Cent Eur J Chem • 10(2) • 2012 • 320-326 DOI: 10.2478/s11532-011-0139-y Central European Journal of Chemistry Binuclear copper complexes with non-steroidal anti-inflammatory drugs as studied by electrospray ionization mass spectrometry Research Article Kaja Kowcun1, Magdalena Frańska2, Rafał Frański1* Adam Mickiewicz University, Faculty of Chemistry, 60-780 Poznań, Poland Poznań University of Technology, Institute of Chemistry, 60-965 Poznań, Poland Received 10 August 2011; Accepted November 2011 Abstract: The solutions containing one of the copper salts (CuCl2, Cu(ClO4)2, Cu(NO3)2, and CuSO4) and one of the non-steroidal anti-inflammatory drugs (NSAIDs, ibuprofen, ketoprofen or naproxen) were analyzed by electrospray ionization mass spectrometry Three of the salts, namely CuCl2, Cu(ClO4)2 and Cu(NO3)2, yielded binuclear complexes of drug:metal stoichiometry 1:2 Existence of the complexes of such stoichiometry has not been earlier observed For copper(II) chloride the complexes (ions of the type [M-HCOOH+Cu2Cl]+ and [M+Cu2Cl]+, M stands for the drug molecule) were formed in the gas phase When copper(II) perchlorate or copper(II) nitrate was used, the observed binuclear copper complexes (ions of the type [M-H+Cu2(ClO4)2+CH3OH]+, [M-H+Cu2(ClO4)2]+ and [M-H+Cu2(NO3)2+CH3OH]+, [M-H+Cu2(NO3)2]+) were observed at low cone voltage, thus these complexes must have already existed in the solution analysed Therefore, such complexes may also exist under physiological conditions Keywords: Ibuprofen • Ketoprofen • Naproxen • Copper complexes • Electrospray ionization mass spectrometry © Versita Sp z o.o Introduction Copper(II) complexes with non-steroidal anti-inflammatory drugs (NSAIDs) as ligands have attracted a lot of attention recently, as stressed in the review papers [1-3] The exemplary NSAIDs whose copper complexes have been studied are aspirin and other derivatives of salicylic acid [4-6], niflumic acid [7], tolfenamic acid [8], oxicams [9,10], ketoprofen derivatives [11], diclofenac [12,13], naproxen [13-17], ibuprofen [18-22] Besides aspirin, ibuprofen seems to be the most commonly applied NSAID (paracetamol is the most common drug but it rather cannot be classified as anti-inflammatory agent) [23] Naproxen and ketoprofen have structures similar to that of ibuprofen, these three drugs are aromatic derivatives of propanoic acid substituted at the position, namely ibuprofen - (RS)-2-(4-(2-methylpropyl)phenyl)propanoic acid, naproxen - (+)-(S)-2-(6-methoxynaphthalen-2-yl) propanoic acid and ketoprofen - (RS)-2-(3-benzoylphenyl) propanoic acid (Scheme 1) It is well known that electrospray ionization mass spectrometry (ESI-MS) is a very good tool allowing detection of metal-organic ligand complexes present in solution Furthermore, ESI-MS allows detection of the species present in solution which are difficult to observe by other methods [24] The knowledge of the ions which exist in solution is important from the practical point of view since they can be expected to exist under physiological conditions On the other hand, a number of processes may occur in the gas phase leading to the formation of ions not earlier present in solution The most common processes occurring under ESI conditions are counter ion attachment, deprotonation of coordinated solvent molecules, reduction of metals, that are easily reduced under ESI conditions (e.g Cu2+) Sometimes, under ESI conditions more complicated processes may occur, namely copper reduction may be accompanied by formation of cluster ions [25] or formation of a helical complex [26] Occurrence of these processes may be * E-mail: franski@amu.edu.pl 320 K Kowcun, M Frańska, R Frański desirable, since they allow studies of the interactions between specific chemical individuals in the gas phase in the absence of a solvent The importance of gas-phase studies is described in detail in the excellent paper by Dearden et al [27] The aim of this study is to identify the copper complexes with NSAIDs (namely ibuprofen, ketoprofen and naproxen) by using ESI-MS, thus in the solution as well in the gas phase We have focused on the complexes which have not been earlier observed by other methods CH3 O CH3 H3C OH (RS)-ibuprofen - Ibup O CH3 O OH Experimental procedure The NSAIDs used ((RS)-ibuprofen, (RS)-ketoprofen and (S)-naproxen), have been obtained from SigmaAldrich (Poznań, Poland) All three drugs are chiral compounds, however, for the presented study (the discussed binuclear copper complexes contains only one drug molecule) the chirality is of no importance The ESI mass spectra were obtained on a Waters/Micromass (Manchester, UK) ZQ2000 mass spectrometer (single quadrupole type instrument, Z-spray, software MassLynx V3.5) The sample solutions were prepared in methanol (or methanol/water mixture), at the concentration of drug and copper salt (CuCl2, Cu(ClO4)2, Cu(NO3)2, and CuSO4) of 10-4 mol dm-3 The sample solutions were infused into the ESI source using a Harvard pump at a flow rate of 80 µL min-1 The ESI source potentials were capillary kV, lens 0.5 kV, extractor V and cone voltage (CV) 10-60 V This latter parameter had the most profound effect on the mass spectra obtained Increase in this voltage leads to the so-called “in-source” fragmentation/dissociation, but a too low cone voltage may cause a drop in sensitivity The pressure in this region is about 1.5 Pa, which is low enough to allow an ion a significant mean free path of travel before collision occurs, yet high enough for the probability of collision to be still significant [28] It is possible that collisions may induce formation of some ions [29] The source temperature was 120oC and the desolvation temperature was 300oC Nitrogen was used as the nebulising and desolvating gas at flow-rates of 100 and 300 L h-1, respectively Results and discussion Among the copper salts used, three of them, namely CuCl2, Cu(ClO4)2 and Cu(NO3)2, yielded the unexpected binuclear complexes with NSAIDs The complexes are unexpected because they have drug:metal stoichiometry (RS)-ketoprofen - Ket CH3 O OH H3CO (S)-naproxen - Napr Scheme Non-steroidal used anti-inflammatory drugs (NSAIDs) 1:2, the complexes of such stoichiometry have not been earlier observed The complexes of copper(II) perchlorate were analogical to those observed for copper(II) nitrate, as shown further The attachment of a bivalent counter ion SO42- could lead to the formation of a neutral complex whose detection by ESI-MS is impossible In the ESI mass spectra obtained there was a number of intense peaks assigned to ions which are not subjects of this work For example in the mass spectrum shown in Fig 1a there were the peaks assigned to [Ibup+Cu]+ at m/z 269, [(Ibup)-H+Ca]+ and/or [(Ibup)+K]+ at m/z 451 (potassium and calcium are common contamination), not rationalized peaks at m/z 226 and 253, in Fig [Cu2(ClO4)3+(CH3OH)3]+ at m/z 519, [Cu2(ClO4)2CH3O+CH3OH]+ at m/z 387 (the m/z values correspond to the 63Cu and 35Cl containing ions), in Fig [Ibup-H+Cu+(CH3OH)2]+ at m/z 332, [Ibup-HCOO+Cu+CH3OH]+ at m/z 256 Only the ions containing a drug molecule and two copper atoms (as it always clearly results from a characteristic isotopic pattern) will be discussed here 3.1 Binuclear copper complexes derived from CuCl2 Fig shows the ESI mass spectrum of methanol solution containing ibuprofen and CuCl2 obtained at cone voltage 40 V (CV=40V, for clarity the spectrum in Fig 1a is shown in the m/z range 200-500) At this value both binuclear copper complexes observed, namely ions [Ibup-HCOOH+Cu2Cl]+ m/z 321 and [Ibup+Cu2Cl]+ 321 Binuclear copper complexes with non-steroidal anti-inflammatory drugs as studied by electrospray ionization mass spectrometry m/z 367 are characterized by the highest abundances (the m/z values correspond to the 63Cu and 35Cl containing ions) Analogically to ibuprofen-containing solution, the observed binuclear copper complexes for ketoprofencontaining solution and naproxen-containing solution were the ions [Ket-HCOOH+Cu2Cl]+ m/z 369, [Ket+Cu2Cl]+ m/z 415 and [Napr-HCOOH+Cu2Cl]+ m/z 345, [Napr+Cu2Cl]+ m/z 391 as shown in supplemental material The question is how the above binuclear ionic complexes (ions of the type [M-HCOOH+Cu2Cl]+ and [M+Cu2Cl]+, Fig 1) were formed They definitely cannot exist in solution Thus, they are formed in the ESI conditions It may be assumed that ions [M-HCOOH+Cu2Cl]+ were formed from ions [M+Cu2Cl]+ as a result of HCOOH molecule loss If it is true, the abundance ratio [M-HCOOH+Cu2Cl]+/[M+Cu2Cl]+ should increase with cone voltage increase, however it was not observed Fig shows the breakdown plots of the abundances of the [Ibup-HCOOH+Cu2Cl]+ and [Ibup+Cu2Cl]+ ions against the cone voltage as an example As can be clearly interpreted from Fig 2, the ratio [Ibup-HCOOH+Cu2Cl]+/[Ibup+Cu2Cl]+ does not increase with the cone voltage increase (although it is not constant) Thus it is reasonable that ions of the type [M-HCOOH+Cu2Cl]+ and [M+Cu2Cl]+ are formed independently in the region where the cone voltage is applied Formation of these ions is a consequence of copper reduction (formally the ions contain two Cu(I) atoms) and ion-molecule collision (reaction) The respective collisions should occur between Cu2Cl+ ion and neutral drug molecule M, as well as between Cu2Cl+ ion and neutral M-HCOOH species The ions Figure 322 observed at lower m/z value confirm this assumption As shown in Fig 3, the cone voltage increase yielded abundant ion Cu2Cl+, thus one of the reagents It is obvious that a considerable part of the drug used is not ionized in the ESI condition, thus at higher cone voltage the collisions between Cu2Cl+ ion and the neutral drug molecule M occur producing [M+Cu2Cl]+ ions As shown in Fig 3, the cone voltage increase leads to the formation of abundant [Ket-HCOO]+ ion at m/z 209 (analogical ions were observed for ibuprofen and naproxen, [Ibup-HCOO]+ m/z 161 and [Napr-HCOO]+ m/z 185, they are typical of NSAIDs [30-32]) One of the possible formation pathways (obviously not the only one) of this ion is a simple reaction: [Ket+H]+ → [Ket-HCOO]+ + HCOOH Theoretically, the competitive reaction is: [Ket+H]+ → Ket-HCOOH + [HCOOH+H]+ Which of these reactions occurs with higher efficiency depends on the proton affinities of Ket-HCOOH and HCOOH but this problem will not be discussed here The m/z value of the ion [HCOOH+H]+ is too low for typical ESI-MS experiment thus this ion was not detected (usually ESI mass spectra are recorded from m/z 100) Anyway, the second reaction leads to the formation of neutral Ket-HCOOH species (analogical reactions lead to the formation of analogical species for ibuprofen and naproxen) Collision/reaction of neutral M-HCOOH species with Cu2Cl+ ions leads to the formation of ions of the type [M-HCOOH+Cu2Cl]+ It has to be stressed that both ions discussed [M-HCOOH+Cu2Cl]+ and [M+Cu2Cl]+ were also formed ESI mass spectrum of methanol solution containing ibuprofen and CuCl2 [Ibup+Cu2Cl]+ m/z 367, [Ibup-HCOOH+Cu2Cl]+ m/z 321 (in all figures captions the m/z values correspond to the 63Cu and 35Cl containing ions) K Kowcun, M Frańska, R Frański Figure The abundances of the [Ibup-HCOOH+Cu2Cl]+ and [Ibup+Cu2Cl]+ ions versus the cone voltage as an example The absolute ion abundances (in arbitrary units) taken from mass spectra recorded were divided by 106 Figure ESI mass spectra of methanol solution containing ketoprofen and CuCl2 at lower m/z range, [Cu2Cl]+ m/z 161, [Ket+H]+ m/z 255, [Ibup-HCOOH]+ m/z 209 323 Binuclear copper complexes with non-steroidal anti-inflammatory drugs as studied by electrospray ionization mass spectrometry Figure ESI mass spectrum of methanol solution containing ibuprofen and Cu(ClO4)2 [Ibup-H+Cu2(ClO4)2 + CH3OH]+ m/z 561, [Ibup- H+Cu2(ClO4)2]+ m/z 529 when a mixture of methanol/water (1/1) was used as a solvent instead of pure methanol (as it is expected for the ions formed in the gas phase) 3.2 Binuclear copper complexes derived from Cu(ClO4)2 and Cu(NO3)2 When copper(II) perchlorate was used instead of copper(II) chloride the binuclear copper complexes were also observed but they were not analogical to that detected for copper chloride The observed ions were of the type [M-H+Cu2(ClO4)2+CH3OH]+ and [M-H+Cu2(ClO4)2]+ Fig shows the ESI mass spectra of methanol solution containing ibuprofen and Cu(ClO4)2 as representative examples The spectra obtained for the other NSAIDs are shown in supplemental material The abundance ratio [M-H+Cu2(ClO4)2]+/ [M-H+Cu2(ClO4)2+CH3OH]+ increases with increasing cone voltage Thus, as expected, ions [M-H+Cu2(ClO4)2+CH3OH]+ lose methanol molecule producing ions [M-H+Cu2(ClO4)2]+ 324 and The ions [M-H+Cu2(ClO4)2+CH3OH]+ + [M-H+Cu2(ClO4)2] formally contain two Cu(II) atoms, thus they are not formed as a result of copper reduction Furthermore, the ions [M-H+Cu2(ClO4)2+CH3OH]+ and [M-H+Cu2(ClO4)2]+ were most abundant at a low cone voltage 10-20 V, thus they are unstable in gas (at cone voltage 40 V and higher they were not detected) It may be assumed that the ions exist already in solution If the assumption is true, the solvent composition should strongly affect their formation As shown in Fig 5, the use of a mixture methanol/water (1/1) as a solvent instead of pure methanol gave the ions [M-H+Cu2(ClO4)2+CH3OH]+ and [M-H+Cu2(ClO4)2]+ characterized by very low signal to noise ratio When copper nitrate was used, the binuclear copper complexes were observed and they were analogical to those detected for copper perchlorate The observed ions were of the type [M-H+Cu2(NO3)2+CH3OH]+ and [M-H+Cu2(NO3)2]+ Fig shows the ESI mass spectrum of a methanol solution containing ibuprofen and Cu(NO3)2 K Kowcun, M Frańska, R Frański Figure ESI mass spectrum of methanol/water (1/1) solution containing ibuprofen and Cu(ClO4)2 [Ibup-H+Cu2(ClO4)2+CH3OH]+ m/z 561, [Ibup-H+Cu2(ClO4)2]+ m/z 529 Figure ESI mass spectrum of methanol solution containing [Ibup-H+Cu2(NO3)2]+ m/z 487 as representative examples The spectra obtained for the other NSAIDs are shown in supplemental material The behaviour of the ions [M-H+Cu2(NO3)2+CH3OH]+ and [M-H+Cu2(NO3)2]+ was analogical to that of ions [M-H+Cu2(ClO4)2+CH3OH]+ and [M-H+Cu2(ClO4)2]+ Ions [M-H+Cu2(ClO4)2+CH3OH]+ lose methanol molecule producing ions [M-H+Cu2(ClO4)2]+, they are unstable in the gas phase and their formation is strongly affected by the solvent composition Conclusion Formation of binuclear copper complexes with NSAIDs strongly depends on the counter ion present For copper(II) chloride the complexes (ions of the type [M-HCOOH+Cu2Cl]+ and [M+Cu2Cl]+) were formed in the gas phase, in the region where the cone voltage is applied Their formation is an effect of collision/reaction ibuprofen and Cu(NO3)2 [Ibup-H+Cu2(NO3)2+CH3OH]+ m/z 455, of neutral drug molecules M or neutral M-HCOOH species with Cu2Cl+ ions This finding is important from fundamental/scientific point of view, since it informs us about the interaction between the drug molecule and copper containing species in the absence of a solvent When copper(II) perchlorate or copper(II) nitrate was used the observed binuclear copper complexes (ions of the type [M-H+Cu2(ClO4)2+CH3OH]+, [M-H+Cu2(ClO4)2]+ and [M-H+Cu2(NO3)2+CH3OH]+, [M-H+Cu2(NO3)2]+) were different from those observed for copper(II) chloride The observed ions ware found to be unstable in the gas phase It is reasonable to assume that the ions exist already in solution, which may be of importance from the practical/pharmaceutical point of view Obviously, the parameters of the solution used for ESI experiments are different from physiological conditions, however, the presence of such ions in physiological solutions cannot be excluded 325 Binuclear copper complexes with non-steroidal anti-inflammatory drugs as studied by electrospray ionization mass spectrometry References [1] C.T Dillon, T.W Hambley, B.J Kennedy, P.A Lay, J.E Weder, Q Zhou, Met Ions Biol Syst 41, 253 (2004) [2] J.E Weder, C.T Dillon, T.W Hambley, B.J Kennedy, P.A Lay, J.R Biffin, H.L Regtop, N.M Davies, Coord Chem Rev 232, 95 (2002) [3] N.E.A El-Gamela, J Coord Chem 62, 2239 (2009) [4] A.L Abuhijleh, Inorg Chem Commun 14, 759 (2011) [5] N.I Jakab, Z Vaskova, J Moncol, B Gyurcsik, J Šima, M Koman, D Valigura, Polyhedron 29, 2262 (2010) [6] V Brumas, B Brumas, G Berthon, J Inorg Biochem 57, 191 (1995) [7] F.T Greenaway, E Riviere, J.J Girerd, X Labouze, G Morgant, B Viossat, J.C Daran, M.R Arveiller, N.-H Dung, J Inorg Biochem 76, 19 (1999) [8] D Kovala-Demertzi, D Hadjipavlou-Litin, A Primikiri, M Staninska, C Kotoglou, M A Demertzis, Chem Biodivers 6, 948 (2009) [9] R Cini, G Tamasi, S Defazio, M B Hursthouse, J Inorg Biochem 101, 1140 (2007) [10] S Roy, R Banerjee, M Sarkar, J Inorg Biochem 100, 1320 (2006) [11] D.K Saha, S Padhye, S Padhye, Metal-Based Drugs, 8, 73 (2001) [12] A Theodorou, M.A Demertzis, D Kovala-Demertzi, E.E Lioliou, A.A Pantazaki, D.A Kyriakidis, BioMetals 12, 167 (1999) [13] F Dimiza, F Perdih, V Tangoulis, I Turel, D.P Kessissoglou, G Psomas, J Inorg Biochem 105, 476 (2011) [14] Y.C Zhu, S.C Yan, X.N Dong, G.F Zuo, J.G Wu, R.W Deng, Chemical Papers 57, 87 (2003) [15] M.Q Zhang, Y.C Zhu, J.G Wu, P Shi, R.W Deng, Z.N Chen, Chemical Papers 55, 202 (2001) 326 [16] A.L Abuhijleh, J Khalaf, European J Med Chem 45 3811 (2010) [17] S Yaqub, Ihsan-Ul-Haqb; S Ali, B Mirza, F Ahmed, S Shahzadi, J Coord Chem 62, 3463 (2009) [18] A Trinchero, S Bonora, A Tinti, G Fini, Biopolymers 74, 120, (2004) [19] A.L Abuhijleh, J Inorg Biochem 55, 255 (1994) [20] C.R Gordijo, C.A.S Barbosa, A.M Da Costa Ferreira, V.R.L Constantino, D De Oloveira Silva, J Pharm Sci 94, 1135 (2005) [21] P.-N Ahn-Thou, P Lemoine, B Viossat, J.-C Chaumeil, A Tomas, Z Kristallogr NCS 222, 93 (2007) [22] A Andrade, S.F Namora, R.G Woisky, G Wiezel, R Najjar, J.A.A Sertie´, D De Oliveira Silva, J Inorg Biochem 81, 23 (2000) [23] J Beausse, Trends Anal Chem 23, 753 (2004) [24] R Frański, B Gierczyk, J Incl Phenom Macrocycl Chem 62, 339 (2008) [25] C Hao, R.E March, J Mass Spectrom 36, 509 (2001) [26] M Wałęsa-Chorab, V Patroniak, G Schroeder, R Frański, Eur J Mass Spectrom 16, 163 (2010) Dearden, Y Liang, J.B Nicoll, [27] D.V K.A Kellersberger, J Mass Spectrom 36, 989 (2001) [28] R.B Cole, J Mass Spectrom 35, 763 (2000) [29] R Frański, Rapid Commun Mass Spectrom 25, 672, (2011) [30] I Ferrer, E.M Thurman, Anal Chem 77, 3394 (2005) [31] K.B Borges, A.R.M De Oliveira, T Barth, V.A.P Jabor, M.T Pupo, P.S Bonato, Anal Bioanal Chem 399, 915 (2011) [32] H Harada, T Endo, Y Momose, H Kusama, Rapid Commun Mass Spectrom 23, 564 (2009) ... [Ibup-HCOOH]+ m/z 209 323 Binuclear copper complexes with non- steroidal anti- inflammatory drugs as studied by electrospray ionization mass spectrometry Figure ESI mass spectrum of methanol solution... physiological solutions cannot be excluded 325 Binuclear copper complexes with non- steroidal anti- inflammatory drugs as studied by electrospray ionization mass spectrometry References [1] C.T Dillon,... both binuclear copper complexes observed, namely ions [Ibup-HCOOH+Cu2Cl]+ m/z 321 and [Ibup+Cu2Cl]+ 321 Binuclear copper complexes with non- steroidal anti- inflammatory drugs as studied by electrospray