Anal. Chem. 2006, 78, 1212-1217
Supercritical Fluid Chromatography-Tandem Mass Spectrometry for the Enantioselective Determination of Propranolol and Pindolol in Mouse Blood by Serial Sampling Jiwen Chen, Yunsheng Hsieh,* John Cook, Richard Morrison, and Walter A. Korfmacher
Drug Metabolism and Pharmacokinetics Department, Schering-Plough Research Institute, Kenilworth, New Jersey 07033
Packed-column supercritical fluid chromatography (pSFC) coupled to an atmospheric pressure chemical ionization (APCI) source and a tandem mass spectrometer (MS/MS) with minimum sample pretreatment was explored for the rapid and enantioselective determination of (R,S)-propranolol in mouse blood. Serial bleeding of mice is advantageous for the reduction of animal usage, dosing errors, and animal-to-animal variation. The effects of the eluent flow rate and composition as well as the nebulizer temperatures on the ionization efficiency of racemic propranolol and pindolol as model compounds in the positive ion mode under pSFC conditions were studied. The fundamental parameters on the proposed hyphenated system such as matrix ionization suppression and chromatographic performances were investigated in improving sensitivity and enantiomeric separation for the detection of the analytes. The proposed chiral pSFC-APCI/MS/MS approach requiring ∼3 min/sample for the determination of (R,S)-propranolol at a low nanogram per milliliter region was partially validated with respect to specificity, linearity, reproducibility, and accuracy and was applied to support a pharmacokinetic study.
ally, most chiral separation-based methods were developed using either UV or fluorescent detection. However, there is a trend toward interfacing enantioseparation technologies with more sensitive mass spectrometry-based methods without losing enantioselectivity.8 Although the tandem mass spectrometric detection provides little selectivity for stereoisomeric drugs, it can provide complete resolution of the dosed drugs from endogenous materials and their metabolites. In this work, we investigated the analytical potential of using pSFC-MS/MS for the quantitation of chiral drug molecules in mouse blood samples. Several factors such as the composition of mobile phase, eluent flow rate used for the normal-phase mode, which might affect both the chromatographic performance and ionization efficiencies of the test stereoisomeric components, were explored. Matrix ionization suppression, one of the common concerns when developing new mass spectrometry-based methods was also investigated. The atmospheric pressure chemical ionization (APCI) mechanisms for the test compounds in the liquid CO2/ methanol mobile phase were also studied. The proposed method for the simultaneous determination of (R,S)-propranolol was partially validated and applied to support a pharmacokinetic study in the mouse by serial bleeding.
According to the U.S. Food and Drug Administration’s (FDA) policy statement for the development of new stereoisomeric drugs, to evaluate the pharmacokinetics of a single enantiomer or mixture of enantiomers, manufacturers should develop quantitative assays for individual enantiomers in in vivo samples early in drug development.1 The analytical throughput of stereoisomeric assays are known to be strongly dependent on the efficiency of chiral stationary phases and the composition of mobile phases. In general, packed-column supercritical fluid chromatography (pSFC) for enantioseparation offers shorter injection-to-injection cycle times and better resolution than high-performance liquid chromatography (HPLC) or capillary electrophoresis.2-7 Convention-
EXPERIMENTAL METHODS Reagents and Chemicals. Carbon dioxide (SFC mode) was obtained from Airgas (Cheshire, CT). Methanol, N,N-dimethylethylamine (DEA), and isopropylamine (IPA) (HPLC grade) were purchased from Sigma-Aldrich Chemical Co., Inc. (Saint Louis, MO). Lysophophatidylcholine and palmitoyl 1,2-diphalmitoyl-snglycero-3-phosphocholine used as phospholipids were purchased from Sigma-Aldrich Chemical Co., Inc. (R)- and (S)-propranolol hydrochloride isomers and racemic (R,S)-pindolol hydrocloride were purchased from Sigma-Aldrich Chemical Co., Inc. and used as a standard. D7Racemic propranolol was purchased from C/D/N Isotopes Inc. (Quebec, Canada) and used as an internal standard (ISTD). Deionized water was generated from a Milli-Q water purifying system purchased from Millipore Corp. (Bedford, MA), and house high-purity nitrogen (99.99%) was used. Drug-free mouse blood samples were purchased from Bioreclamation Inc. (Hicksville, NY).
* To whom correspondence should be addressed. E-mail: yunsheng.hsieh@ spcorp.com. Phone: 908-7405385. Fax: 908-7402966. (1) http://www.fda.gov/cder/guidance/stereo.htm. (2) Hoke, S. H.; Pinkston, J. D.; Balley, R. E.; Tanguay, S. L.; Eichhold, T. H. Anal. Chem. 2000, 72, 4235-4241. (3) Terfloth, G. J. Chromatogr., A 2001, 906, 301-307. (4) Harris, C. M. Anal. Chem. 2002, 74, 87A. (5) Johannsen, M. J. Chromatogr., A 2001, 937, 135-. (6) Villeneuve, M. S.; Anderegg R. J. J. Chromatogr., A 1998, 826, 217-.
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(7) Chen, J.; Korfmacher, W. A.; Hsieh, Y. J. Chromatogr., B 2005, 820, 1-8. (8) Desai M. J.; Armstrong D. W. J. Chromatogr., A 2004, 1035, 203-210. 10.1021/ac0516178 CCC: $33.50
© 2006 American Chemical Society Published on Web 01/13/2006
Equipment and Instrumentation. SFC was performed on a Berger SFC system (Newark, DE) equipped with a SFC pump (pump A), a modifier pump (pump B), and a column oven as described previously.9 Tandem mass spectrometric detection was performed using a PE Sciex (Concord, ON, Canada) model API 4000 triple quadrupole mass spectrometer equipped with heated nebulizer (APCI) probes. The pSFC-APCI/MS/MS system consisted of a Leap autosampler with a refrigerated sample compartment (set to 10 °C) from LEAP Technologies (Carrboro, NC). A Chiralcel OD-H (250 × 4.6 mm, 5 µm) column purchased from Chiral Technologies, Inc. (West Chester PA) was used as the chiral column for normal-phase mode and was maintained at 45 °C. The effluent from the pSFC systems was connected directly to the mass spectrometer without splitting or makeup flow. Pump A and pump B were used for neat carbon dioxide and modifier, respectively. Outlet pressure was regulated to 100 bar by a pressure regulator. The amount of modifier that was added into the carbon dioxide mobile phase was expressed in volume. The peak resolution, Rs, were calculated as described elsewhere.10-11 In this work, the schematic diagram of the postcolumn infusion system for the matrix effect studies is similar to that used for the regular HPLC-MS/MS system shown elsewhere.10 The test analyte isomers dissolved in modifier solvent, used for normalphase pSFC, were continuously infused into Peek tubing between the chiral column and mass spectrometer through a tee using a Harvard Apparatus model 2400 (South Natick, MA) syringe pump. Either a supernatant extract of blank rat plasma or a modifier solvent (10 µL) (as a reference signal) was injected into the chiral column for comparison of ionization responses. Effluent from the analytical column mixed with the infused compounds and then entered the APCI interface. Mass Spectrometric Conditions. The mass spectrometer was operated in the positive ion mode. The heated pneumatic nebulizer probe conditions were as follows: 500 °C temperature setting, 80 psi nebulizing gas pressure, 1.0 L min-1 auxiliary gas flow, 0.9 L min-1 curtain gas flow rate. The MS/MS reaction selected to monitor propranolol, pindolol, and ISTD were the transitions from m/z 260, 249, and 267, the [M + H]+ ions, to the same product ion at m/z 116, respectively. The protonated molecules were fragmented by collision-activated dissociation with nitrogen as the collision gas at a pressure of instrument setting 5. The collision offset voltage was 25 V. Data were acquired and calculated using Analyst 1.4.1 software (PE Sciex). Pharmacokinetic Studies by Serial Sampling. CD/1 mice were obtained from Charles River Laboratories (Raleigh, NC) and were housed in a vivarium with a 12-h light/dark cycle. Food and water were available ad libitum. The mice were treated with propranolol at an oral dose of 30 mg/kg of body weight. A 4-µL sample of blood was withdrawn at certain time intervals from the tail vein from two animals using a serial bleeding design and then was mixed in an Eppendorf tube with 40 µL of 90:10 methanol/ H2O solution containing the internal standard at a concentration of 50 ng/mL for each isomer. After centrifugation, the supernatant (9) Hsieh, Y.; Favreau, L.; Schwerdt, J.; Cheng, K.-C. J. Pharm. Biomed. Anal., in press. (10) Hsieh, Y.; Chintala, M.; Mei, H.; Agans, J.; Brisson, J.; Ng, K.; Korfmacher, W. A. Rapid Commun. Mass Spectrom. 2001, 15, 2481-2487. (11) Hsieh, Y.; Wang, G.; Brisson, J.; Ng, K.; Korfmacher W. J. Pharm. Biomed. Anal. 2003, 33, 251-261.
(30 µL) was transferred to a 384-well plate. Stock solutions of (R,S)propranolol and (R,S)-pindolol were prepared as 1 mg/mL solutions in methanol. Standard solutions were prepared by serial dilution in methanol/water. Analytical standard samples for mouse PK studies were prepared by spiking known quantities of the standard solutions into extracted blood supernatant from 5 to 1000 ng/mL levels. The mouse blood samples were processed using a protein precipitation technique. Aliquots of 10-µL supernatants were injected for pSFC-APCI/MS/MS analysis. RESULTS AND DISCUSSION Integrating pSFC to the Mass Spectrometer. Chiral HPLC was the most popular method for the determination of propranolol and pindolol, β-blocking drugs for the treatment of cardiovascular disorders.12-16 However, the SFC mobile phase possessing low viscosity and high diffusivity has the potential for fast chiral separations at higher flow rates. There have been a few literature reports on interfacing SFC with mass spectrometry.17-27 Supercritical fluid mobile phases ease solvent removal and disposal concerns. Also, the unique CO2/methanol mobile phase with acidic or basic additives makes the hyphenation of pSFC to the APCI sources compatible. Polysaccharides derivatives (Chiralcel OD-H) are one of the most popular chiral selectors for enantioseparation separations under normal-phase conditions due to their versatility, durability, and loading capacity.7,28 Normally, the column employed in HPLC could be used in SFC.29-30 As demonstrated previously, the Chiralcel OD-H yielded higher column efficiency with SFC than HPLC.29,31 The SFC retention mainly is governed by the polarity of the mobile phase. The modifier, methanol, employed in this study is a major polar elution solvent under the SFC conditions. The lower modifier content would decrease the retention of analytes. The decrease of retention factor, k′, of the analytes by increasing modifier concentration is due to the increase of solvent (12) Rumiantsev, D. O, Ivanova, T. V. J. Chromatogr., B 1995, 674, 301-305. (13) Pham-Huy, C.; Radenen, B.; Sahui-Gnassi, A.; Claude, J. J Chromatogr., B 1995, 665, 125-132. (14) Egginger, G.; Lindner, W.; Brunner, G.; Stoschitzky, K. J. Pharm. Biomed. Anal. 1994, 12, 1537-1545. (15) Santoro, M. I. R. M.; Cho, H. S.; Kedor-Hackmann, E. R. M. Drug Dev. Ind. Pharm. 2001, 27, 693-697. (16) Xia, Y.; Bakhtiar, R.; Franklin, R. B. J Chromatogr., B 2003, 788, 317-329. (17) Bakhtiar, R, Tse F. L. S. Rapid Commun. Mass Spectrom. 2000, 14, 11281135. (18) Wang, T, Barber, M, Hardt, I, Kassel, DB. Rapid Commun. Mass Spectrom. 2001, 15, 2067-2075. (19) Zhao, Y.; Woo, G.; Thomas, S.; Semin, D.; Sandra, P. J. Chromatogr., A 2003, 1003, 157-166. (20) Crowther, J. B.; Henion, J. D. Anal. Chem. 1985, 57, 2711-2716. (21) Dost, K.; Jones, D. C.; Auerbach, R, Davidson, G. Analyst 2000, 125, 17511755. (22) Dost, K.; Davidson, G. Analyst 2003, 128, 1037-1042. (23) Jones, D. C.; Dost, K.; Davidson, G.; George, M. W. Analyst 1999, 124, 827-831. (24) Dost, K.; Jones, D. C.; Davidson, G. Analyst 2000, 125, 1243-1247. (25) Sjoberg, P. J. R.; Markides, K. E. J Chromatogr., A 1999, 855, 317-. (26) Zhao, Y.; Sandra, P.; Woo, G.; Thomas, S.; Gahm, K.; Semin, D. Pharm. Discovery 2005, 5, 30. (27) Tuomola, M.; Hakala, M.; Manninen, P. J Chromatogr., B 1998, 719, 2530. (28) Hoke, S. H.; Pinkston, J. D.; Bailey, R. E.; Tanguay, S. L.; Eichhold, T. H. Anal. Chem. 2000, 72, 4235-4241. (29) Hsieh, Y.; Favreau, L.; Cheng, K.-C.; Chen, J. Rapid Commun. Mass Spectrom. 2005, 19, 3037-3041. (30) Berger, T. A. J Chromatogr., A 1997, 785, 3. (31) Smith, R. M. J Chromatogr., A 1999, 856, 83-115.
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Figure 2. Relative APCI responses of propranolol and pindolol as a function of percentage of modifier in the presence of 0.2% DEA.
Figure 1. Effect of (A) composition and (B) flow rate of mobile phase on peak resolution. pSFC conditions: 45 °C oven temperature; 500 °C probe temperature in the presence of 0.2% DEA.
strength of the mobile phase and the deactivation of the stationary phase.29,32 The retention in SFC is also dependent on other factors such as column temperature, pressure, and flow rate of the mobile phase. The resolution (Rs) between two peaks for both (R,S)propranolol and (R,S)-pindolol in a pSFC-MS/MS chromatogram is calculated as 2∆Z/(Wa + Wb) where ∆Z is the separation between peaks A and B and Wa and Wb are the widths at the base of peaks A and B, respectively. The resolutions (Rs) for both (R,S)propranolol and (R,S)-pindolol mixtures as a function of modifier percentages and flow rates in the presence of additive of 0.2% DEA were plotted in Figure 1A and B, respectively. DEA and IPA are recommended as an amine additive and a strong base, for the analysis of basic compounds on polysaccharide phases. The resolution patterns of both peak pairs with DEA shown in Figure 1 were similar to those with IPA.29 As shown in Figure 1A, the improved resolution at lower ratios of modifier appears to be a column residence or capacity effect. However, the flow rate has little effect on resolution as given in Figure 1B. These results are in a good agreement with a previous study.33 Experimental conditions such as the compositions and the flow rates of eluent may also affect the ionization efficiencies of the analytes when hyphenating pSFC to the APCI source.29 Figure 2 indicates that the relative APCI responses of all test compounds decreases gradually as the modifier amount increases. This phenomenon was assumed likely due to incomplete vaporization at higher modifier. Previously, an increase in solvent flow rates resulting in appreciable negative impact on the relative sensitivities of the test compounds due to less effective heat transfer at higher flow rates was observed in the presence of IPA.29 However, in the presence of DEA, there is no substantial effect on the ionization efficiencies of both propranolol and pindolol on increasing the flow rate of mobile phase as shown in Figure 3. This indicated that the additive of DEA may have effect on the ionization efficiencies of the analytes. Normally, the probe tem(32) Zou, W.; Dorsey, J. G.; Chester, T. L. Anal. Chem. 2000, 72, 3620-3626. (33) Wilson, W. H. Chirality 1994, 6, 216-219.
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Figure 3. Relative APCI responses of propranolol and pindolol as a function of flow rate of the mobile phase in the presence of 0.2% DEA.
Figure 4. Relative APCI responses of propranolol and pindolol as a function of the temperature of the heated nebulizer in the presence of 0.2% DEA.
perature of the APCI source has a positive effect on the sensitivity of the analytes due to the high volatility of SFC eluent, which efficiently assists the nebulization process.29 However, the loss in APCI responses of the analytes in the presence of DEA was observed as the temperature of the heated nebulizer increased from 300 to 500 °C (Figure 4). This indicated that DEA as an additive in the SFC eluent for improving chromatographic separation may alter or compete with the ionization process of the analytes. An increase with the probe temperature may enhance the proton-transfer efficiency of DEA, resulting in lower ionization efficiencies of the analytes. The comparison of the APCI responses of the analytes with DEA or IPA was achieved by flow injection analysis. For propranolol and pindolol, the APCI signals were around 10- and 2-fold higher, respectively, when DEA was replaced with IPA as the mobile-phase additive. To improve APCI performance, a better understanding of fundamental processes of its ionization mechanism under the SPC
conditions is desirable. In this work, the formation of major reactant ions was recorded from the mass spectra of the liquid CO2/methanol solvent systems used for chiral separation. These data should provide fundamental evidence for proposing ionization mechanisms of analytes in the SFC-APCI system. For the APCI interface, the column effluent was vaporized through the heated nebulizer inlet probe, which consists of a concentric pneumatic nebulizer and a heated quartz tube.34-39 The heated mixture of solvent and vapor is then introduced into the APCI reaction chamber. The plasma of air, solvent, and sample components then encounters a cloud of electrons emitted from the tip of a corona electrode pin held at 3-5 kV on atmosphere. The electric field is sufficiently strong to ionize nitrogen, oxygen, and solvent vapor by removal of an electron through electron impact ionization. In the positive ion mode, these abundant reagent ions react in turn with surrounding solvent molecules through collision in the gas phase producing intermediate protonated charged clusters. The analytical ions could be produced by the proton-transfer process with the reagent gas plasma if the acidity of the analytes is less than that of protonated charged clusters. The protonation reactions of a given analyte, M, under SFC conditions with IPA additive were demonstrated previously.29 In this work, the ionization mechanism of analytes in the SFC-APCI interface with DEA additive was proposed as follows:
e- + N2 f N2+• + 2eCH3OH + e- f CH3O+•H + 2eCH3O+•H + n(CH3OH) f [(CH3OH)n + H] + + CH3O. CH3O+•H + (C2H5)2NH f (C2H5)2NH2+ + CH3O. [(CH3OH)n + H] + + M (analyte) f n(CH3OH) + [M + H]+ Here, a fashion similar to the HPLC-APCI operation was observed where the formation of the methanol-dimer cluster ion, [(CH3OH)2 + H]+, m/z 65] was detected (data not shown). The protonated additive molecules of DEA, [(C2H5)2NH2+, m/z 74] were also produced. The additive to the modifier in the pSFC system is normally required to improve the peak shape and enantioselectivity. The APCI responses of both pindolol and propranolol through flow injection analysis using CO2/methanol solvent with DEA were found to be less than those without DEA. The APCI signals of propranolol and pindolol were reduced by a factor of 10 and 2, respectively. However, The APCI responses of both pindolol and propranolol using CO2/methanol solvent with or without addition of IPA were found to be comparable.29 These data indicated that the production of all test analyte ions was (34) Niessen, W. M. A.; Tinke, A. P. J. Chromatogr., A 1995, 703, 37. (35) The API Book; PE Sciex, Thornhill, ON, Canada, 1989. (36) Willoughby, R. C.; Sheehan, E. W.; Mitrovich, S. A Global View of LC/MS; Global View Publishing: Pittsburgh, 1997. (37) Harrison, A. G. Chemical Ionization Mass Spectrometry; CRC Press: Boca Raton, FL, 1983. (38) Parker, C. E.; Bursey, M. M.; Smith R. W.; Gaskell, S. J. J. Chromatogr. 1985, 347, 61-74. (39) Mitchum, R. K.; Korfmacher. W. A. Anal. Chem. 1983, 55, 1485A-1497A.
primarily through the proton-transfer process with the protonated methanol clusters. The DEA molecules are often in large access relative to the analytes and might compete with the analytes for ionization. In the gas phase, the proton-transfer reaction occurs when the proton affinity of the neutral solvent molecules is greater than that of the donors. The fundamental of ion source chemistry is of importance in the SFC-APCI/MS system. It was also reported that the intensities of various analytes were found to be significantly affected by the solvents and the corona needle restrictor tip distance when APCI was interfacing with the SFC-MS system.25 No evidence was found for the CO2 to be involved in the proton-transfer reactions for ionization. Therefore, IPA was chosen as an additive to the SFC mobile phase for further quantitation of propranolol in mouse blood samples. Matrix Effects. A well-understood concern about assay reliability when developing new MS-based methods is the possibility of encountering matrix ionization suppression problems.9-10,40 One aspect that might be overlooked is the matrix ionization suppression effect when the retention time window for the two enantiomers is accounting for more than a considerable ratio of the total LC run times. In general, the matrix effect is dependent on ionization sources, sample preparation and treatment, analytes, and chromatographic conditions. The continuous postcolumn infusion experiments are the standard method for monitoring the ionization suppression for any new hyphenated approaches developed in our laboratory. The infusion chiral pSFC-APCI/ MS/MS chromatograms of propranolol and pindolol after a 10-µL injection of either mobile phase or mouse blood extract are given in Figure 5. The affected area of the chromatographic run was evaluated according to the differences in the infusion chromatograms between the mobile-phase injection and the mouse blood extract injection. The loss of APCI sensitivity was considered to be caused by the matrix ion suppression effects due to blood sample extract constituents eluting from the chiral column. Figure 5 demonstrates that the loss of APCI response of propranolol and pindolol was not observed. Phospholipids are commonly found in mammalian blood and animal tissue as structure components in membranes and have been identified to be a major source of ion suppression.41 Due to their hydrophobicity, phospholipids tend to be strongly retained on reversed-phase HPLC. As a result, they can remain on the HPLC column after a sample is analyzed and may elute in a very unpredictable manner in one of the following injections. To examine the possible contribution of phospholipids to the matrix effect on the SFC-APCI/MS system, the elution of the two most abundant phospholipids42 (lethicin C16:0 and lysolecithin C16:0), sample was monitored as surrogate markers after injection of the extracted blank blood. Both marker compounds had little retention on the column and were well separated from the analyte (data not shown). Moreover, no lipid markers were observed in 10 subsequent injections of solvent blanks (data not shown), indicating complete elution of both lipids. These results suggested that phosolipids were unlikely to compromise the performance of the assay in the proposed pSFC-APCI/MS system. In addition, pSFC (40) Mei, H.; Hsieh, Y.; Nardo, C.; Xu, X.; Wang, S.; Ng, K.; Korfmacher, W. A., Rapid Commun. Mass Spectrom. 2003, 17, 97-103. (41) Shen, J. X.; Motyka, R. J.; Roach, J. P.; Hayes, R. N. J. Pharm. Biomed. Anal. 2005, 37, 359-367. (42) Phillips, G. B.; Dodge, J. T. J. Lipid Res. 1967, 8, 676.
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Figure 5. Reconstructed postcolumn pSFC-APCI/MS/MS chromatograms of (A) propranolol and (B) pindolol after injection of supernatant of blank mouse blood (solid line) and methanol (dot line) in the presence of 0.3% IPA.
Figure 7. Blood concentration vs postdosing time profiles of propranolol isomers from a single mouse.
Figure 6. Reconstructed pSFC-APCI/MS/MS chromatograms of racemic propranolol from (A) blank, (B) standard at 25 ng/mL, and (C) study mouse blood samples and the reconstructed pSFC-APCI/ MS/MS chromatograms of D7-racemic propranolol from study mouse blood samples.
may be an attractive alternative if the pronounced ion suppression of the analyte due to phospholipids is observed in the reversedphase HPLC system. Application to a Mouse Pharmacokinetic Study. As an example, the proposed chiral pSFC-APCI/MS/MS method was applied for the simultaneous determination of propranolol isomers in mouse blood using a serial bleeding approach to demonstrate the realistic suitability of analysis. For a 20-g mouse, total blood loss was less than 10 µL from each blood collection and less than 5% of the total blood volume after 10 samples over a 24-h period. Consequently, there was no adverse reaction resulting from 1216 Analytical Chemistry, Vol. 78, No. 4, February 15, 2006
excessive blood loss. Figure 6 shows the pSFC-APCI/MS/MS chromatograms for the analytes and the internal standard in mouse blood samples. In Figure 6A, the APCI signals of propranolol from the blank mouse blood sample indicated that the endogenous peak from blood samples did not interfere with the propranolol peak (Figure 6B) under experimental conditions. Protein precipitation with methanol gave clean chromatograms with no other interfering peaks present under normal-phase chromatographic modes. Also, six lots of blank mouse whole blood were screened to determine the selectivity of the assay. No detectable signal was observed at the retention times of the propranolol enantiomers. This suggested a good specificity of the methods. The retention times and peak shape, as shown in Figure 6B-D for both (R,S)-propranolol and D7-racemic propranolol in standard and study blood samples, were found to be reproducible during the course of the study. The calibration curves from the same transition ranges were constructed by plotting the analyte/internal standard peak-to-area ratios against individual analyte concentrations in mouse blood. The calibration curves for both isomers using the APCI interfaces from standard mouse blood samples at each concentration level were linear with a correlation coefficient, r2, greater than 0.99 (graph is not shown). Accuracy (% bias) was less than 15% at all concentrations, 5-5000 ng/mL. The standard and study mouse blood samples were independently analyzed for propranolol using the pSFC-APCI/MS/MS method. The blood concentrations of two analytes following oral administration at 30 mg/kg were
Table 1. Validation Results for (R,S)-Propranolol in the QC Samples
day 1, R-form mean % deviation CV (n ) 4) day 1, S-form mean % deviation CV (n ) 4) day 2, R-form mean % deviation CV (n ) 4) day 2, S-form mean % deviation CV (n ) 4) day 3, R-form mean % deviation CV (n ) 4) day 3, S-form mean % deviation CV (n ) 4)
low QC, 15 ng/mL
middle QC, 150 ng/mL
high QC, 3750 ng/mL
7.0 3.8
-1.8 3.7
3.0 0.7
6.5 4.8
0.6 2.8
5.8 2.9
11 3.0
-2.0 3.8
9.4 3.3
5.0 9.8
-1.8 5.4
4.5 4.4
8.6 10
-1.7 3.3
5.9 0.9
9.6 4.1
-1.2 1.5
1.1 0.5
plotted against postdosing times (Figure 7). The area under the curve, AUC(0f24), and maximum blood concentration, Cmax, of (R)-propranolol were 1008 ng‚h/mL and 285 ng/mL, respectively, which were greater than those of (S)-propranolol at 563 ng‚h/mL and 172 ng/mL, respectively. The time at which the maximum blood concentration (Tmax) for (R)- and (S)-propranolol was achieved was the same at 1 h. The accuracy and precision for the assay was evaluated from quality control (QC) samples at 15, 150, and 3750 ng/mL. To examine the susceptibility of the method for lot-to-lot matrix variations, QCs were prepared from the same spiking solution but using different lots of mouse whole blood for each run. The interday precision and accuracy in the measurement of low, medium, and high QC samples obtained for
both analytes were in the range of 0.5-10% relative standard deviation and in the range of 98-111% of the nominal values, respectively, which were within the current FDA-recommended acceptance criteria of 15% at the low, medium, and high QC levels. (Table 1). CONCLUSIONS A rapid and sensitive pSFC-API/MS/MS method was developed for simultaneously monitoring chiral drugs in mouse blood samples. The described methodology demonstrated the potential of performing pharmacokinetic studies in a serially bled mouse model and a reduction in the amount of the test compounds required to undertake pharmacokinetic studies in support of drug discovery and more robust intraanimal pharmacokinetics. The use of pSFC coupled to tandem mass spectrometry has facilitated the analysis of small-volume blood samples. The coupling of CO2/methanol normal-phase pSFC to the APCI source showed good potential for maximum enantiomeric separation efficiency and the detection of chiral drugs. The proton-transfer reactions in the gas phase were responsible for the ionization of analytes under the SFC condition. The addition of basic additives to the mobile phase had a marginal impact on the selectivity for chiral separation but a strong impact on the ionization efficiency using the APCI source. The ionization suppression in the proposed chiral pSFC-APCI/MS/MS system was found to be negligible. ACKNOWLEDGMENT The authors thank our animal dosing group for planing PK studies presented in this work.
Received for review December 13, 2005.
September
9,
2005.
Accepted
AC0516178
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