Anal. Chem. 1995, 67,3608-361 1
Cisplatin, Transplatin, and Their Hydrated Complexes: Separation and Identification Using Porous Graphitic Carbon and Electrospray Ionization Mass Spectrometry Hans C. Ehrsson,* lnger 6. Wallin, and Anita S. Andemson Karolinska Pharmacy, P.O. Box 160, S-171 76 Stockholm, Sweden
Per Olof Edlund Pharmacia AB, Biopharmaceuticals, S-1 12 87 Stockholm, Sweden
A new liquid chromatographic technique for the separation of the anticancer drug cisplatin, its trans isomer, and their corresponding cytotoxic hydrated complexes is presented. The separation is performed on porous graphitic carbon using an alkaline aqueous phase, where the hydrated complexes are present in their less reactive, uncharged form. The electrospray mass spectrum of cisplatinMered from that of transplatin, giving a sodiated adduct ion. The monohydrated complex of cisplatin showed [M - H201+,M+, and [M - HzO + MeCNl+ clusters starting at mlz 263,281, and 304, respectively, using a solvent mixture containing acetonitrile. The identi@of mlz 304 was established by MS/MS. Cisplatin (Scheme 1) is one of the most important drugs for the treatment of solid tumors, e.g., testicular 'and ovarian carcinomas. It is generally assumed that cisplatin is present as intact drug in extracellular fluids because of the high chloride concentration.' However, the chloride concentration is considerably lower inside the cell, which favors the formation of the hydrated complexes. The monohydrated complex is supposed to be the important cytotoxic species, its effect being mediated by reaction with DNA.2-4 The hydrated complexes have also been suggested to cause renal t~xicity.~Liquid chromatographic analysis of cisplatin has frequently been performed on strong anionexchange columns or reversed phase columns with a mobile phase containing a lipophilic quaternary ammonium compound (for a review, see ref 6). Isolation of the mono- and dihydrated complexes by liquid chromatography has so far been carried out at low pH, where the complexes are present as cations. Strong cation exchanger^^-^ or solvent-generated cation have (1) Rosenberg, B. Cancer Treat. Rep. 1 9 7 9 , 63, 1433-1438. (2) Johnson, N. P.; Hoeschele. J. D.; Rahn. R 0. Chem. Biol. Interact. 1980, 30. 151-169. (3) Knox, R J.; Friedlos, F.; Lydall, D. A; Roberts, J. J. Cancer Res. 1 9 8 6 , 46, 1972-1979. (4) Bancroft, D. P.; Lepre, C. A; Lippard, S. J. J. Am. Chem. SOC.1 9 9 0 , 112, 6860- 687 1. (5) Daley-Yates,P. T.;McBrien, D. C. H. Biochem. Phamzacol. 1984,33,30633070. (6) De Waal, W. k J.; Maessen, F. J. M. J.; Kraak, J. C. J. Pharm. Biomed. A n d . 1990,8, 1-30. (7) Safirstein, R; Daye, M.; Guttenplan, J. B. CancerLett. 1983,18,329-338. (8) Gonnet, F.; Lemaire, D.; Kozelka, J.; Chottard, J. C. J. Chromatogr. 1993, 648, 279-282.
3608 Analytical Chemistry, Vol. 67, No. 19,October I, 1995
in most cases been utilized. The hydrated complexes are highly reactive when protonated, while the corresponding uncharged forms are considerably less reactive.13 The aqua complexes react with frequently used mobile phase constituents like acetonitrile and phosphate b~ffers.'~J~ The diaqua complex has also been found to interact with sodium per~hlorate,'~ usually regarded as a nonreactive salt in this respect.8 In the present study, we have for the first time separated cisplatin and its uncharged hydrated complexes on porous graphitic carbon (PGC) using aqueous sodium hydroxide as a mobile phase. We have also included transplatin and its hydrated complexes, putative impurities in commercial preparations of cisplatin.16 Furthermore, the identity of the compounds was established by electrospray ionization mass spectrometry. EXPERIMENTAL SECTION Chemicals. Cisplatin and transplatin were purchased from Sigma Chemical Co. (St. Louis, MO). Solutions containing cisand trans-diammineaquachloroplatinum(ID ion were prepared from a hydrolysis equilibrium obtained by dissolving cis- and transplatin in distilled water (1 mM). The solution was left at room temperature and protected from light for 24 h. cis- and trunsdiamminedihydroxoplatinum(ID were prepared by hydrolysis of cis- and transplatin (0.2-1 mM) in 0.003 M NaOH for 24 h at room temperature (protected from light). Apparatus. The LC system consisted of a Valco Model C6W injector (Houston, TX) with a fixed loop volume of 100 pL, a Kontron Model 420 pump (Rotkrenz, Switzerland), and an LDC Spectromonitor I11 photometric detector. The wavelength monitored was 283 nm. The platinum complexes were separated on a Hypercarb S (particle size, 7 pm) column 100 x 4.6 mm i.d. (Shandon,Runcom, U.K,). The mobile phase was 0.001 M NaOH, with a flow rate of 0.5 mL/min. The analysis was carried out at 20 "C (room temperature) and at 0 "C (mobile phase and column (9) Anderson, A; Ehrsson, H. J. Chromatogr. B 1 9 9 4 , 652, 203-210. (10) De Waal, W. A J.; Maessen, F. J. M. J.; Kraak, J. C. J. Chromatogr. 1987, 407, 253-272. (11) Mistry, P.; Lee, C.; McBrien, D. C. H. Cancer Chemother. Phamacol. 1989, 24, 73-79. (12) Macka, M.; Borak, J.; Kiss, F. J. Chromatogr. 1991, 586, 291-295. (13) Lim, M. C.; Martin, R. B. J. Inorg. Nucl. Chem. 1976, 38, 1911-1914. (14) Segal, E.: Le Pecq, J. B. Cancer Res. 1 9 8 5 , 45, 492-498. (15) Vrana, 0.;Kleinwachter, V.; Brabec, V. Ezpen'entia 1984, 40, 446-451. (16) Arpalahti, J.; Lippert, B. Inorg. Chim. Acta 1987, 138, 171-173.
0003-2700/95/0367-3608$9.00/0 0 1995 American Chemical Society
Scheme la H3N\
,a R
H3N'
'Cl
+
pK, 6.56''
H3N\R/0H
-
+
pK, 7.21"
H3N\ /OH I
PI\
H ~ N ' 'OH
Diaqua a
pKa values from refs 17 and 18.
B
I "0
5
10
time (min) Figure 1. Chromatogram of cisplatin (A) and its mono- (B)and dihydrated (C) complexes. The mixture was prepared by adding cisdiamminedihydroxoplatinum(l1)in 0.001 M NaOH to a hydrolysis equilibrium solution of cisplatin in distilled water. The concentrations were (A) 0.2, (B) 0.5, and (C) 0.3 mM. Column temperature, 0 "C.
placed in an ice bath). The void volume of the system was determined by the front disturbance in the chromatograms on injection of water. Electrospray mass spectra were obtained using a Quattro triple quadrupole instrument from Fison Instruments (Altrincham, U.K.). Cisplatin, transplatin, and the hydrated complexes were isolated using the liquid chromatographic conditions described above and collecting narrow fractions of the apex of the peaks (final concentration, 50-500 pM). The solutions were mixed with an equal volume of acetonitrile containing 0.5% acetic acid and injected by a 20 p L loop injector into a carrier stream of 1:l acetonitrile-acetic acid (0.02% in water), with a flow of 10 pL/ min. The spectrum of cis-diamminedihydroxoplatinum(II) was also obtained after dilution of the chromatographiceffluent with methanol and using a carrier stream of methanol. The spectra were recorded by accumulation of 6-8 scans at unit mass resolution in the mass range of 10-500 m/z. The ion source was
time (min) Figure 2. Chromatogram of cis- (A) and transplatin (D).I and II represent di- and monohydrated complexes, respectively. Initial concentrations: cisplatin, 0.1 mM; transplatin, 0.25 mM. Column temperature, 20 "C. Table I.Influence of Temperature (T, "C) on the Capacity Factors
T
KcisPt
KtransPt
k'monwis
k'monetrans
ydicis
k'di-trans
20 0
1.88 2.48
5.48 8.24
0.89 0.97
0.95 1.09
0.39 0.31
0.40 0.33
tuned using the sodiated ion of cisplatin, giving the best response at conditions typical for low in-source fragmentation with a sampling cone voltage between 19 and 40 V. Daughter ion spectra were recorded after collision-induced fragmentation with argon at a cell pressure of 0.2 pbar and a collision energy of 80 eV. RESULTS AND DISCUSSION
This study shows that cisplatin, transplatin, and their corresponding hydrated complexes can be separated on PGC with a strongly alkaline (0.001 M NaOH) mobile aqueous phase, where Analytical Chemistry, Vol. 67,No. 19,October 1, 1995
3609
Table 2. Influence of Temperature (T, "C) on the Resolution between the Mono- and Dlhydrated Complexes of Cis- and Transplatin
T
Rs mono/di(cis)
Rs mono/di(rrans)
20 0
1.50 2.07
1.72 2.48
A
100 ra15
IMtNal+
\ ";
Table 3. Theoretical Isotopic Distribution in Molecules Containing PtCl,, Qiven as Relatlve Abundance, Where n = 0, 1, or 2
B M*
Aa A + 1 A + 2 A + 3 A + 4 A + 5 A + 6 A S 7 A S 8 P t 9 7 1 0 0 7 5 PtCl 92 94 100 F'tC12 71 73 100
0 30 47
Corresponds to the isotope
21 43 58
0 0 7
0 6 16
0 0 0
0 0 2
lg4Pt.
the hydrated complexes are present in a less reactive, uncharged form (Scheme 1).17J8 A chromatogram illustrating the separation of cisplatin and its corresponding mono- and dihydrated complexes is given in Figure 1. It can be calculated that ~ 1 of%the monohydrated complex is converted to the dihydrated complex during the chromatographicrun (25 "C) using the rate constants given in ref 19. Hence, it is possible to isolate the pure monohydrated complex, supposed to be the ultimate cytotoxic agent of cisplatin, from an equilibrium mixture of cisplatin in distilled water for subsequent use in in vitro and in vivo studies. Cisplatin has a planar structure and is highly polar.zo PGC has previously been found to strongly interact with planar structures by dispersion and charge transfer interactions.21*2z The excellent separation of cis- and transplatin is illustrated in Figure 2. The shorter retention of cisplatin as compared to transplatin is most probably attributed to its dipolar character. The capacity factors increased for cis- and transplatin when the analysis was carried out at 0 "C as compared to room temperature (Table l),while the capacity factors for the hydrated complexes were virtually unaffected. However, the resolution between the mono- and dihydrated complexes was improved at the lower temperature (Table 2). A similar effect on resolution has previously been observed on pyrocarbon containing supportsz3and on PGC.z4 Previous mass spectrometric studies of platinumcontainiig compounds involve ionization by electron i m p a ~ t , 2fast ~ * ~atom ~ b0mbardment,2~*~~ field d e s o r p t i ~ nlaser , ~ ~ desorption,3° and 25zCf (17) Bemers-Price, S. J.; Frenkiel, T. A; Frey, U.; Ranford, J. D.: Sadler, P. J.J. Chem. Sot., Chem. Commun. 1992, 789-791. (18) Andersson, A; Hedenmalm, H.; Elfsson, B.; Ehrsson, H. J. Pham. Sci. 1994,83,859-862. (19) Miller, S. E.; House, D. A Inorg. Chim. Acta 1989, 166, 189-197. (20) Souchard, J. P.; Ha, T. T. B.; Cros, S.; Johnson, N. P. J. Med. Chem. 1991, 34, 863-864. (21) Bassler, B. J.; Kaliszan, R; Hartwick, R A J. Chromatogr. 1989,461,139147. (22) Tanaka, N.; Kimata, IC;Hosoya, IC;Miyanishi, H.; Araki, T.J. Chromatogr. A 1993, 656, 265-287. (23) Colin, H.; Diez-Masa, J. C.; Guiochon, G.; Czajkowska, T.; Miedziak, I. J. Chromatogr. 1978, 167, 41-65. (24) Stefansson, M.; Hoffman, K. J. Chirality 1992,4, 509-514. (25) Haake. P.; Mastin, S. H.J. Am. Chem. SOC.1 9 7 1 , 93, 6823-6828. (26) Weller, R. R; Eyler, J. R; Riley, C. M. /. P h a m . Biomed. Anal. 1985,3, 87-94. (27) Siegel. M. M.; Bitha, P.; Child, R G.; Hlavka, J. J.; Lin, Y.; Chang, T. T. Biomed. Environ. Mass Spectrom. 1986, 13,25-32.
3610 Analytical Chemistty, Vol. 67,No. 79,October 7, 7995
-
"]304
C
10-400
[M-NHs-MeCNIt
[M-MIgl+
'2480
2MI
5
m/Z 0 180
1W
2W
210
220
230
240 250
260
270
200
2W
300 310 120 330 340
D 0
52 2 246.0
157 t
','270.1
E
"'1
I \
[M-CY +MeCNI*
2 180
180
2W
220
240
260
280
Mo
320
340
Figure 3. Electrospray mass spectra of cisplatin (A) and the monohydrated complex of cisplatin (B), together with daughter ion mass spectra of m/z 304 produced from the monohydrated complex of cisplatin (C), the dihydratedcomplex of cisplatin (D), and transplatin (E). Spectra A, B, and E were obtained using water-acetonitrileacetic acid; spectrum D was obtained using water-methanol-sodium hydroxide.
plasma desorption.'8 Electrospray is one of the softest ionization techniques available for nonvolatile and thermally unstable compounds, e.g., cisplatin, iproplatin, and tetra~latin.~~ Electrospray
mass spectrometry was used in the present work to confirm the identity of the hydrated forms of cisplatin and to compare the mass spectra of cis- and transplatin. Electrospray ionization usually gives ions that reflect the analyte ions present in the solution infused to the interface, while analytes without protolytic properties can be detected as adducts with alkali metal salts or Ion evaporation is thought to be the most important ionization mechanism during ESI, but gas phase reactions must also be considered. The analytes are declustered by low-energy collisions with gas molecules in the intermediate pressure region of the ES source and adduct ions in the solvent, or fragment ions can be formed, depending on the experimental conditions. Cisplatin gave a sodiated adduct ion [M Nal+ (Figure 3A) showing an isotope cluster starting at m/z 321, with the distribution 67:68:100:41:607:12:1:3. This isotope pattern is in excellent agreement with the theoretical values given in Table 3 for an ion containing one Pt and two C1 substituents. In contrast to cisplatin, the spectrum of transplatin (Figure 3E) does not show the [M + Na]+ ion. The monohydrated complex of cisplatin shows [M H20]+,M+, and [M - H2O MeCN]+ clusters (Figure 3B), starting at m/z 263,281, and 304, respectively. Collision-induced dissociation of the ion at m/z 304 by MS/MS was performed to
+
+
(28) Claereboudt, J.; De Spiegeleer, B.; Lippert, B.; De Bruijn, E. A; Claeys, M. Spectrosc. Int. J 1989,7, 91-112. (29) Dalietos, D.; Furst, A; Theodoropoulos, D.; Lee, T. D. Int. J Mass Spectrom. Ion Processes 1984,61, 141-148. (30) Claereboudt, J.; De Spiegeleer, B.; De Bruijn, E. A,; Gijbels, R; Claeys, M. /. Pharm. Biomed. Anal. 1989,7,1599-1610. (31) Poon, G. IC; Mistry, P.; Lewis, S. Bioi. Mass Spectrom. 1991,20,687692. (32) Kebarle, P.; Tang, L. Anal. Chem. 1993,65, 972A-986A
confirm the adduct with acetonitrile ( F i r e 3C). Daughter ions were found at m/z 287,263,246,228, and 211, and the proposed structures of the fragment ions are given. The dihydrated complex showed no M2+ion (m/z 132) but did show another doubly charged ion at m/z 155, corresponding to the substitution of both aqua groups with acetonitrile. The charge state at m/z 155 was assigned to 2 since the isotope peaks were 0.5 Da apart (data not shown). The [M HI+ ion at m/z 263 was detected when the same sample was diluted with methanol instead of acidified acetonitrile (Figure 3D), showing the isotope cluster expected for an ion consisting of one Pt atom. Good sensitivity was obtained when the samples were infused in a wateracetonitrile mixture acidified with acetic acid, but fewer gas-phase reactions occurred at slightly alkaline conditions (0.5 mM NaOH) with methanol as the organic modifier. A careful tuning of the kinetic energy of the analyte ions within the ion source (cone voltage) was important in order to be able to produce a spectrum of the hydrated Pt complexes. In conclusion, it has been shown that cisplatin, transplatin, and their hydrated complexes can be separated on porous graphitic carbon using an alkaline mobile phase. Furthermore, the importance of electrospray mass spectrometry in the structure elucidation of these platinum-containing compounds has been demonstrated.
+
Received for review January 25, 1995. Accepted July 12, 1995.a AC9500904 @Abstractpublished in Adounce ACS Abstracts, September 1, 1995.
Analytical Chernistty, Vol. 67, No. 19,October 1, 7995
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