Anal. Chem. 1994,66, 23562361
Determination of h w g r m n Lev- of Peptide Drug in Rabbit and Human Plasma Using High-Performance Lwld Chromatography Coupled wlth Electrospray Ionlzatlorr Mass Spectrometry J. Crowthw,' V. A d U W " , 1.MukhwJoe, K. Jordan, P. Abuaf, N. Corkum, a. addrtdn, and J. Tolad Immunobbbgy Research Institute, Annat&&, New Jersey 0880 I
High-perforarrace liquid cbmatognphy (WE) coupled to masa spectrometry (MS)ushg an ekctrospny ioaizrtion (ESI) interface proridcs a seasitiremetbodfor the quantitative analysis of pepti& drugs in complex biologbl umtrixm. ESI HPLC-MS w e d to tbe W SOf~ 8 p~nfrpcptMc S (IRI-514)ia rabbit d h m @SOIL Prior to inrlysiS, tbe plasma samples were prepued using protein precipitation followed by solid-phe extraction. Tbe lower Uplit of quantitation using selected ioa mMlitoringwas determined to be 2 ng/mL, w k 8 mLof human plrsma spikedwith 1-40 ng/mL was extracted. Rabbit phsma (1 mL) samples spiked with 10-4 OOOngof authentic drug/mL gave a h e a r respoaoe when a deuterated peptide analog was employed as an intend sturdard. A commercial ESI interface was modifiedto permit higher flow rates (10-20 p l l n r i a ) to enter tbe mass spectrometer source. The revised interface provided a 10-fold llmeaseinsemitivitiesd permittedtheuseof standardHPLC colt" (2.0-mm i.d.) and HPLC instrumentation. ESI HPLC-MS analysis was automated to provide pruttetnle4 precise, and senoitivedetectionof small peptides in both human a d rabbit plasma. Ushq this methoddogy, a toldcdrinetic study of i n t r a v m l y adminisIRI-514 at threedose levels indicated that tbe area under tbe curve values were dose proportional.
Electrospray ionization ( S I ) mass spectrometry (MS) has been used extensively recently for the characterization of small proteins'-5 and peptides.@ In addition to qualitative studies, this technique can be adapted to the quantitative determinationof low levels of peptide due to the inherent high sensitivity of ESI MS. By interfacing high-performanceliquid chromatography (HPLC) directly to ESI MS instrumentation, trace levels of peptide drug can be measured in relatively
complex samples such as plasma, serum, and urine."' Fouda et al.12 reported excellent sensitivities for the quantitative LC-MS analysis of a renin inhibitor in serum. This derivatized hydrophobic dipeptide was analyzed by usc of a heated nebulizer interface. Peptides which are larger or more hydrophilic, however, are more readily analyzed using
ESI MS. ESI LC-MS for the analysis of peptide drug in plasma provides an accurate, sensitive, and reproducible technique, features that are necessary for the determinationof low levels of peptide in bioavailability and toxicokinetic studies.l3 Previous approaches to peptide analysis in plasma at these low levels involved derivitization followed by quantitation of the fluorescent derivative, but these methods suffered from endogenous interferences and reproducibility problems.14 Radiochemical technique& lack sufficient sensitivities in addition to the inconvenience of synthesis and handling of radiolabeled drug, When peptide drugs are dosed orally, highly specific and particularly sensitive methodology must be used, since oral peptide formulations are known to have limited bioavailability,'618 which results in low levels of the drug in plasma. Natural peptides are quickly degraded through acid and enzymatic hydrolysis in the stomach and gut, obviating the absorption of effective quantities of drug into the circulatory system. To improve absorption, peptide analogs resistant to enzymaticand acid hydrolysis may be synthesized. IRI-514 (Ac-Arg-Pro-AspPro-Phe-NHz), a pentapeptide (9) Schncider, R. L.; Cole, M.J.; Foudr, H. 0 . Proceedings of the 41st ASMS
Conference on Mass Spectrometry and Applied Topics, San Francisco,CA, May 31-June 4, 1993; p 53. (10) LeBlanc, M. D.;Quillirm, M. A.; Bailey. M. R.; North, D.H. Prarcdlngs of the 4lst Confemnce on Mass Speciromrtry and Applied Topics, S&I Francisco, CA, May 31-June 4, 1999; p 837. (ll)Kayc,B.;Clark,M.W.H.;C~,N.J.;Macrpc,P.V.;Stopher,D.A.Biol. Mass Spectrom. 1991. 21, 585-589. (12)Fouda,H.;Nomini,M.;Sc~a.R.;Gedutic,C.I.AmSoc.MwSpmnmr 1991, 2, 164167. t Current addrerc/Affiliition: Affymax Pharmaauticala, 3410 Cmtral Ex(13) P a t C. C.; Barr, W. H.; &act, L. Z.; C M h . J.; D a j a b , R. E.; Funt, pressway, S n t a Clara, CA 95051. D.E.; Hartcr, J. G.; Lcvy, G.; Luddm T.;R o d " , J. H.; Smrtham, L.; (1) Bruins. A. P.; Covey, T. R.; Hmioa. J. D.A d . Chem. 1987,59,2642-2646. Schcntag, J. J.; Shah, V. P.; Sheiner, L. B.; Skclly, J. P.; Staaoki, D. R.; (2) Fenn, J. B.; Mann, M.;M a g , C. K.;Woag. S. F.; Whitehoure, C. M.Science Tmpl+R. J.;Virwanathan,C.T.; Whinger, J.;Yacobi,Av.CIin.Phunnacd. 1989, 246.64-71. Ther. 1992, 51, 465473. (3)Loo,J.A.;Ubeth,H.R.;Smith.R.D.Anol.Biochem.1989,179,40C412. (14) Newcomb. R. LC-GC 1993, 10 (l), 34-39. (4) Feng, R.; Konbhi, Y . AMI. Chem. 1992,64.2090-2095. (15) Digmir,G.A.;Sadcfa.E.P.;Pur,A.F.;&ihaR.M.;McCLisC.;~thL ( 5 ) Gallagher. R. T.;Chapman, J. R.; Barton,E. C. Proceeding of the39th ASMS B. M.;Ghebre-Scllasie, I.; Lya. U.; Nabitt, R. U.; Randinitia. E. 1. C l f n Conferem on Mass Spectrometry and Allied Topics, Nashville, TN,1991; Pharwwcol. 1990, 30. 621. Elsevier: New Ywk,1991; pp 244-243. (16) Wood,J.M.;C~ne,L.;dc~pmM.;Buhlnulya.P.;Ruqler,H.;Stlntoa (6) Chait, B. T.; Kent,S. B. H. Science 1992, 257, 1885-1884. J. L.; JUPP, R. A.; Kay. J. 1. Cordkpacc. Pihormocol. 1989, f4,221-226. (7) Gwzctta, A. W.; Baa, L. J.; Hancock, W. S.; Keyt, B. A.; Bennett, W. F. ( 17) k, V. H. L. In Peptides, a Target for New Drug Developmnt; Bloom, S.R., AMI. Chem. 1993,65,2953-2962. Bumstock, G., Lis.;IBC Technical Smim: W o n . 1991; pp 120-134. (8) Scindlcr, P. A.; Van Dorsaelaer. A.; Falick, A. M. AMI. Blochem. 1993,213, (18) Kompella. U. B.; Lee, V. H. L. In Peptides and Protein Drug Oclkry; Lee, 256263. V. H.L., Ed.; Marcel Dckker: New York, 1991; pp 391-486.
2S66
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undergoing evaluation in the treatment of anxiety and depression, was found to be stable in several in vitro enzymatic models (pepsin, carboxypeptidase, prolidase) and in 0.1 M HCl. ESI HPLC-MS was used to analyze plasma samples to determine the concentration of this peptide analog following oral dosing. Present methodology and its application for the assay of IRI-514 in rabbit and human plasma is presented. Samples of rabbit plasma were collected following the intravenous administration of IRI-5 14 to determine the toxicokinetics of the drug. Similarly,samples of human plasma were analyzed to estimate the bioavailability and dose proportionality of IRI-5 14 upon oral administration. Prior to analysis, the plasma samples were processed using protein precipitation followed by solid-phase extraction (SPE) to remove high molecular weight contaminants and particulates. Significant modification of commercial ESI MS instrumentation was necessary to provide the required sensitivities and system reliability. The concentration ranges studied for the rabbit and human plasma using SPE-ESI-HPLC-MS were 1 0 4 0 000 ng/mL and 2 4 0 ng/mL, respectively. ESI HPLC-MS is ideally suited for the target analysis of peptides and proteins in complex biological matrixes due to the ability of the mass spectrometer to selectively monitor only specifiedmolecular weights corresponding to the peptides of interest. The mass spectrometer thus serves as a very sensitive HPLC detector with tunable selectivity. The analysis of plasma samples, in particular, provides a means to determine a drug’s bioavailability despite low plasma levels, which is useful in comparing various dosage forms, in comparing routes of administration, and in correlating plasma levels with pharmacodynamic results.
EXPER IMENTAL SECTION Materials, Reagents, and Peptide Standards. HPLC grade acetonitrile and methanol were obtained from Burdick and Jackson (Muskegon, MI). Trifluoroacetic acid (HPLC spectro grade) was obtained from Pierce Chemical Co., Rockford, IL. ACS grade ammonium formate and formic acid were purchased from Eastman Kodak, Rochester, NY, HPLC grade water was obtained using a Millipore (Super 2) water purification system (Milford, MA). Phosphate-buffered saline (PBS) used in standard dilutions was purchased through Sigma Chemical Co., St. Louis, MO, and prepared according to the manufacturer’s specifications. Supelclean LC-18 SPE cartridges of 3- and 8-mL capacity were used with disposable flow control valve liners in a Visiprep DL manifold (Supelco, Inc., Bellefonte, PA). Blank rabbit and human plasma were obtained to use as controls. IRI-5 14 was synthesized by Bachem (Bubendorf, Switzerland) as the citrate salt. The deuterated analog, IRI-5 14d5 (Ac-Arg-Pro-Asp-Pro-ds-Phe-NHz),was synthesized as the acetate salt at IRI using solid-phase synthesis and t-BOC synthesis strategies. The deuterated phenylalanine (MSD isotopes, Toronto, ON,Canada) was tested by separate ESI HPLC-MS experiments to determine the isomeric purity (98%+ D5 and no detectable Do) prior to synthesis to ensure that no undeuterated IRI-514 was present in the internal standard.
Preparation of Standards. The internal standard (IS) solution, 2.0 pg/mL, was prepared by dissolving 1.2 mg of IRI-5 14-d5,equivalent to 1.Omgof the peptide (peptidecontent determined by amino acid analysis), in 500 mL of water. The stock solution A of IRI-514, 100.0 pg/mL, was prepared by dissolving 14.0 mg of IRI-514, equivalent to 10.0 mg of the peptide, in 100 mL of HPLC grade water. The stock solution B of IRI-5 14, 1.O pg/mL, was prepared by diluting 1.O mL of stock solution A to 100 mL with HPLC grade water. IRI-5 14 standards were prepared in both water and blank rabbit and human plasma by spiking with an appropriate volume of the IRI-514 stock solutions A and B. An 8-mL aliquot of plasma was spiked with 100 pL of a 2000 ng/mL solution of deuterated IRI-5 14 (Phe-d5) to serve as an internal standard. Pretreatment of Plasma Samples. Prior to ESI HPLCMS analysis, plasma samples were pretreated to “desalt” the sample and remove both very hydrophilic and hydrophobic impurities. Internal standard, preferably a deuterated analog of the peptide drug, was first added to the plasma samples. The internal standard served both to compensate for the recovery of drug from plasma and to reduce the variability in the sensitivity of the electrospray process run to run. Samples were then pretreated to precipitate proteins and extracted using reversed-phaseSPEcartridgesto remove excess salts and interfering substances. Upon removal of the elution solvent, the dried residue was dissolved in 0.5 mL of HPLC mobile phase. IRI-514 was concentrated 2-16-fold during pretreatment. Solid-Phase Extraction of Rabbit Plasma Samples and Standards. Briefly, 0.100 mL of a 2.0 pg/mL IS solution and 2 mL of acetonitrile were added to 1.0 mL of each IRI-514 spiked rabbit plasma. The sample was vortexed for 1 min and centrifuged at 3000 rpm for 15 min at 4 OC. The supernatant obtained was evaporated and dried under nitrogen at room temperature. Before use, the SPE c18 cartridges from a single lot were preequilibrated by washing sequentially with 3.0 mL each of 100% acetonitrile, 75% (v/v) acetonitrile in water, 50% (v/v) acetonitrilein water, 25% (v/v) acetonitrile in water, 100% water, and finally 5.0 mL of 0.1% (v/v) TFA in water. The dried acetonitrile extract was redissolved in 1.0 mL of PBS by vortex mixing for 1 min and applied to the resin bed of a 3-mL capacity preequilibrated SPE cartridge for IRI5 14 extraction. The cartridges were initially eluted with 3.0 mL of 0.1% TFA and then with 3.0 mL of 3% acetonitrile in 0.1% TFA to wash impurities from the column. Finally, IRI514 was eluted from the cartridges with 3.0 mL of 30% acetonitrile in 0.1% TFA. For the elution of IRI-514 from the cartridges, many compositions of acetonitrile in 1% TFA aqueous solution were tried, and the final elution solvent mixture for SPE, 30% acetonitrile in 1% TFA solution, was found to be optimal for the acceptable recovery of IRI-514 and IS,while coelution of plasma interferences was avoided. Solid-Phase Extraction of Human Plasma Samples and Standards. In the same manner, 8.0 mL of IRI-514 spiked human plasma standards or actual samples had 0.100 mL of IS added prior to precipitation with 13 mL of acetonitrile, and the resultant mixture was processed as described above. The dried residue was redissolved in 3.OmL of PBS and further processed using a 6-mL-capacity preequilibrated SPE cartridge Am&ticalChemistry, Vol. 66,No. 14, July 15, 1994
2357
AOOCO ORGANIC 'UAKNP'
REMOVABE CONE
125um X 4 0 u m
Gou) COATED CAPILLARY
1/32" VALCO TEE
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"PLc/spuT TO WASTE 1:20 USING UNEAR RfsTRlCTOR TO
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Ground Atmospheric Pressure
as described above. The solvent mixtures used for the l-mL method, as described above, were also applied to this 8.0-mL method, however, at higher volumes (5.0 mL). Concentration of Extracts Prior ta ESI HPLC-IWS Analysis. The eluents were brought to dryness in a Savant (Farmingdale,NY) Speed-Vacunder vacuum. Each residue was redissolved in 0.500 mL of the mobile phase and vortexed. The solution was transferred to autosamplervials. Standards and samples were processed similarly. Instnmentation. HPLC. TheHPLC consistedof a Waters (Milford, MA) 717 autosampler (temperature controlled at 10 "C), a 600MS pump, and a 486 detector. An Applied Biosystems 400 HPLC pump was used to deliver additional methanol postcolumn. All Waters gguipment was controlled and UV data recorded using the Waters 860 data system. Isocratic analysis of sampleswas performed using a mobile phaseconsisting of 1% (v/v) methanol, 20% acetonitrile in 10 mM ammonium formate pH adjusted to 5.2 with 1% formic acid. A Y MC reversed-phase (Wilmington, NC) Basic HPLC column (5-pm silica, 2.0 mm X 250 mm) was used throughout all experiments. The flow rate was 0.25 mL/min and the detector set at 220 nm. A 30 pL aliquot of each sample was injected onto the HPLC column using the autosampler; run time was set at 15 min. Since many samples (45-96) were often analyzed in a single run, the autosampler was programmed to chill the samples and limit sample degradation. MS. The mass spectrometerwas a Nermag RlOlOC with an Analytica of Branford (Branford, CT) electrospray interface modified for nebulization and a postcolumn split flow. Instrument control and data analysis was accomplished using Spect420software modified (Mass Evolutions, Houston, TX) to permit unattended and multiplesample analyses using a Waters autosampler. The resolution was optimized using a software-controlledand updated microprocessor board (Mass Evolutions). The instrument's ion optics were adjusted to maximize sensitivity at approximately monthly intervals by infusion of a 100 pM solution of IRI-5 14 dissolved in mobile phase. The mass spectrometer was programmed to monitor ions m / z 672 (IRI-514) and 677 (IRI-514-ds internal standard) with a total cycle time of 3 s (dwell time of 1.5 s). Since the HPLC effluent into the source was at ground potential, a -3.3-kV potential was applied to the interfaces's 600 pm4.d. capillary tube to provide electrospray conditions. 2368 A&yti&l
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Two 1010 L/min capacity rotary pumps (No. D045, Galileo, East Granby, CT) were used to evacuate the electrospray chamber, and two additional pumps were used to provide rough pumping for the instrument diffusion pumps. EsESource Modification. The inlet to the Analytica source was modified to accept the 0.25 mL/min flow rates which were compatiblewith the chromatographiccolumn diameter (2.0") used in this study. Smaller diameter columns (
