were filtered prior to analysis through a 0.45 μm nylon filter, and a 10 μL aliquot was injected in triplicate. The logarithm of the relative retention factor was recorded; this is defined as:
HPLC) as listed by the Organisation for Economic Cooperation and Development (OECD).16 These compounds are well-characterized, and there is high confidence in their respective published log KOW values. One of the test compounds, benzoic acid, was ionized under the experimental conditions of the optimized IAM-HPLC method.
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⎡ t − t0 ⎤ log kIAM(pH7.4) = log⎢ r ⎥ ⎣ t0 ⎦
EXPERIMENTAL SECTION Materials. Methanol (HPLC gradient grade), sodium chloride (99.9 %), potassium chloride (99 %), dibasic sodium phosphate heptahydrate (99 %), and potassium phosphate (99 %) were purchased from Fischer Scientific (Loughborough, UK). Water was deionized with a Triple red system to 18.2 mΩ. All samples were obtained from commercial sources and were of 99 % purity or greater and used without further purification. All mobile phases were filtered through a 0.45 μm Millipore nylon filter, adjusted to pH 7.4, and degassed prior to use. Instrumentation. Two Agilent HPLC 1100 systems were used. The first system consisting of a G1379A degasser, G1311A quaternary pump, G1313A autosampler, G1316A column heater, and G1362A refractive index detector (1200). The second consisting of a G1310A isocratic pump, G1367A autosampler, G1362A refractive index detector, and a Jones chromatography 7955 external column heater. The five columns were all IAM.PC.DD2 (4.6 × 100 mm; Regis Chemical Company, Morton Grove, IL, USA). The chromatograms were recorded by Agilent Technologies ChemStation for LC systems, version B.03.01-SR1. Chromatographic Conditions for the Optimized IAMHPLC Method. The HPLC analysis used isocratic elution. The mobile phase was 10 mM phosphate-buffered saline (PBS) at pH 7.4 or mixtures of PBS and methanol (30 to 60 % v/v methanol premixed at pH 7.4). At a flow rate of 1 mL·min−1. The column temperature was 25 °C. The mobile phases were premixed (30 to 60 % v/v of methanol), and analysis was performed at 5 % increments. The refractive index detector was set to 40 °C. The void elution volume t0 was determined using water. PBS was used as the mobile phase to maintain a constant pH for the analysis of ionized compounds and compounds requiring extrapolation. The use of a buffer at a physiologically relevant pH allows for the generation of partition coefficients with a greater biological relevance than the traditional log KOW value. Determination of Method Robustness. The robustness analysis was performed on five columns (two columns contained stationary phase from one batch, a further two columns contained stationary phase from a second batch, and the final column contained stationary phase from a third batch). Analysis was performed on one column on three separate days to establish the effect of day-to-day variability. One of the five columns was analyzed on a second system to establish the effect of system variability. As two HPLC systems were used during the analysis, the system time was determined by injecting a sample, while a zero volume connector was in the place of the column; this proved that water is unretained on the column. The dead time was subtracted from all retention times recorded to give an on column retention time. This takes into account differences between systems. All samples were prepared at a concentration of 10−2 M, except 3-nitroaniline which was prepared at 5·10−3 M due to solubility in diluent. Butanone, benzoic acid, and 3-nitroaniline
where tr and t0 are the retention times for the sample and an unretained compound (water), respectively. The unretained compound recommended by the column manufacturer is citric acid. However, Demare et al.18 reported that this led to irreproducible results. For this reason water was used as the unretained compound and was included with each run sequence. The analysis of bibenzyl and p-terphenyl used varying percentages of methanol in the mobile phase. The log kIAM (pH 7.4) value is determined by extrapolation to the aqueous mobile phase. Extrapolation was required due to long elution times for highly hydrophobic compounds. Extrapolation consisted of analysis at 5 % increments of methanol in the mobile phase, and the log kIAM (pH 7.4) values obtained were plotted against % methanol and extrapolation to 0 % methanol, performed to determine log kIAM (aq). All extrapolations were linear and had an r2 of 0.99 or greater. Statistical Analysis. The Minitab (v.15)19 statistical package was used for the analysis of the data. Two-way analysis of variance (ANOVA) was performed where the data were balanced, and the general linear model (GLM) was used to perform the same analysis when the data were unbalanced. Balanced and unbalanced refer to the number of data points compared for each condition (conditions investigated were the effect of system, batch and column on measured log kIAM (pH 7.4)). If the same number of data points are considered for each condition, the data are balanced. Similarly, if a different number of data points is considered for each condition, the data are unbalanced. Two-way ANOVA was used as the five substances analyzed cover a range of log KOW and log kIAM (pH 7.4) values. The first variable in the two-way ANOVA is the compound, where variability is expected due to the deliberate selection of compounds with a range of hydrophobicities. The second variable in the two-way ANOVA was either the day, the column, the batch, or the system of analysis. Variability in the second variable is undesirable. ANOVA/GLM analysis can test for differences, alternatively, as in this case, it can test for similarity. When testing for similarity, the Tukey test, which is a post hoc test, is applied to the output from the ANOVA/GLM test. The Tukey reports an upper and lower limit for difference which has to be within a predetermined range to demonstrate similarity; overall the Tukey test has a test wide 95 % confidence interval.20 For results reported in this analysis the acceptable variation in log kIAM (pH 7.4) values is taken to be 0.3 log units. This is the same experimental variability considered acceptable for the determination of log KOW.21 For the determination of log KOW by RP-HPLC, the OECD guideline no. 117 reports repeatability criteria of ± 0.1 log units (0.2 log units) (for repeat measurements under identical conditions using the same reference compounds to construct a calibration graph) and reproducibility criteria of ± 0.5 log units (1.0 log units; for analysis using different reference materials).16 The more stringent 0.2 log units from the OECD guideline has also been applied to the log kIAM (pH 7.4) results. However, it is noted 3698
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Table 2. Arithmetic Mean Experimental log kIAM (pH 7.4) Values Obtained for the Five Samples, Using Five IAM-HPLC Columns, Containing Three Batches of Stationary Phase and Analyzed on One of Two Systems arithmetic mean log kIAM (pH 7.4) column
batch
system
1 1 1 2 2 1 3 3 2 4 3 1 5 1 1 arithmetic mean (standard deviation) across all data log kIAM (pH 7.4) range
aqueous plate count (max and min) 1126−1156 1297−1350 614−663 714−768 1978−2184
3-nitroaniline
2-butanone
benzoic acid
bibenzyl
p-terphenyl
0.800 0.785 0.785 0.820 0.846 0.807 (0.026)
−0.390 −0.465 −0.363 −0.330 −0.484 −0.406 (0.066)
−1.060 −0.945 −0.880 −0.840 −0.804 −0.906 (0.101)
3.52 3.63 3.49 3.60 3.63 3.57 (0.07)
4.79 4.93 4.85a 5.06 4.93 4.93 (0.11)
0.061
0.154
0.256
0.140
0.270
a
Value not included in calculation of method robustness, value calculated using extrapolation from two mobile phase compositions. Column 3 produced a poorly defined peak for one of the required concentrations, extrapolation is not valid. Refer to the text for further details. Batches 1, 2, and 3 refer the batch of stationary phase the column is packed with.
greater than intraday variability, as demonstrated by the smaller standard deviations intraday compared to the overall standard deviation for interday variability. Two-way ANOVA analysis of the data was performed, which identified the effects of substance and day and also the interaction between substance and day as being significant. Intercompound variability is expected and is, therefore, discounted. The Tukey test was applied to day-to-day variability, comparing values measured on a separate day against all other days. All Tukey test confidence intervals for log kIAM (pH 7.4) values show there to be no significant difference between days of analysis with the maximum variance being 0.03 (All Tukey test upper and lover limits for difference are reported in the Supporting Information, SI Table 4). System-to-System Variability. The log kIAM (pH 7.4) values determined on different HPLC systems were analyzed using the GLM, due to the data being unbalanced (single preparation, injected in triplicate for four columns and triplicate preparation, injected in triplicate for one column). The two variables were substance (five levels, five compounds) and system (two levels, two systems). The Tukey test was applied to assess the effect of the system upon the analysis; the upper and lower limits for difference were −0.06 and 0.04, respectively, which has a range of 0.1 and is within the predetermined range of acceptability. Column-to-Column Variability. The log kIAM (pH 7.4) values determined using different IAM-HPLC columns were analyzed using the GLM (due to the data being unbalanced, single preparation, injected in triplicate and triplicate preparation, injected in triplicate), and the two variables were substance (five levels, five compounds) and column (five levels, five columns). The Tukey test was applied to investigate the effect of column-to-column variability. The maximum range for upper and lower limits for difference was between columns 3 and 4 with a range of 0.15, which is within the predetermined range of acceptability. The five columns used in the analysis contain the stationary phase from three different batches; for this reason the effect of batch-to-batch variability was investigated. Batch-to-Batch Variability. The GLM (due to the data being unbalanced, single preparation, injected in triplicate and triplicate preparation, injected in triplicate) was applied to the log kIAM (pH 7.4) values to assess the effect of batch-to-batch variability, followed by the Tukey test. The Tukey test demonstrates that the maximum variance in upper and lower limit for difference was a range of 0.1, which is within the predetermined range of acceptability.
that OECD criterion requires the use of six reference materials for the determination of log KOW, whereas the robustness testing considers five compounds independently, without the use of additional reference materials. Therefore, this criterion is not strictly applicable to this analysis. Together, these criteria were used to determine the robustness (or otherwise) of the IAM-HPLC method.
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RESULTS AND DISCUSSION Determination of log kIAM (pH 7.4). Table 2 shows the arithmetic mean log kIAM (pH 7.4) values obtained for five compounds analyzed using five columns (full data are available as Supporting Information, including Tukey test results, SI Tables 2 to 4). These values were used to investigate the robustness of the IAM-HPLC method due to variation in the column, stationary phase batch, and system of analysis. The range of log kIAM (pH 7.4) values obtained for each compound is within the predetermined range of 0.3 log units. In addition three of the compounds are within the more stringent range of 0.2 log units. The fact that the standard deviation for each compound is small demonstrates that the results obtained for each individual compound are similar in response across the five columns, and therefore the data are comparable. The value reported for p-terphenyl using column 3 has not been included in the determination of method robustness. The value for log kIAM for p-terphenyl was calculated from extrapolation of log kIAM values measured using 50 %, 55 %, and 60 % MeOH in the mobile phase. A peak was identified using both 55 % and 60 % MeOH. When using a mobile phase of 50 % methanol, a peak for p-terphenyl can be identified above the background noise. However, without overlaying previous chromatograms obtained on the other columns, it would not be possible to confidently recognize this peak. Extrapolation of the two values obtained for 55 % and 60 % MeOH gives a log kIAM value within the range obtained for the remaining four columns. Despite this, this value has not been used in the analysis as extrapolation from two points is not valid. Assessment of Method Robustness. Intraday and Interday Variability. Intraday and interday variability were determined for the five compounds using column five only, by injecting a single preparation of each sample in triplicate, on three separate days. The three chromatograms for each preparation of each compound were comparable when overlaid (refer to SI Figure 3). Interday variability in log kIAM (pH 7.4) is 3699
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(9) Kotecha, J.; Shah, S.; Rathod, I.; Subbaiah, G. Relationship between immobilized artificial membrane chromatographic retention and human oral absorption of structurally diverse drugs. Int. J. Pharm. 2007, 333, 127−135. (10) Taillardat-Bertschinger, A.; Barbato, F.; Quercia, M. T.; Carrupt, P. A.; Reist, M.; La Rotonda, M. I.; Testa, B. Structural Properties Governing Retention Mechanisms on Immobilized Artificial Membrane (IAM) HPLC Columns. Helv. Chim. Acta 2002, 85, 519−532. (11) Luo, H.; Zheng, C.; Cheng, Y. K. The retention properties of nucleobases in alkyl C8-/C18- and IAM-chromatographic systems in relation to log POW. J. Chromatogr., B 2007, 847, 245−261. (12) Ledbetter, M. R.; Gutsell, S.; Hodges, G.; Madden, J. C.; O’Connor, S.; Cronin, M. T. D. A Database of Collated Retention Factors for Immobilised Artificial Membrane HPLC and an Assessment of the Effects of Experimental Variability. Environ. Toxicol. Chem. 2011, 30, 2701−2708. (13) Ledbetter, M. Development of Analytical Methods to Derive Hydrophobicity Parameters for use as Descriptors for the Prediction of the Environmental and Human Health Risk of Chemicals. PhD thesis, Liverpool John Moores University, Liverpool, UK, 2012. (14) Valko, K.; Bevan, C. D.; Reynolds, D. P.; Abraham., M. H. Rapid-Gradient HPLC Method for Measuring Drug Interactions with Immobilized Artificial Membrane: Comparison with Other Lipophilicity Measures. J. Pharm. Sci. 2000, 89, 1085−1096. (15) Valko, K.; Du, C. M.; Bevan, C.; Reynolds, D. P.; Abraham, M. H. Rapid Method for the Estimation of Octanol/Water Partition Coefficient (Log Poct) from Gradient RP-HPLC Retention and a Hydrogen Bond Aciditiy Term (Σα2H). Curr. Med. Chem. 2001, 8, 1137−1146. (16) OECD Guidelines for the Testing of Chemicals, No. 117: Partition coefficient (n-octanol/water), High performance liquid chromatography (HPLC) method; The Organisation for Economic Cooperation and Development OECD: Paris, 1994. (17) US EPA. Estimation Programs Interface Suite for Microsoft Windows, v 4.10; United States Environmental Protection Agency: Washington, DC, 2010. (18) Demare, S.; Roy, D.; Legerdre, J. Y. Factors Governing the Retention of Solutes on Chromatographic Immobilized Artificial Membranes: Application to Anti-Inflammatory and Analgesic Drugs. J. Liq. Chromatogr. Relat. Technol. 1999, 22, 2675−2688. (19) Minitab Inc. Minitab Statistical Software version 15; Minitab Inc.: Coventry, 2007. (20) Rowe, P. H. Essential Statistics for the Pharmaceutical Sciences; Wiley: New York, 2007. (21) Dearden, J. C. The Measurement of Partition Coefficients and Lipophilicity. Quant. Struct.-Act. Relat. 1988, 7, 133−144.
CONCLUSIONS An IAM-HPLC assay has been optimized using compounds covering a wide range of hydrophobicities (KOW of −1.35 to 6.03 and a log kIAM (pH 7.4) of −1.92 to 4.93). The range of log kIAM (pH 7.4) values obtained for each compound across five columns (Table 2) is within the acceptable range of experimental variability for the determination of hydrophobicity;21 in addition the standard deviation for each compound is small. The robustness of the method has been confirmed, using GLM analysis followed by post hoc Tukey tests. The Tukey test output from each analysis (variability due to day, column, batch, or system of analysis) demonstrates that the upper and lower limit for difference is within the predetermined range of acceptability. The method has been demonstrated to provide reproducible results and to be robust across the system, column, and stationary phase batch. An IAM-HPLC method that is robust has increased confidence in the log kIAM (pH 7.4) values obtained for compounds using this method and any models that are subsequently developed based on this descriptor.
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ASSOCIATED CONTENT
S Supporting Information *
Reported log kIAM (pH 7.4) values for 66 compounds analyzed using the robust method and both log kIAM (pH 7.4) values and statistical analysis for the detemination of method robustness. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel.: + 44 (0)1234 264730. E-mail: Moira.Ledbetter@ unilever.com. Funding
The funding of the Safety and Environmental Assurance Centre, Unilever Research, Sharnbrook, England is gratefully acknowledged. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Dearden, J. C. Partitioning and Lipophilicity in Quantitative Structure-Activity Relationships. Environ. Health Perspect. 1985, 61, 203−228. (2) Leo, A. J.; Hansch, C.; Elkins, D. Partition coefficients and their use. Chem. Rev. 1971, 71, 525−616. (3) Madden, J. C. Introduction to QSAR and Other In Silico Methods to Predict Toxicity. In In Silico Toxicology Principles and Applications; Cronin, M. T. D., Madden, J. C., Eds.; Royal Society of Chemistry: London, 2010. (4) Cronin, M. T. D. The role of hydrophobicity in toxicity prediction. Curr. Comput.-Aided Drug Des. 2006, 2, 405−413. (5) Caron, G.; Ermondi, G.; Scherrer, R. A. Lipophilicity, Polarity, and Hydrophobicity. In Comprehensive Medicinal Chemistry II; Taylor, J. B., Triggle, D. J., Eds.; Elsevier: New York, 2007. (6) Taillardat-Bertschinger, A.; Carrupt, P.; Barbato, F.; Testa, B. Immobilized Artificial Membrane HPLC in Drug Design. J. Med. Chem. 2003, 46, 655−665. (7) Giaginis, C.; Tsantilil-Kakoulidou, A. Alternative Measures of Lipophilicity: From Octanol-Water Partitioning to IAM retention. J. Pharm. Sci. 2008, 97, 2984−3004. (8) Pidgeon, C.; Venkatarum, U. V. Immobilized Artificial Membrane Chromatography: Supports Composed of Membrane Lipids. Anal. Biochem. 1989, 76, 36−47. 3700
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