BIOLOGICAL MASS SPECTROMETRY, VOL. 20, 687-692 (1991)

Electrospray Ionization Mass Spectrometry of Platinum Anticancer Agents G. K. Poont and P. Mistry Drug Development Section, Institute of Cancer Research, Belmont, Sutton, Surrey, SM2 SNG, UK

s.Lewis Finnigan MAT, 355 River Oaks Parkway, San Jose,California 95134, USA

Quantification and identification of platinum drugs and their metabolites in biological samples has always been difficult because tbese compounds are thermally unstable, non-volatile and insoluble. We have demonstrated that electrospray ionization mass spectrometry can be a valuable technique for direct mass spectral analysis of platinum anticancer agents and for obtaining structural information as a result of fragmentation. Full-scan spectra were obtained with approximately 10 pmol samples. These results show the potential of applying this technique in pharmacokinetic studies of platinum anticancer drugs.

INTRODUCTION Cisplatin (cis-dichlorodiammineplatinum(I1)) (Fig. 1) and its less toxic analogue, carboplatin (cis-diamminecyclobutane-1,l-dicarboxylatoplatinum(I1)) are exceptionally useful anticancer agents in the treatment of various solid tumours, including testicular and However, intrinsic and acquired ovarian cancers.

'-'

"3N

H3N

'

a'

CISPLATIN M W 298

IPROPLATIN MW

416

c1

TETRAPLATIN M W 448 * Platinum isotope cornsponds to Ig4R

Figure 1 . Structures of platinum complexes studied by ESI mass

spectrometry.

t Author to whom correspondence should be addressed. 1052-9306/91/11068746 $05.00

0 1991 by John Wiley & Sons, Ltd.

resistance to these agents remains a major problem. There is a clear need to develop more effective platinum anticancer drugs and/or develop methods to modulate the sensitivity of tumours to the currently available drugs. A better understanding of platinum drug pharmacokinetics and biochemical determinants of action would aid in the achievement of these aims. Measurement of these parameters is dependent on the availability of reliable analytical techniques by which the parent drug and its metabolites can be quantified in biological samples. A number of methods are currently available for detection of platinum drugs, including radiochemical labelling with 195mPt,4*5 X-ray fluorescence (XRF),6 proton-induced X-ray emission (PIXE),' inductively coupled plasma (ICP)? flameless atomic absorption (FAA)' and inductively coupled plasmaatomic emission spectrometry (ICP-AES)." All of these methods have some drawbacks such as poor sensitivity, the short half-life of 195mPt radionuclide, matrix problems, and, in addition, they only provide information on total platinum levels in the biological samples. The use of high-performance liquid chromatography (HPLC) in conjunction with ultraviolet (UV) detection, with or without post-column derivatization,"*12 or electrochemical d e t e ~ t i o n , ' ~ . ' or ~ some of the abovementioned techniques can possibly identify the platinum species present following drug treatment. However, none of these techniques can provide definitive structural information on the parent drugs or their metabolites. Such information can be obtained by lg5Pt, or 15N or 'H nuclear magnetic resonance spectroscopy,' '*16 but this technique has several limitations. At least several milligrams of material is required for each investigation, while platinum complexes are only slightly soluble in most organic solvents and are not very stable in solution. Furthermore, for these techniques, isolation of relatively pure fractions of analytes prior to analysis is often required, and therefore they are not applicable to the study of unstable complexes. Recently mass spectrometry has been applied to detect platinum complexes using fast atom bombardment Received 20 February 1991 Revised 31 July 1991

688

G . K. POON, P. MISTRY AND S. LEWIS

(FAB) i~nization,’~-’’ field desorption ionization,” electron impact (EI)23 with Fourier transform (FT) mass ~pectrometry,’~ or laser microprobe mass analysis (LAMMA) with time-of-flight (TOF) mass spectrometry.” These involatile and thermally labile compounds are best investigated using FAB, FD or LAMMA ionizations. Of these three, direct introduction of the samples via FAB prot; into the mass spectrometer seems to be the most commonly used method because this technique is generally available to most laboratories. The FAB mass spectrometric technique requires the complexes to be dissolved in a suitable organic solvent (e.g. dimethylsulphoxide (DMSO), dimethylformamide (DMF), methanol-water), before being introduced onto the FAB probe tip which has previously been coated with a thin layer of matrix. The matrix may vary and can be glycerol, thioglycerol, 3-nitrobenzylalcohol, a 5 : 1 w/w mixture of dithiothreitol and dithioerythritol, triethanolamine, and others.” Sometimes a mixedsolvent system such as DMSO-thioglycerol is used in order to enhance the solubility of the platinum compounds.” Platinum complexes behave differently towards various solvents and matrices. Quite often, ligand exchange is observed in the spectra, or matrix adducts are formed, making interpretation of the spectra difficult. In addition, if more than one component is present in the sample, there may be ion suppression effects, while the high matrix backgrounds often hinder the detection of the compounds of interest and severely decrease the sensitivity of detection. The use of tandem mass spectrometry (MS/MS) can eliminate some of these difficulties.’0.21 Electrospray ionization (ESI) mass spectrometry was first investigated by Dole and co-w~rkers.’~~’~ Fenn, Whitehouse and c ~ - w o r k e r s ’ ~recently -~~ demonstrated that this soft ionization technique can be used to analyse large biomolecules. The principle design of their ESI interface involved introducing samples through a stainless steel open tubular needle into a region of high potential gradient at atmospheric pressure. Charge is deposited onto the emerging liquid surface and disperses the liquid by Coulombic forces into a fine spray. Evaporation of solvent from the drops is hastened by a countercurrent of heated bath gas. As the drop size decreases, the charge density increases and results in an electric field strength strong enough to desorb the ions into the mass spe~trometer.’~ This technique often produces multiply charged ions, which provides the opportunity to determine the molecular weights of compounds with masses higher than the upper limit of mass-to-charge ratio of a quadrupole mass spectrometer. Mass spectra of compounds with molecular weights of 66000 Da are readily ~btained.~’.~’ In fact Loo et al.33 have reported an ESI mass spectrum of bovine albumin ‘native’ dirner, which has a molecular weight in excess of 130000. Apart from the applications of ESI mass spectrometry to other materials such as high-molecular-weight biomolecules, several laboratories have applied this technique to study ionic transition-metal complexes with low molecular eight.^^.^' One of the advantages of ESI is that it is possible to transfer the ions from solution to the gas phase with little decomposition, thus making it ideal for

analysing organo-metallic complexes. This paper describes investigations into the analysis of cisplatin, iproplatin (cis-dichloro-bis-isopropylamine-trans-hydroxyplatinum (IV)) (Fig. 1) and tetraplatin (d,l-transtetrachloro-1,2-diaminocyclohexaneplatinum(IV)) (Fig. 1) with ESI mass spectrometry and suggests that in conjunction with HPLC it should be an important analytical technique for quantification and structural elucidation of platinum complexes in biological samples.

EXPERIMENTAL Cisplatin and iproplatin were provided by Johnson Matthey Technology Centre (Reading, UK) and tetraplatin was a gift from Dr M. Wolpert-Defilippes (NCI, Bethesda, Maryland, USA). All three compounds were purified by HPLC using a PLRP-S reverse phase column (250 x 4.6 mm, Polymer Laboratories, Shropshire, UK). The samples were eluted by a linear gradient of 15-90% acetonitrile in water. The appropriate peaks containing the platinum complexes were identified by UV absorption at 214 nm and platinum analysis by flameless atomic absorption. The corresponding fractions were collected and dried under vacuum. Prior to analysis by ESI mass spectrometry the compounds were dissolved in methanol-water (50: 50 v/v) containing 1% acetic acid at the following concentrations: 10 pmol p1-’ of cisplatin, 5 pmol pl-’ of iproplatin and 30 pmol pl-’ of tetraplatin. The mass spectrometer used for the analyses was a TSQ 700 triple-quadrupole system (Finnigan Mat, San Jose, California, USA) equipped with an electrospray ion source (Analytica, Branford, Connecticut, USA) which has been previously de~cribed.~’ Samples were infused into the electrospray source at 1 pl min-’ using a Harvard infusion pump (model no. 22, Harvard Apparatus Inc., Cambridge, Massachusetts, USA). A fused-silica uncoated capillary (100 pm id.; 3 feet long) was used to connect the delivery syringe with the stainless steel spray needle. A potential difference of 3700-4000 V was applied between the grounded needle and the metalized ends of the glass capillary tube that passes ions into the analyser. Air, heated to about 60°C,was used as the drying gas. Mass analysis was carried out in the third quadrupole Q 3 . Profiled data were averaged for a time sufficient to obtain good signal to noise, generally about 1 min. The amount of sample consumed in the analysis was calculated from the sample concentration, the flow rate and the time of analysis. The lowest level of detection for individual compounds was determined from single centroided scans. Tandem mass spectra were obtained by opening the resolution in Q1 so that the entire isotope cluster was transmitted to the collision cell in the second quadrupole Qz.Argon (0.5 mtorr) was used to perform collisionally activated dissociation (CAD) and daughter ions were detected in the third quadrupole Q 3 . CAD was also performed in the electrospray source in the high-pressure region between the glass capillary tube and skimmer by increasing the voltage on the capill a r ~Full-scan . ~ ~ mass spectra in the positive mode were

689

EIMS OF PLATINUM ANTICANCER AGENTS

acquired from m/z 100 to m/z 500, at 5 s per scan. Data analysis was performed on a DEC 2100 data system, which is part of the TSQ 700 system.

of spectrum observed. When analysing an unknown component, it is difficult to know whether [M + HIf or [M + Na]' is the ion observed on the spectrum. This ambiguity may be overcome by adding acetic acid to the solution, to enhance the [M HI' ion, or by adding sodium salt to enhance the [M Na] ion. In addition the results from two modes of CAD experiments are reported : classical MS/CAD/MS, performed in the collision cell of the second quadrupoie, and CAD/MS, which is performed in the region between the capillary and skimmer in the electrospray source. The mass spectrum of iproplatin is shown in Fig. 2. The base peak of this compound in the mass spectrum is [M + HI' at m/z 417. A spectrum with good signal to noise was obtained from 5 pmol of the sample consumed. Platinum-containing fragment ions were observed at m/z 345 [MH - 2HCl]', m/z 306 [(CH,),CH(NH,)Pt(OH)Cl + HI+, m/z 291 [(CH,)CH (NH,)Pt(OH)Cl HI+ and m/z 252 [(CH,),CHNH Pt]', respectively. MS/MS analysis of the m/z 417 cluster gave a strong daughter ion at m/z 58 [Fig. 2(b)].

+

RESULTS AND DISCUSSION ~~

All platinum anticancer agents that were studied by ESI mass spectrometry contained chlorine. They therefore all showed characteristic cluster ions in their mass spectra due to the isotopic peaks of both platinum and chlorine ions, and the m/z values listed in the text refers to the first peak of an ion cluster which contained 194Pt and "Cl isotopes. Two types of spectra were observed for a number of platinum complex studies: one contained predominantly the protonated molecule [M + H I + while the other contained the natriated molecule [M + Na]'. Results presented were obtained from ESI of cisplatin and its analogues and provides examples for each type

+

+

+

419 [M+H]+

80-

(CH,bCH-NH,/

u

f

kE'07 1.53

Pt IlC' OH

60-

B E Da

232 251

100

200

I

34.7 I

I

300

.+E*05 2.75

33

293

nlz Figure 2. (a) ESI mass spectrum of iproplatin. Insert: expansion of the [M + H I + ion cluster; (b) CAD daughter ion spectrum of [M + H I + of iproplatin.

G. K. POON, P. MISTRY AND S. LEWIS

690

323

'E.05 7.26

0

x

3

Jt

U

60-

L

.C

CI

,2

40

8 I

232

Figure 3. ESI mass spectrum of cisplatin. Insert: expansion of the [M + Na]+ ion cluster.

This ia originates from the [(CH3)2C=NH2]+ alkyl side chain. In addition the fragment ions m/z 345, 306, 291 and 252 described above were observed. The mass spectrum of iproplatin has previously been reported by using EI/FT mass spectrometry. FragWeller et mentation products, similar to those observed in the ESI spectrum, were present. However, the molecular ion was not observed in the EI spectrum, rendering it less useful. When samples contain sodium salts, a natriated molecule cluster is often formed and will be the predominant ion in the spectrum. An example of this is the spectrum of cisplatin (Fig. 3). The most prominent ion in the mass spectrum is the [M + Na]+ cluster starting at m/z 321. The isotopic distribution (76 :76 : 100 :54 :62) is in good agreement with the theoretical values for PtCl, compounds (Table 1). No [M + H I + molecular ion was observed in the spectrum. The two ion clusters at m/z 210 and 194 are the result of cisplatin fragmentation and correspond to the structures [Pt(NH,)]+ and Pt +, respectively. The cluster at m/z 337 is the kaliated molecule [M + K]'. The ion at m/z 304 was derived from loss of NH, from the natriated ion. Some of the other peaks in the spectrum such as m/z 150, 232 and 301 could not be identified. Often the appearance of natriated rather than protonated ions is a function of pH during sample processing and can be controlled to suit

the needs of the analyst. This mass spectrum is corn arable to those analysed by LAMMADOF mass spectrometry2' or EI/FT mass s p e ~ t r o m e t r y . ~ ~ The results from the analysis of tetraplatin provides an excellent example of the utility of CAD mass spectrometry in the electrospray source. Figure 4(a) is the ESI spectrum of tetraplatin obtained with 120 V applied to the capillary. The major constituent is the [M + Na]+ ion at m/z 471. The protonated molecule at m/z 449 is absent. If the voltage at the end of the capillary is increased to 175 V, CAD occurs. The resulting spectrum is shown in Fig. 4(b) and indicates that substantial fragmentation has occurred under these conditions. The following ions are present in the spectrum: m/z 471 ([M Na]+), m/z 341 ([M - 3C1- 2H]+), m/z 377 ([M - 2C1- HI'), m/z 413 ([M - Cl]+), m/z 400 ([M + Na - 2Cl]+), m/z 303 ([M - 4HCll'). The proposed losses are based on comparison of the measured isotope ratios with theoretical values. It should be noted that a sample which had probably deteriorated in solution gives a spectrum which is similar to the CAD mass spectrum at 120 V except that the natriated ion is absent. The mechanism of this observation is beyond the scope of this paper. However, one can speculate that the sample deterioration could have occurred by replacement of C1 with OH in solution and subsequently loss of water under ESI condition. Finally,

+

Table 1. Theoretical value distribution for molecular ion cluster observed in molecules containing PtCI, ,where II = 0, 1, Pt PtCl RCI, RCI, PtCI, Cisplatin lproplatin Tetraplatin O A

...,4

A.

(A+1)

(A + 2)

(A + 3)

W+4)

97 92 72 59 49

100 94 73 59 50

75 100 100 100 100

0 30 47 57 64

21 43 58 73 88

0 7 18 31

0 6 15 27 42

298 (72%) 416 (6.8%) 448 (48%)

299 (73%) 417 (74%) 449 (52%)

300 (100%) 418 (100%) 450 (100%)

301 (47%) 419 (52%) 451 (69%)

302 (58%) 420 (58%) 452 (90%)

303 (8%) 421 (12%) 453 (36%)

304 (15%) 422 (16%) 454 (44%)

corresponds to the isotope la4Pt.

(A + 5 )

0

(A+6)

EIMS OF PLATINUM ANTICANCER AGENTS

691

473

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a.

inn.

*E+ 05 3.55

P n

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m/z

b. loo

I+0 5 !.44

41 343

8a

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5 60

9

305

I

%!

.r

p a

I

c,

3Q l 3

n

379 @1

91 I

453

20

VZ

C. 100-

473

I+ 0 4 9.50

80Y

305

I

1

60402

Y .r

c,

343

40n

I

,,,. .,,, 100

,

400

200

,

,

,

I

,

.

,

!

n/z

Figure 4. (a) ESI mass spectrum of tetraplatin showing [M + Na] ion cluster. Insert: expansion of the [M + Na]+ ion cluster; (b) CAD spectrum of tetraplatin obtained by increasing the capillary voltage to 175 V and resulting in CAD mass spectrometry at the ESI source; (c) classical tandem mass spectrum of tetraplatin. +

692

G. K. POON, P. MISTRY A N D S. LEWIS

results of a classical MS/MS experiment of the daughters of the cluster at m/z 471 has a similar fragmentation pattern, with ions at m/z 400, 341 and 303 [Fig. qc)]. This spectrum contained additional structural information; low-mass ions associated with the ring structure were observed at m/z 96 ([C6Hl,N]+) and m/z 112 ( C C 6 H d J +). Better sensitivity was obtained for these three platinum compounds using ESI than by FAB ionization mode. The detection limit (with signal to noise level of the order of 20: 1 or better) on a full-scan mass spectrum in the ESI/MS ionization mode is