Anal. Chem. 2000, 72, 4235-4241
Comparison of Packed-Column Supercritical Fluid Chromatography-Tandem Mass Spectrometry with Liquid Chromatography-Tandem Mass Spectrometry for Bioanalytical Determination of (R)- and (S)-Ketoprofen in Human Plasma Following Automated 96-Well Solid-Phase Extraction Steven H. Hoke, II,*,† J. David Pinkston,‡ Ruth E. Bailey,† Suzanne L. Tanguay,† and Thomas H. Eichhold†
Health Care Research Center, The Procter & Gamble Company, P.O. Box 8006, Mason, Ohio 45040, and Miami Valley Laboratories, The Procter & Gamble Company, P.O. Box 538707, Cincinnati, Ohio 45253
The popularity of packed-column supercritical fluid, subcritical fluid, and enhanced fluidity liquid chromatographies (pcSFC) for enantiomeric separations has increased steadily over the past few years. The addition of a significant amount (typically 20-95%) of a viscosity lowering agent, such as carbon dioxide, to the mobile phase provides a number of advantages for chiral separations. For example, higher mobile-phase flow rates can often be attained without a concomitant loss in chromatographic efficiency since diffusion coefficients, and optimum velocities, are typically higher in pcSFC. Ultratrace enantioselective quantitation of drugs in biomatrixes is an ideal application for these chromatographic attributes. To demonstrate the utility of this approach, a pcSFC tandem mass spectrometry (pcSFC-MS/MS) method was compared to a LC-MS/MS method for quantitation of the (R)- and (S)-enantiomers of ketoprofen (kt), a potent nonsteroidal, anti-inflammatory drug, in human plasma. After preparation using automated solid-phase extraction in the 96-well format, kt enantiomers were separated on a Chirex 3005 analytical column using isocratic conditions. Validation data and study sample data from patients dosed with either orally or topically administered ketoprofen were generated using both pcSFC and LC as the chromatographic methods to compare and contrast these analytical approaches. Generally, most analytical attributes, including specificity, linearity, sensitivity, accuracy, precision, and ruggedness, for both of these methods were comparable with the exception that the pcSFC separation provided a roughly 3-fold reduction in analysis time. A 2.3-min pcSFC separation and a 6.5-min LC separation provided equivalent, near-baseline-resolved peaks, demonstrating a significant time savings for analysis of large batch pharmacokinetic samples using pcSFC. * Corresponding author: (e-mail:)
[email protected].) † Health Care Research Center. ‡ Miami Valley Laboratories. 10.1021/ac000068x CCC: $19.00 Published on Web 07/22/2000
© 2000 American Chemical Society
(R,S)-2-(3-Benzoylphenyl)propionic acid (ketoprofen, kt; Figure 1), is a potent nonsteroidal, anti-inflammatory drug that is commonly formulated as a tablet for oral administration or, in some countries, as a topical gel or cream. In both cases, the active is formulated as a racemic mixture. It is well known that the (S) form of kt contains the intrinsic pharmacologic activity,1 and for this reason, it is desirable to determine the plasma concentrations of the individual enantiomers. The estimated bioavailability of orally dosed ketoprofen is g92%,2 and the maximum plasma concentrations reach low-microgram per milliliter levels after a typical 25-mg dose.2,3 With peak concentrations in that range, LC with UV detection has been established to provide adequate sensitivity for obtaining full pharmacokinetic (PK) curves.4-10 Typical chiral LC-UV methods reported in the literature have analysis times of approximately 10-25 min and lower limits of quantitation (LLOQ) of 25 ng/mL. Other approaches for bioanalytical determination of ketoprofen are GC/MS based with reported 10-20-min analysis times.11-15 One of these methods14 (1) Hutt, A. J.; Caldwell, J. Clin. Pharmacokinet. 1984, 9, 371-373. (2) Jamali, F.; Brocks, D. R. Clin. Pharmacokinet. 1990, 19, 197-217. (3) Foster, R. T.; Jamali, F.; Russell, A. S.; Alballa, S. R. J. Pharm. Sci. 1988, 77, 70-73. (4) Carr, R. A.; Caille, G.; Ngoc, A. H.; Foster, R. T. J. Chromatogr., B: Biomed. Sci. Appl. 1995, 668, 175-181. (5) Lovlin, R.; Vakily, M.; Jamali, F. J. Chromatogr., B: Biomed. Sci. Appl. 1996, 679, 196-198. (6) Grubb, N. G.; Rudy, D. W.; Hall, S. D. J. Chromatogr., B: Biomed. Sci. Appl. 1996, 678, 237-244. (7) Boisvert, J.; Caille, G.; McGilveray, I. J.; Qureshi, S. A. J. Chromatogr., B: Biomed. Sci. Appl. 1997, 690, 189-193. (8) Yagi, M.; Shibukawa, A.; Nakagawa, T. Chem. Pharm. Bull. 1990, 38, 25132517. (9) Wanwimolruk, S.; Wanwimolruk, S. Z.; Zoest, A. R. J. Liq. Chromatogr. 1991, 14, 3685-3694. (10) Rifai, N.; Lafi, M.; Sakamoto, M.; Law, T. Ther. Drug Monit. 1997, 19, 175-178. (11) Leis, H. J.; Leis, M.; Windischhofer, W. J. Mass Spectrom. 1996, 31, 486492. (12) Alkatheeri, N. A.; Wasfi, I. A.; Lambert, M. J. Vet. Pharmacol. Ther. 1999, 22, 127-135. (13) Gonzalez, G.; Ventura, R.; Smith, A. K.; de la Torre, R.; Segura, J. J. Chromatogr., A 1996, 719, 251-264.
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Figure 1. Full-scan product ion spectra of (A) ketoprofen and (B) SIL ketoprofen. The asterisk denotes the chiral center.
is stereoselective but requires derivatization and has a LLOQ of 1 ng/mL for each enantiomer. Because the circulating levels of kt after topical administration are much lower than those observed following an oral dose, the sensitivity of the methodologies listed above is not adequate to define PK curves following a topical dose. For this reason, a method was developed to lower the limit of quantitation using liquid chromatography coupled with tandem quadrupole mass spectrometry (LC-MS/MS).16 The resulting method has a LLOQ of 50 pg/mL per enantiomer and relatively short analysis time of 6.5 min/sample. In addition, automation of the sample preparation was performed using robotic solid-phase extraction (SPE) in the 96-well plate format, which allowed the processing of a 96-well plate every 50 min. Though the LC-MS/MS methodology was quite reliable, accurate, and precise, it possessed the drawback that the separation still required a 6.5-min analysis time to yield adequate resolution of the enantiomers. While that throughput represented an advance over existing methodologies, it was still much slower than analysis times typically used for racemic bioanalytical quantitation which range from a few minutes to less than 1 min/analysis.17-20 The popularity of packed-column supercritical fluid, subcritical fluid, and enhanced fluidity liquid chromatographies for enantio(14) Jack, D. S.; Rumble, R. H.; Davies, N. W.; Francis, H. W. J. Chromatogr., B: Biomed. Sci. Appl. 1992, 584, 189-197. (15) DeJong, E. G.; Kiffers, J.; Maes, R. A. A. J. Pharm. Biomed. Anal. 1989, 7, 1617-1622. (16) Eichhold, T. H.; Bailey, R. E.; Tanguay, S. L.; Hoke, II, S. H. J. Mass Spectrom. 2000, 35, 504-511. (17) Janiszewski, J.; Schneider, R. P.; Hoffmaster, K.; Swyden, M.; Wells, D.; Fouda, H. Rapid Commun. Mass Spectrom. 1997, 11, 1033-1037. (18) Davies, I. D.; Allanson, J. P.; Causon, R. C. J. Chromatogr., B: Biomed. Sci. Appl. 1999, 732, 173-184. (19) Joyce, K. B.; Jones, A. E.; Scott, R. J.; Biddlecombe, R. A.; Pleasance, S. Rapid Commun. Mass Spectrom. 1998, 12, 1899-1910. (20) Zweigenbaum, J.; Heinig, K.; Steinborner, S.; Wachs, T.; Henion, J. Anal. Chem. 1999, 71, 2294-2300.
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meric separations has increased steadily over the past few years.21-26 While these three techniques are different, they are closely related and require the same instrumentation. All three will be referred to as “pcSFC” in this report. The addition of a significant amount, typically 20-95%, of a viscosity lowering agent, such as carbon dioxide, to the mobile phase provides a number of advantages for chiral separations.27,28 Lower viscosity allows the use of higher mobile-phase flow rates (i.e., faster analyses) and/ or longer columns (i.e., higher efficiencies29) than when traditional liquid mobile phases are used. Compared to liquid chromatography, these higher mobile-phase flow rates can often be attained without a concomitant loss in chromatographic efficiency since diffusion coefficients, and optimum velocities, are typically higher with pcSFC. While CO2 itself is relatively nonpolar, mixtures of CO2 and polar organic solvents retain the polarity and solvating power of the polar organic until significant levels (40-60%) of CO2 are added.30 These mixtures can often, though not universally, be used to replace existing chiral LC mobile phases, without loss of the chiral recognition mechanism of the analyte. However, each separation must be tested on an individual basis.31 Although rarely performed, the ultratrace quantitation of drugs in biological fluids is an ideal application of this separation technique when coupled with tandem mass spectrometric detection. Previous reports have described the development and survey application of the pneumatically assisted electrospray source for pcSFC-MS/MS that was used in the present study.32,33 Here, a thorough assessment of the “real world” utility of the pcSFCMS/MS technique for high-throughput bioanalytical quantitation of drugs is provided, using enantiospecific quantitation of ketoprofen as an example. This is accomplished by validation and application of a pcSFC-MS/MS method for quantitation of the (R)- and (S)-enantiomers of ketoprofen in human plasma and comparison of the results of this method to results generated with an earlier LC-MS/MS method. EXPERIMENTAL SECTION Chemicals and Reagents. (R,S)-Ketoprofen was purchased from the United States Pharmacopeial Convention (Rockville, MD) while the internal standard, [13C1,2H3]-(R,S)-ketoprofen (SIL kt) was synthesized at P&G Pharmaceuticals (Norwich, NY). Blank human plasma was obtained from volunteers at Procter & Gamble (Mason, OH) or from Golden West Biologicals (Temecula, CA). (21) Phinney, K. W.; Sander, L. C.; Wise, S. A. Anal. Chem. 1998, 70, 23312335. (22) Maftouh, M. Spectra Anal. 1997, 26, 25-28. (23) Smith, R. In Supercritical Fluid Chromatography with Packed Columns, Techniques and Applications; Anton, K., Berger, C., Eds.; Chromatography Science Series 75; Marcel Dekker: New York, 1998; pp 223-249. (24) Sun, Q.; Olesik, S. V. Anal. Chem. 1999, 71, 2139-2145. (25) Terfloth, G. LC-GC 1999, 17, 400-405. (26) Wolf, C.; Pirkle, W. H. LC-GC 1997, 15, 352-363. (27) Anton, K., Berger, C., Eds. Supercritical Fluid Chromatography with Packed Columns, Techniques and Applications; Chromatography Science Series 75; Marcel Dekker: New York, 1998. (28) Berger, T. Packed Column SFC, RSC Chromatography Monographs; The Royal Society of Chemistry: Cambridge, 1995. (29) Berger, T. A.; Wilson, W. H. Anal. Chem. 1993, 65, 1451-1455. (30) Yuan, H.; Olesik, S. V. Anal. Chem. 1998, 70, 1595-1603. (31) Bargmann-Leyder, N.; Tambute, A.; Caude, M. Chirality 1995, 7, 311325. (32) Pinkston, J. D.; Baker, T. R. Rapid Commun. Mass Spectrom. 1995, 9, 10871094. (33) Baker, T. R.; Pinkston, J. D. J. Am. Soc. Mass Spectrom. 1998, 9, 498-509.
Sodium heparin was used as the anticoagulant with both sources of plasma. Methanol for SPE was purchased from J. T. Baker (Phillipsburg, NJ), and formic acid (98%) for SPE was obtained from EM Science (Gibbstown, NJ). Ammonium acetate, methanol (HPLC grade), and formic acid (Reagent grade) for preparation of the mobile phases were purchased from J. T. Baker. Carbon dioxide for pcSFC was obtained from Air Products and Chemicals, Inc. (Allentown, PA). Standard Solutions, Calibration Standards, and Quality Control (QC) Samples. Standard solutions of (R,S)-ketoprofen were prepared (as the racemate) at 0.01, 0.10, 1.0, 10, and 100 µg/mL in 80:20 H2O/MeOH. For each solution, 10, 20, or 50 µL was added to 1 mL of plasma to prepare calibration standards at 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10, 20, 50, 100, 200, 500, 1000, 2000, and 5000 ng/mL (as the racemate). Quality control samples were prepared similarly at levels of 1.0, 10, 200, and 1000 ng/mL (as the racemate). SIL ketoprofen solution was prepared at 2 µg/mL in 80:20 H2O/MeOH, and a 25-µL aliquot was spiked into 1 mL of plasma for calibration standards, QC samples and study samples to yield a final concentration of 50 ng/mL. Further details regarding standards and QC samples are provided elsewhere.16 Sample Preparation and Solid-Phase Extraction. Prior to analysis, ketoprofen was isolated from the sample matrix by performing automated SPE in the 96-well format. The sample preparation procedures were performed using a Biomek 2000 equipped with a vacuum manifold and a Multimek 96 channel pipettor, both from Beckman Coulter, Inc. (Fullerton, CA). SPE was performed using Oasis HLB sample extraction plates (Waters Corp., Milford, MA) with each column containing 30 mg of sorbent. HTS deep well tubes were purchased from Matrix Technologies Corp. (Lowell, MA). Plasma samples, calibration standards, and QC samples were prepared for analysis as described elsewhere.16 LC- and pcSFC-MS/MS Instrumentation. The pcSFC solvent delivery system was composed of a Gilson (Middletown, WI) modular system, that included a model 308 control pump, designed to deliver CO2, two model 306 auxiliary pumps, for the delivery of conventional organic and aqueous mobile phases, a model 811C dynamic mixer, a model 821 pressure regulator, a model 831 temperature regulator (column compartment), and a model 234 autosampler. The system was configured using a series of two- and three-way valves such that it could be converted in 5-10 min from LC to SFC mode and vice versa. The packed chiral stationary phase used with both modes of separation was Chirex 3005 (Phenomenex, Torrance, CA) consisting of (R)-1-naphthylglycine and 3,5-dintrobenzoic acid. The dimensions of the guard and analytical columns used for the LC separation were 2 × 30 mm and 2 × 250 mm, respectively, while the dimensions for the guard and analytical columns used for the pcSFC work were 4 × 50 mm and 4 × 250 mm, respectively. The mobile phase for the LC analysis was 30 mM ammonium acetate adjusted to pH 3.5 with formic acid in 5:95 H2O/MeOH delivered at 0.5 mL/min. The mobile phase for the pcSFC analysis consisted of a mixture of carbon dioxide/methanol (45:55) that was maintained at a rate of 5.0 mL/min. For the pcSFC work, a makeup flow was added to the effluent after the separation but prior to the MS analysis. The makeup flow consisted of 30 mM ammonium acetate, adjusted to pH 3.5 with formic acid in 5:95
H2O/MeOH, and was maintained at 0.5 mL/min. The makeup flow for this interface between the pcSFC and mass spectrometer can be used to enhance ionization and to control the pressure drop across the column.34 In this work, the flow and dimensions of the interface transfer line32,33 were such that the postcolumn pressure was maintained at a level to ensure the CO2/MeOH mixture was one phase throughout the column.34 The makeup flow was delivered by a model D series 260 syringe pump (Isco, Lincoln, NE). For both separation schemes, the entire chromatographic effluent was passed into the mass spectrometer interface for nebulization/ionization and subsequent detection. The mass spectrometer used with both modes of separation was a PE Sciex API III+ (Thornhill, ON, Canada). For the LC separation, the mass spectrometer was operated in the TurboIonSpray configuration, consisting of the articulated IonSpray inlet used in conjunction with the heated TurboProbe desolvation unit. In the case of the pcSFC, a modified TurboIonSpray source was used which allowed the addition of a makeup flow using a tee junction. The details of this modified source are described elsewhere.32,33 No sheath flow liquid was used in this work. For both effluents, the TurboProbe temperature and nitrogen gas flow rate were 450 °C and 8 L/min, respectively, and the nebulizer gas pressure was 60 psi (nitrogen). Protonated analyte ions were generated using ESI and orifice potentials of 3800 and 65 V, respectively. Collisional activation was achieved using argon as the collision gas at a thickness of 270 × 1013 molecules/cm2 and a collision energy of 17 eV. The selected reaction monitoring (SRM) transition m/z 255-209 was monitored for detection of (R)-kt and (S)-kt, while the SRM transition m/z 259-213 was monitored for SIL (R)-kt and SIL (S)-kt. Dwell time for each transition was 400 ms for the LC analysis and 200 ms for the pcSFC analysis because the pcSFC peaks were considerably sharper than the LC peaks. Quantitation of (R)- and (S)-kt. Peak areas for the chromatographic peaks were determined using the PE-Sciex software package, MacQuan version 1.4. Calibration curves for (R)-kt were constructed by plotting peak area ratios of (R)-kt/SIL (R)-kt versus (R)-kt concentrations and fitting these data to a 1/x2 linear regression plot. For the LC data, the calibration curve was segmented into two separate curves ranging from 0.05 to 100 ng/ mL and from 100 to 2500 ng/mL to obtain better accuracy of calibration standards and quality control samples. The pcSFC calibration range was also segmented into two curves, the first ranging from 0.05 to 2.5 ng/mL and the second from 2.5 to 2500 ng/mL, again for better accuracy. Calibration of the instrumentation for (S)-kt was performed analogously. Pharmacokinetic Study. Four male volunteers received a 25mg topical dose of ketoprofen using Oruvail Gel. This product, which is commercially available in the United Kingdom, was applied on the bicep region of the left arm and remained on the skin for the duration of the sampling period. Four additional volunteers received a 25-mg oral dose of ketoprofen, in the form of two 12.5-mg Actron caplets. For both dose forms, venous blood (10 mL) was drawn prior to dosing and at 20, 40, and 80 min and 2.5, 4.5, 8.5, 12.33, 16, and 24 h post-treatment. Plasma was harvested and stored frozen at -70 °C in polypropylene cryovials until the time of analysis. (34) Chester, T. L.; Pinkston, J. D. J. Chromatogr., A 1998, 807, 265-273.
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Method Comparison. For both LC and pcSFC validation and study sample analysis, the following analytical attributes were examined and compared: (1) specificity, (2) linearity, (3) sensitivity, (4) accuracy, (5) precision, (6) ruggedness, (7) sample throughput, and (8) utility for analysis of PK study samples. For the purpose of comparing pcSFC to LC accuracy with actual study samples, pooled samples were prepared by mixing four 250-µL aliquots from samples collected at the same time after administration of a given dose to obtain 1-mL aliquots. These pooled samples were then prepared for SPE as described above and each resulting extract was analyzed by both LC-MS/MS and pcSFC-MS/MS. RESULTS AND DISCUSSION Mass Spectra. The product ion MS/MS spectra of protonated kt and SIL kt are shown in Figure 1A and B, respectively. By evaluating all of the available transitions for quantitation, it was determined that monitoring the loss of the acidic functionality from the protonated molecular ion provided the best sensitivity and selectivity in the plasma matrix and was used for method development. While this is not a particularly selective transition, it was shown to provide good results for quantitation of (R)- and (S)-kt in human plasma. Separation Optimization. Chromatographic conditions for both modes of separation were investigated to optimize the sensitivity, specificity, and resolution of the two enantiomers as well as sample throughput. For the LC separation, normal-phase options were explored including the use of a Chiralpak AD column (Chiral Technologies, Exton, PA) with a hexane/2-propanol/TFA mobile phase. These conditions produced a very nice separation with a resolution (Rs) of 2.33. The sensitivity, however, was very poor using this mobile phase and the mass spectrometer signal was unstable without the addition of a makeup flow. Therefore, chiral separations with reversed-phase conditions were explored. An excellent separation was obtained with a Chirex 3005 4.0-mmdiameter column with a resolution of 2.09. Similar conditions using a Chirex 3005 2.0-mm-diameter column provided a ∼4-fold increase in sensitivity and faster run times, but resulted in reduced resolution of 1.42. A practical compromise between sensitivity, resolution, and analysis time resulted in the selection of the 2.0mm column at a flow rate of 0.5 mL/min for the LC conditions. The pcSFC separation of (R)- and (S)-kt was attempted with a variety of conditions and columns. Various combinations of CO2 and MeOH were used with Chiralcel OD, Chiralcel OJ, and Chiralpak AD columns (Chiral Technologies), but no acceptable separation of the kt enantiomers was obtained. Also, the 2.0-mm Chirex 3005 column was used with pcSFC and produced a separation with a 45% valley between the enantiomers (Rs ) 0.78). This result was judged unacceptable for further quantitative method development. The 4.0-mm column, however, did produce a very good separation. Figure 2A shows the separation with a resolution of 1.47 achieved with a 5 mL/min flow rate and 50% MeOH in CO2 in less than 2 min. More recent results have been obtained using 10 mL/min pump heads on the Gilson pcSFC instrument. Under the conditions described above for the 5 mL/min result, but using a 10 mL/min flow rate, a separation could be obtained in
