matrix-assisted laser desorption/ionization, nanostructure-assisted laser desorption/ionization and...

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ISSN: 1469-0667 © IM Publications LLP 2014doi: 10.1255/ejms.1302 All rights reserved

EUROPEAN JOURNALOFMASSSPECTROMETRY

Matrix-assisted laser desorption/ionization, nanostructure-assisted laser desorption/ionization and carbon nanohorns in the detection of antineoplastic drugs. 1. The cases of irinotecan, sunitinib and 6-alpha-hydroxy paclitaxelEleonora Calandra,a,b Sara Crotti,a,b,c Marco Agostini,a,c,d Donato Nitti,c Marco Roverso,e Giuseppe Toffoli,b Elena Marangon,b Bianca Posoccob and Pietro Traldia,f*aIstituto di Ricerca Pediatrica, Città della Speranza, Corso Stati Uniti 4, 35127 Padova, Italy. E-mail: [email protected] and Clinical Pharmacology Unit, Centro di Riferimento Oncologico, IRCCS National Cancer Institute, Via Franco Gallini 2, 33081 Aviano, ItalycSurgical Clinic, Department of Surgical, Oncological and Gastroenterological Sciences, University of Padova, Via Nicolo Giustiniani 2, 35128 Padova, ItalydDepartment of Nanomedicine, The Methodist Hospital Research Institute, 6670 Bertner Avenue, Houston, 77030 Texas, USAeDipartimento di Medicina, Università degli Studi di Padova, Via Giustiniani 2, I-35100 Padova, ItalyfIENI CNR, Corso Stati Uniti 4, 35127 Padova, Italy

The development of surface-assisted laser desorption/ionization (SALDI) methodologies in mass spectrometry allows, in principle, the development of new analytical approaches to qualitative and quantitative measurements on small molecules. Some of these methods have been applied to characterize two antineoplastic drugs: irinotecan (1) and sunitinib (2), and also 6-a-hydroxy-paclitaxel (3), the main metabolite of paclitaxel. Three different SALDI approaches have been tested employing nanostructure-assisted laser desorption/ionization (NALDI), carbon nanohorns (NHs) and carbon NHs covered by liquid additives. The results so obtained have been compared to those observed under matrix-assisted laser desorption/ionization (MALDI) conditions. Compounds 1 and 2 show the easy formation of protonated molecular species under all the experimental conditions, but the highest absolute intensity was achieved by NALDI. On the contrary, ionic species of low intensity are present for 3, among which are those that exhibit the highest intensity caused by [M + K]+ ions. After a critical evaluation of the obtained data, the linear response of the [M + H]+ ion intensity of 1 versus different deposited sample amounts was investigated, and the best results (R2 = 0.9889) were obtained under MALDI conditions. The analysis of plasma samples spiked with 1 showed, again, that the MALDI approach was the best one (R2 = 0.9766). The failure of NALDI measurements could be rationalized by the presence of ion-suppression effects.

Keywords: matrix-assisted laser desorption/ionization; surface-assisted laser desorption/ionization; nanostructure-assisted laser desorption/ionization; carbon nanohorns; therapeutic drug monitoring

E. Calandra et al., Eur. J. Mass Spectrom. 20, 445–459 (2014)Received: 14 November 2014 ■ Revised: 13 January 2015 ■ Accepted: 13 January 2015 ■ Publication: 21 January 2015

446 MALDI, NALDI and Carbon Nanohorns in the Detection of Irinotecan, Sunitinib and 6-a-Hydroxy Paclitaxel

IntroductionTherapeutic drug monitoringAntineoplastic drugs are usually dosed according to the patient’s body surface area, without considering the possible simultaneous presence of other drugs or individual factors that affect absorption, distribution, metabolism and excretion. As a consequence, a wide variation in drug plasma concen-tration can occur, leading to an over- or under-drug dosage in some patients. The effectiveness of antineoplastic drugs in relation to the tumor response to treatment or to patient overall survival remains low1 and the possibility to achieve effective drug plasma concentrations remains a key issue in anticancer chemotherapy. To overcome intra- and inter-individual variability in drug plasma concentrations, for a real personalization of the drug treatment, therapeutic drug moni-toring (TDM) is required.2 Nevertheless, TDM usage in clinical practice is limited by the complexity of this analysis, which is time consuming, expensive and needs dedicated resources. Then new strategies for TDM are requested.

Enzyme-linked immunosorbent assay (ELISA)3 should be the most rapid and inexpensive way to perform TDM. However, the low specificity of ELISA frequently causes cross-reactions among similar structures (e.g., the parent drug and its active/inactive metabolites) and gives rise to alterations in the quan-titative results.4 To our knowledge, only an anti-taxol antibody is commercially available (Abcam, Cambridge, UK), giving an evaluation of taxol and its metabolite levels.

Actually, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is used to perform pharmacoki-netic studies thanks to its sensitivity and specificity.5 Anyway, this technique suffers some limitations because of the neces-sity for sample preparation before analysis (e.g., pre-analytical steps including internal standard introduction, sample extrac-tion, purification procedures and the development of suit-able chromatographic methods) and requires a high operator expertise for optimization of the instrumental parameters and for data interpretation.

Consequently, the development of fast and alternative analytical methods for TDM is surely of interest. In this frame, laser desorption/ionization (LDI) techniques seem to be highly promising, in particular when based on surface-activated methods using nano substrates [surface-assisted laser desorption/ionization (SALDI)].6 It is known that under matrix-assisted laser desorption/ionization (MALDI) conditions the low mass range of the spectrum is covered, to some extent, by matrix ions and consequently the detection/quantification of low molecular weight compounds, such as antineoplastic drugs, becomes a difficult task. To overcome this problem, two possible approaches have been proposed. The first, proposed by van Kampen et al.7 is based on the use of a high molecular weight matrix, [meso-tetrakis(pentafluorophenyl)porphyrin], which under laser irradiation does not lead to any fragment ions at low m/z values. For the ion formation of the drug, cati-onization with Li+ was employed. After the development of an exhaustive protocol and of a suitable purification procedure,

the method was successfully employed for the quantifica-tion of human immunodeficiency virus protease inhibitors in peripheral blood mononuclear cells.7

The second approach, based on laser irradiation of the drug deposited on nanostructures without the presence of “classic” matrices used in MALDI experiments, has led to a really wide scientific production, proving its effectiveness.8–12 The chem-ical and physical aspects of these methods were recently reviewed by Stolee et al.6 Summarizing, it has been shown that when the nanostructure dimensions are commensurate with the laser wavelength, a series of phenomena is observed that reflects an enhanced desorption/ionization of organic and inorganic compounds. These phenomena (optical, elec-tronic, vibrational, thermal, mechanical and chemical ones) originate by rapid energy deposition inducing highly non-linear processes. In other words, the analogous dimensions of photon wavelength and nanostructures lead to a severe reduc-tion in the diffusive phenomena present in “classic” MALDI experiments: the laser energy in the case of the nanosystem remains “trapped” inside the nanostructure, leading to the above-cited phenomena which are absent, or at least strongly reduced, under MALDI conditions. Furthermore, considering the nanostructure dimensions (in the range of 1–500 nm) and the applied extraction voltages (in the order of 104 V), this results in strong electrical fields (E = 104 V/100 × 10–7 cm = 104 V/10–5 cm = 109 V cm–1) being present that are able to induce field ionization/desorption phenomena.13

Nanosystems of different chemical nature and with different morphologies have been tested. Thus, nanostructures based on porous silicon (desorption/ionization on silicon)9,10,12,14,15 structured as microcolumns,16,17 nanodots,18 nanowires,9,19 nanoparticles,20 and nanofilms21 have been proposed. Alternatively to porous silicon, many other materials have been studied, among which are Au,21,22 Ag,23 Ge,24,25 C26–29 and Pt.30 In particular, graphite-assisted laser desorption/ioniza-tion26–29, 31 found interesting applications.

Besides avoiding low molecular weight ionic species origi-nating from “classic” MALDI matrices, in some cases with nanostructures a significant increase of sensitivity (detection limit of 800 × 10–24 mol!)32 has been observed.

A further point that has to be stressed is that in some studies effective results have been obtained by covering the nanostructures with suitable compounds, such as glycerol (G) and thioglycerol (T).8,33 In this framework, the role of water acidity and surface morphology has been discussed by Chen et al.34 We emphasize that these approaches are similar to that employed by Tanaka et al. in an early paper on MALDI:35 in that case a mixture of glycerol and powdered cobalt was employed as the matrix and the cobalt crystals could be, in principle, considered as the “nano” substrate. Furthermore, it must be considered that the thin layer of glycerol and thioglycerol on the nanosystem could induce ionization mechanisms analo-gous to those present under fast atom bombardment (FAB) conditions.36

E. Calandra et al., Eur. J. Mass Spectrom. 20, 445–459 (2014) 447

The aim of this investigation was to evaluate the results that can be achieved by some of the above-described approaches in the characterization of two antineoplastic drugs, irinotecan (1), employed in presence of colorectal cancer37 (see Figure 1), and sunitinib (2), employed for the treatment of gastrointes-tinal stromal tumors and advanced and/or metastatic renal cell carcinoma37 (see Scheme 1), and also of 6-a-hydroxy paclitaxel (3), the main metabolite of paclitaxel, which is employed in the presence of advanced ovarian cancer, breast cancer and non-small cell lung cancer37 (see Scheme 2).

The measurements were performed under the following conditions:(1) MALDI, by using a-cyano-4-hydroxycinnamic acid (CHCA)

as matrix;(2) NALDI (nanostructure-assisted laser desorption/ioniza-

tion), using a commercially available silicon target;(3) SALDI on carbon nanohorns (NHs), whose structure and

dimension are described in the experimental section;(4) SALDI on carbon NHs covered with different compounds

[G, T, polyphenyl ether (PPE)].As will be seen, different compounds exhibit different ionic

yields and different fragmentation phenomena under the different conditions, and the investigation allowed us to iden-tify the best experimental conditions for the development of analytical procedures devoted to their detection. This inves-tigation can be considered the preliminary step necessary to develop a TDM method for the evaluation of the concentrations of 1, 2 and 3 in plasma of patients treated with 1 and 2 and paclitaxel (3).

ExperimentalMaterialsIrinotecan (1) and sunitinib (2) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA), while 6-a-hydroxy pacli-taxel (3) was supplied by Toronto Research Chemicals, Inc. (North York, Ontario, Canada). For each standard, 0.7 mg mL–1 solutions were prepared. For 1 and 2, water was used as the solvent, and for 3 acetonitrile was used in order to overcome the problem of its poor solubility in water.

InstrumentationMALDI, NALDI and SALDI MS measurements were performed using an UltrafleXtreme MALDI-TOF instrument (Bruker Daltonics, Bremen, Germany) equipped with a 1 kHz Smartbeam II laser (l = 355 nm) operating in the reflectron positive- and negative-ion mode. For the positive-ion mode the instrument conditions were: ion source 1 (IS1) = 25.00 kV, ion source 2 (IS2) = 22.40 kV, lens = 8.00 kV, reflectron poten-tial = 26.45 kV, reflector 2 = 13.45 kV, delay time = 120 ns. External mass calibration was based on the monoisotopic values of [2M + H]+ of CHCA at m/z 379.0925 and the [M + H]+ of bradykinin (1-7), angiotensin II, angiotensin I, substance P and bombesin at m/z 757.3992, 1046.5420, 1296.6853, 1347.7361 and 1619.8230, respectively (Peptide Calibration Standard, Bruker Daltonics, Bremen, Germany). For the negative-ion mode, the instrumental conditions were IS1 = 20.00 kV, IS2 = 17.85 kV, lens = 6.15 kV, reflectron potential = 21.15 kV, reflector 2 = 10.75 kV, delay time = 80 ns. The external mass calibration was based on the monoisotopic values of [2M – H]– of CHCA at m/z 377.0779 and the [M – H]– of bradykinin (1-7), angiotensin II, angiotensin I, substance P and bombesin at m/z 755.3834, 1044.5272, 1294.6702, 1345.7208 and 1617.8077, respectively (Peptide Calibration Standard, Bruker Daltonics, Bremen, Germany).

Figure 1. Structure of irinotecan (1).

Scheme 1. Structure of sunitinib and fragments produced by laser irradiation.

Scheme 2. Structure of 6-a-hydroxy paclitaxel and fragments produced by laser irradiation.


MALDI matricesThree different MALDI matrices were evaluated: CHCA (saturated solution), sinapinic acid (SA, saturated solution) and 2,5-dihydroxybenzoic acid (DHB, 10 mg mL–1). For the matrices the same solvent was employed: water/acetoni-trile (50:50, v/v) containing 0.1% (v/v) trifluoroacetic acid. Matrices were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

NALDI targetsNALDI targets were steel plates covered by a layer of inorganic silicon nanostructures, commercially available from Bruker Daltonics (Bremen, Germany).

SALDI surfacesSALDI surfaces were prepared by depositing 1 µL of a 10 mg mL–1 suspension in water/methanol (50:50, v/v) of single-wall carbon NHs (Carbonium, Padova, Italy), with the shape and dimensions reported in Figure 2, on the stainless steel sample holder and left to dry at room temperature. For the 10 µg so deposited as a 4.2 mm2 spot and considering a monolayer distribution of NH, a maximum active surface of 25-30 cm2 becomes available.

NH coating proceduresFor NH coating, 0.7 µL of solutions containing glycerol (Sigma-Aldrich, St. Louis, Missouri, USA), thioglycerol (Sigma-Aldrich, St. Louis, Missouri, USA) or 1,3-bis(3-phenoxyphenoxy)benzene (PPE) (Santovac 5, Santo Lubes LLC, St. Charles, Missouri, USA) at 3% (v/v) were deposited on the previously formed NH surface. For the first two compounds methanol was used as solvent, while for the third diethyl ether was employed.

Analytical sample preparationFor MALDI MS analysis 5 µL of 700 µg mL–1 of irinotecan, suni-tinib and 6-a-hydroxy paclitaxel (1, 2 and 3, respectively) solu-tions were added to 5 µL of a saturated solution of CHCA. Two microliters of these solutions were deposited on the sample holder and left to dry at room temperature.

For NALDI MS analysis, 1 µL of 70 µg mL–1 of irinotecan, sunitinib and 6-a-hydroxy paclitaxel (1–3) were deposited on the sample holder and left to dry at room temperature.

For NH SALDI MS analysis, 10 µg of NH were deposited on the sample holder; on the surface so obtained 1 µL of solution of each drug was deposited.

For the NH-covered SALDI MS experiments, 0.7 µL of glyc-erol, thioglycerol or PPE solutions, prepared as described above, were deposited on the NH surface and left to dry at room temperature. On the surfaces so obtained, 1 µL of solu-tion of 1–3 were deposited and left to dry.

Analyses of spiked plasma samplesFasting blood samples (5 mL) from healthy subjects were collected in BD Vacutainer K2-EDTA tubes and centrifuged at 1800g for 10 min. The plasma samples so obtained (about 2.7 mL) were divided into aliquots (100 µL each) and frozen at −80°C until analysis. All the healthy subjects gave their fully informed consent.

Each plasma aliquot (100 µL) was spiked with 5 µL of diluted solutions of 1 to obtain final concentrations of irinotecan of 500, 1000, 1500, 2000, 2500 and 3000 ng mL–1. The samples so prepared were incubated for 1 h at 4°C.

For plasma deproteinization, 400 µL of methanol containing 0.1% formic acid (v/v) was mixed with 105 µL of spiked plasma samples and vortexed for 10 s, followed by centrifugation at 15,000g for 10 min at 4°C. The supernatant was then trans-ferred to another vial. To perform the MALDI analyses, each deproteinized spiked plasma sample was mixed in a 1:1 (v/v) ratio with a saturated solution of CHCA, and 1 µL of this mixture was deposited on the sample holder and left to dry at room temperature.

Results and discussionThe development of fast and reliable detection methods for a semi-quantitative assay of the TDM of antineoplastic drugs is necessary to optimize the therapeutic dose. For this aim we tested different mass spectrometric approaches, based on laser irradiation of the sample, leading to a fast analytical method.

Compounds 1–3 were firstly analyzed by MALDI using CHCA, DHB and SA as matrices. The best results in terms of the abso-lute intensity (a.i.) of molecular ionic species were obtained using CHCA; the spectra so obtained for 1–3 are reported in Figure 3. We emphasize that equal weights of 1–3 were depos-ited on the sample holder in the presence of equal amounts of matrix. Considering the dimensions of the MALDI sample spot [see Figure 4(b)], the surface sample densities of 1, 2 and 3

Figure 2. Graphic representation and dimensions of single wall carbon nanohorns (NH). (Carbonium, Padova, Italy, technical literature).


(considering a homogeneous sample distribution on the matrix surface) are 70 ng mm–2, corresponding to 118 pmol mm–2 for 1, 174 pmol mm–2 for 2 and 80 pmol mm–2 for 3. However, it is well known that the MALDI surface obtained by drying the matrix:analyte solution is not homogeneous because of the different solubility product constant (Kps) of the matrix and the analyte.38 What is generally (but not always!) observed is that the best matrix/analyte molar ratio, leading to the best signal of the analyte molecular ionic species, is present in the outer circle close to the sample drop limit. Consequently, in order

to obtain a “mean” spectrum of the different compounds, a manually driven “roman grating” method was employed by irradiating the samples in the nine points shown in Figure 4. In order to evaluate the reproducibility of the a.i., the measure-ments were performed in triplicate (three different samples prepared and analyzed on three different days). The data so obtained are reported in Table 1 together with the coefficients of variation [CVs (%)].

It is at first sight evident from Figure 3 that 1 and 2 lead to intense [M + H]+ ions at m/z 587 and m/z 399, respectively, with an a.i. of about 9 × 104 and 4 × 104, respectively. Only fragment ions with very low intensities (in the order of 103 a.i.) are present. In the case of 1 the ion at m/z 543 results from the primary loss of CO2. More intense are the ions at m/z 586 and m/z 585, which could result from the primary losses of H• and H2 from the [M + H]+ ion; alternatively, it can be consid-ered that they originate during the ionization phase, with the formation of an odd electron molecular ion, M+• (m/z 586) and a further H• (m/z 585) loss. For compound 2 the ions at m/z 283 and m/z 326 have been already described in the literature39 as originating by cleavages 1 and 2 of Scheme 1. Both these fragmentation pathways involve the loss of neutral moieties, in agreement with Mandelbaum’s “even electron” rule,40 indicating that from even electron ionic species, as [M + H]+ ions are, the loss of neutrals is highly favored from the thermodynamic point of view. For the ions at m/z 306 and m/z 340 a structural assignment cannot be given, suggesting:

Figure 3. MALDI mass spectra of irinotecan (1), sunitinib (2) and 6-a-hydroxy paclitaxel (3) in positive-ion mode using CHCA as the matrix. Seven hundred nanograms of each compound were deposited.

Figure 4. Graphic representation with the area and the imagi-nary roman grating of sample spots for (a) NALDI, NH (and NH + additives) conditions and (b) the MALDI condition.

Table 1. Absolute intensities and CVs of molecular ionic species obtained under MALDI conditions, using CHCA as the matrix and depositing 700 ng of compounds 1–3.

Compounds Ionic species m/z values a.i. CV (%)Irinotecan (1) [M + H]+ 587 88,636 7

[M + Na]+ 609 1016 4 [M + K]+ 625 106 9

Sunitinib (2) [M + H]+ 399 36,272 5 [M + Na]+ 421 326 7 [M + K]+ 437 135 11

6-a-hydroxy paclitaxel (3) [M + H]+ 870 – –[M + Na]+ 892 759 12 [M + K]+ 908 1001 9


(1) the presence of contaminants or (2) the presence of severe structural rearrangements.

Compound 3 behaves in a completely different way. In the related MALDI spectrum (see Figure 3) the most intense ion at m/z 568 results from the [3CHCA + H]+ cluster of the matrix,41 undetectable in the spectra of 1 and 2, indicative of an ion suppression phenomena. As molecular species of 3, only the [M + Na]+ and [M + K]+ ions are present (which, in the cases of 1 and 2, are also present, but at very low inten-sity) at m/z 892 and m/z 908, respectively. Fragment ions at m/z 485 and m/z 607 originate, as described in literature,42 from cleavage 1 of the [M + Na]+ ion (see Scheme 2). The Na+ ion can be “trapped” in different sites of the molecule. In fact, cleavage 1 gives rise to the two complementary ions at m/z 308 and m/z 607. The former corresponds to [a + Na]+ species, while the latter is from the [M – a + Na]+ ion. The ionic species at m/z 607 is the precursor of another fragmentation pathway through the loss of benzoic acid, leading to the ion at m/z 485. We emphasize that the a.i. of the molecular species of compound 3 is lower than those observed for 1 and 2. At first sight, this behavior could be justified considering that the same surface density in the weight of 1–3 (70 ng mm–2) was present and consequently the molar surface density (i.e. moles per mm2) of 3 is lower than those of 1 and 2. This can surely contribute to the lower intensity of molecular species of 3, but cannot justify the difference in a.i. of one order of magnitude. The better results obtained for 1 and 2, in particular for the production of the protonated species, might be justified by the high number of basic sites present in their structures, higher than those present in 3. On the other hand, 3, because of its high complexity, could act as an effective chelating species for K+ and Na+ ions.

Considering the interesting results obtained by the SALDI methods for the analysis of many different compounds, the effectiveness of these approaches was investigated for the qualitative and quantitative determination of 1–3.

The first measurements were based on the commercially available silicon surface NALDI. This approach was first described by Go et al.,9 and showed that a dense array of single-crystal silicon nanowires is a suitable substrate on which to perform SALDI experiments. Valid results were obtained in the characterization of peptides produced by tryptic digestion, as well as synthetic compounds such as tenoxicam, cocaine and endogenous compounds (e.g., oleoylethanolamide) present in biological substrates (human serum and mouse spinal cord tissues).9 This method was called desorption/ionization on silicon; NALDI can be considered as a development based on the Go et al. findings.9,43

Equal amounts of samples 1–3 (70 ng) were deposited on the NALDI silicon surface and irradiated at the same laser power. The results so obtained are reported in Figure 5. Even if the deposited sample quantities were one order of magni-tude lower than those used for MALDI measurements, an increase in the intensity of the molecular species of about one order of magnitude was observed for 1 and 2, while for 3 it was of a lower extent (from 103 to 3 × 103 a.i.; see Table 2).

Interesting results, quite different to those achieved by MALDI, have been obtained. Aside from 3, both 1 and 2 do not show any further formation of cationized ionic species and, for these compounds, [M + H]+ are produced in high yield (compare the data reported in tables 1 and 2). These results are good evidence of the power of NALDI. However, a question arises at this point: how is it possible that a better ionic yield of [M + H]+ of 1 and 2 is achieved in the absence of protonating media in comparison to the MALDI matrices? This result can be justified by the presence of water on the silicon surface. This aspect has been studied by Chen et al.,34 showing that the SALDI yields in [M + H]+ production are strongly reduced only after the removal of physisorbed and chemisorbed water. In the present case, owing to the deposition of water solu-tions of 1 and 2, the presence of quite large amounts of water sorbed on the surface is expected, and are responsible for the [M + H]+ formation from 1 and 2. Compound 3 behaves as in MALDI: [M + H]+ ions are not produced and only [M + K]+ species are detectable; however, also for this compound an increase of the related signal is observed, as reported above (compare the data reported in Tables 1 and 2). The fragmenta-tion processes observed under the MALDI conditions are still present, but in NALDI a new decomposition product is detect-able at m/z 547, originating from the further loss of acetic acid from the species at m/z 607, as described in Scheme 2.

The development of dahlia-structured carbon NHs (see Figure 2) seems to be highly interesting for SALDI experi-

Figure 5. NALDI mass spectra of 1, 2 and 3 in positive-ion mode. Seventy nanograms of each compound were deposited on the NALDI surface.


ments. In fact, the dimension of the dahlia structure makes available a surface for sample deposition greater than that present in nanowires and nanoholes, and this would, in prin-ciple, be reflected in the production of molecular species at a higher yield. In a first set of experiments, the NH surface was

used as it was, without any additives and/or acidification. The partial LDI spectrum of a NH sample is reported in Figure 6(a). As already observed for graphene samples,8 the spec-trum is highly complex, with the presence of wide numbers of ionic species from m/z 600 up to m/z 1300. The series of

Figure 6. LDI mass spectra of NH, in positive-ion mode, without drugs and/or additives in (a) the range between m/z 380 and m/z 1320; (b)–(d) Zooms of the previous spectrum in the ranges related to each tested compound (1–3).

Table 2. Absolute intensities and CVs of molecular ionic species obtained under NALDI conditions. 70 ng of compounds 1–3 were directly deposited on the NALDI plate.

Compounds Ionic species m/z values a.i. CV (%)Irinotecan (1) [M + H]+ 587 307,370 15

[M + Na]+ 609 – –[M + K]+ 625 – –

Sunitinib (2) [M + H]+ 399 631,342 21 [M + Na]+ 421 – –[M + K]+ 437 – –

6-a-hydroxy paclitaxel (3) [M + H]+ 870 – –[M + Na]+ 892 – –[M + K]+ 908 3116 17


ionic clusters, separated by 24 Da, is typical of graphenes and remind one of the data obtained in the case of asphaltenes,44 i.e. the high boiling fraction of oil samples. The m/z ranges of interest for the detection of molecular ionic species of 1–3 are also reported in Figure 6(a). As can be seen, in the case of 1 no interfering species from NH are present [see Figure 6(b)], while for both 2 and 3 the NH sample leads to possibly inter-fering ions [see Figure 6(c) and (d)]. Then, in principle, the NH method seems to be ineffective for the analysis of 2 and 3. But, interestingly, the presence of the analytes strongly reduces the intensities of the ions coming from the NH sample, as shown by the spectra of 1–3 reported in Figure 7, obtained by depos-iting 700 ng of 1–3 on a 10 µg spot of NH.

For compound 1 the [M + H]+ ion is detectable at m/z 587, with an intensity of about 9000 a.i. (see Table 3), while [M + Na]+ and [M + K]+ ions become present at lower intensities (about 120 and 90 a.i., respectively). Some fragment ions are also detectable, such as the species at m/z 543, already detected under MALDI and NALDI conditions, and originating from primary CO2 loss. It is interesting to observe that the [M + H – CO2]+/[M + H]+ ratio is 3/100 in the MALDI, 33/100 in the NALDI and 50/100 in the NH spectra. These results suggest that the CO2 loss can be considered to originate from a thermal process: in the case of MALDI the phonon energy activated by laser irradiation is dissipated on the matrix lattice, while under both NALDI and NH conditions it is concentrated inside the nano system. The fragments at m/z 565 and m/z 579 most probably result from the loss of CO2 from the [M + Na]+ ion and to the loss of H2CO2 from [M + K]+ species, respectively. In the case of compound 2, a behavior different from those observed under MALDI and NALDI conditions occurs. The most intense molecular ionic species is no longer the protonated molecule, but the [M + Na]+ ion. On the contrary, for 3 the same behavior as observed under MALDI and NALDI conditions is present, the most intense ion being caused by [M + K]+ species.

The data obtained for 1 and 2 indicate that in the NH system a protonating medium must be present, responsible for the [M + H]+ production from 1; furthermore, Na and K salts must also be present in quite high concentrations. In fact, the

privileged formation of [M + Na]+ for 2 can be explained by this aspect, together with a Na+ affinity of 2 greater than that of 1, which exhibits a basicity higher than that of 2. In the case of 3, for which the high affinity with respect to K+ was already proposed on the basis of MALDI and NALDI data, the forma-tion of [M + K]+ is further enhanced.

What is to be considered is that under NH conditions a decrease of molecular ionic species intensity is observed for compounds 1 and 2 (see the “NH” column of Table 3). This

Figure 7. SALDI mass spectra of 1, 2 and 3 in positive-ion mode. Seven hundred nanograms of each compound were deposited on the NH surface without additives.

Table 3. Absolute intensities and CVs of molecular ionic species obtained under NH (and NH + additives) conditions. 700 ng of compounds 1–3 were deposited with and without the previous covering of the NH with glycerol, thioglycerol and PPE.

Compounds Ionic species

m/z values

NH NH + G NH + T NH + PPEa.i. CV (%) a.i. CV (%) a.i. CV (%) a.i. CV (%)

Irinotecan (1) [M + H]+ 587 8273 22 5661 49 21,991 33 856 31 [M + Na]+ 609 120 27 1310 37 338 27 193 35 [M + K]+ 625 86 19 1142 41 130 41 643 34

Sunitinib (2) [M + H]+ 399 2272 17 5900 32 144,463 43 1759 37 [M + Na]+ 421 8393 12 141,620 35 116,864 49 166,304 42 [M + K]+ 437 2386 20 30,001 30 15,326 47 23,005 45

6-a-hydroxy paclitaxel (3) [M + H]+ 870 – – – – – – – –[M + Na]+ 892 65 18 128 27 116 45 67 39 [M + K]+ 908 3094 16 5420 26 3473 37 8537 41


behavior may result from the lower surface density of the analytes: the calculated surface for a NH is 250–300 m2 g–1 (carbonium, technical literature), orders of magnitude higher than that typically present on the sample holder surface. This reflects a “dilution” of the sample in different directions on the surface (because of the dahlia structure of NHs) and conse-quently lower quantities of the sample interact with the laser beam.

The NH cationized species intensities of 1–3 are higher than those observed under MALDI conditions, indicating that NHs are more effective in activating cationization reactions. This behavior could be explained by the presence of high amounts of sodium and potassium salts in the NH sample. However, we emphasize that the producer does not use K+ salt treatment, sometimes employed in carbon-based nanosystem prepa-ration, in the production of NH.45 In order to rationalize the observed behavior some inductively coupled plasma–optical emission spectrometric (ICP-OES) measurements were performed on NH samples and the results are summarized in Table 4. Two different batches were analyzed and in both cases the levels of Na and K were quite high, and the potassium level was one order of magnitude higher with respect to that of sodium. These data account for the results obtained under NH conditions and the different behavior observed for 1–3 suggests that sample 3 exhibits a higher affinity for K+ ions, reasonably because of its chelating properties. Under these conditions, compound 1 also leads to intense [M + H]+ species and consequently the presence of protonating agents must also be considered. This could probably result from water present at a trace level in the NH sample or, alternatively, to the reaction of disproportion originating from the 1 dimer, a process observed for MALDI matrix molecules.46

2M M H M Hnhv - +é ù é ù- + +ë û ë û

However, that the [M – H]– ions are completely undetectable in the negative ion mode seems to exclude the latter hypoth-esis and protonation by the reaction with [H3O]+ seems more reasonable.

NH plus additivesThe early papers of Tanaka et al.35 showed that the laser irra-diation of mixtures that constituted powdered cobalt, glycerol and traces of analytes leads to the desorption of molecular ionic species of the analyte itself. Looking at the SALDI data, produced ten years later, it appears that the Tanaka approach is quite similar to the SALDI one, with the powdered cobalt representing, in that case, the nano system. Recently, the

use of glycerol and other additives on the nano surface has been proposed,33 leading to very interesting results, justified by the activation of new chemical channels that result in the analyte ion production. Consequently, it remained of interest to undertake a series of measurements performed by adding different additives on the NH surface, which would reflect different chemical phenomena that lead to ionization. For this aim three different additives were used: glycerol, thioglycerol and PPE (see the Experimental section). In the first two cases what would be expected is a privileged formation of proto-nated species through chemical mechanisms analogous to those obtained under FAB36 conditions. Glycerol and thioglyc-erol exhibit different acidities (pKa values of 14.447 and 9.51,48 respectively) and this necessarily reflects different interactions with analytes. The data obtained under the different experi-mental conditions are summarized in Table 3 [see columns

“NH + G” (NH and glycerol), “NH + T” (NH and thioglycerol) and “NH + PPE”]. The presence of glycerol does not lead to any wide change of the a.i. of the molecular species [M + H]+ of 1. In fact, as reported in Table 3, the [M + H]+ species intensity is practically the same as that observed in the case of NH and lower than those present under MALDI and NALDI conditions. Quite surprisingly, an increase of the [M + Na]+ and [M + K]+ ion intensities appears; this behavior could be caused by the solubility in glycerol of the K and Na salts present in the NH sample (see Table 4) and the consequent higher availability of alkaline ions to react with analyte. This aspect is further confirmed by the behavior of 2, which shows a wide increase of the [M + Na]+ ion intensity (from 8393 a.i. with NH to 141,620 a.i. with NH + G). For compound 3 only a small increase of [M + K]+ ions was observed in the presence of glycerol, while [M + H]+ ions were still completely undetectable.

The results obtained with NH + G can be rationalized by considering that the first step in the experimental set-up consists of the solubilization of Na and K salts present in NH. Then the analytes are solubilized in a glycerol solution rich in Na+ and K+ ions. The cationization reactions, which reasonably occur in the glycerol solution, exhibit a yield higher than that of protonation, which originates in the presence of protonated glycerol. This hypothesis is well supported by the increased production of [M + Na]+ and [M + K]+ for 1, while 2 (whose Na+ affinity was discussed above) shows an intensity increase of more than one order of magnitude. A further increase is also observed for the [M + K]+ ion intensity of 3.

The deposition of thioglycerol on NH leads to quite different results for 1 and 2, showing that the acidity of compounds that cover the surface plays an important role. Thus, for 1 an inten-sity increase of [M + H]+ from 8273 to 21,991 a.i. is present, while for 2 the intensity of [M + H]+ becomes higher than that of [M + Na]+ (144,463 and 116,864 a.i., respectively). The higher acidity of thioglycerol does not lead to significant changes in the spectra of 3, the cationization by K+ remaining the most favored process.

Alternatively to glycerol and thioglycerol, it was considered of interest to perform some experiments using, as the additive to NH, PPE (Santovac 5). We emphasize that PPE was chosen as

Table 4. Composition in Na and K of NH resulting from ICP-OES analyses.

K Namg g–1 mmol g–1 mg g–1 mmol g–1

Batch 1 1.84 0.047 0.27 0.012Batch 2 1.72 0.044 0.21 0.009


a non-protonating agent, with a low ionization energy (IE) (as an example, the IE of diphenyl ether is 8.09 eV49): in this case the MPPE

+• molecular ion should be produced, able to activate charge-exchange reactions; furthermore, the slow electron produced by PPE primary ionization would be available to activate an electron-attachment reaction with the analyte:

+

PPE PPEM M -+<nh

e

A A- -+ <e

In fact, NH + PPE under positive-ion conditions shows mainly the production of MPPE

+•, with a minor contribution of [M + H]+, whereas under negative-ion conditions it shows the formation of odd electron anions MPPE

–•. Hence, by laser irradiation either basic or acidic species can be generated. However, the presence of PPE seems to inhibit the ionization phenomena of 1–3 described above for NH + G and NH + T. For 1, a general decrease of the intensity of molecular ionic species is observed and any odd electron ion (M+•) production is not observed. For 2 and 3 an increase of the intensities of the [M + K]+ ions occurs (see Table 4).

In negative-ion mode only compound 2 gives rise to the [M – H]– ion at m/z 397, whereas for 1 and 3 the possible molecular species (either [M – H]– or M–• ions) are undetectable (see Table 5 and Figure 8).

The results obtained by NH + PPE are not particularly exciting and the processes hypothesized above seem not to be operative. This behavior can be justified, in our opinion, by the lower solubility of 1–3 in PPE, which inhibits the chemical processes obtained in solution with glycerol and thioglycerol.

In conclusion, the above-described data, summarized in the histograms of Figure 9, indicate that:(1) For compound 1 [Figure 9(a)] the operative conditions in

terms of molecular ionic species intensities are achieved under “classic” MALDI conditions, employing CHCA as the matrix; however, the highest [M + H]+ intensity is obtained under NALDI conditions (88,636 a.i. in MALDI and 307,370 a.i. in NALDI spectra). For this compound the formation of [M + Na]+ and [M + K]+ ions is practically negligible.

(2) Compound 2 [Figure 9(b)] shows a highly intense [M + H]+ ion under both MALDI and NALDI conditions. However, under NH, NH + G, NH + T and NH + PPE conditions the most intense species are the [M + Na]+ ones, the [M + H]+ ions being intense only in the case of NH + T. These results indicate that 2 exhibits a high affinity either to H+ or to Na+.

The yields of the related molecular species result from the stoichiometry of the reactant ions: in the case of MALDI, NALDI and NH + T, protonating agents prevail on the Na+ and K+ concentrations. Under negative-ion conditions, by using NH + PPE, compound 2 gives rise to an intense [M – H]– ion and this result could be interesting for its analysis in complex matrices.

(3) Compound 3 [Figure 9(c)] exhibits the highest intensity of [M + K]+ in the case of NH + PPE. However, this cation-ized molecular species is also present in lower intensities under all the other experimental conditions.

What needs to be emphasized is that in the case of an NH substrate a general increase of the molecular ionic species is observed, but also some fragmentation processes are acti-vated. This aspect, already described in the literature,6,17,50,51 is surely of interest from the qualitative point of view, but it must

Table 5. Absolute intensities and CVs of [M – H]–, obtained under SALDI conditions (covering the NH with PPE) are reported. 700 ng of compounds 1–3 were deposited.

Compounds Ionic species m/z values NH + PPEa.i. CV (%)

Irinotecan (1) [M – H]– 585 – –Sunitinib (2) [M – H]– 397 23,980 33

6-a-hydroxy paclitaxel (3) [M – H]– 868 – –

Figure 8. SALDI mass spectra of 1, 2 and 3 in negative-ion mode. Seven hundred nanograms of each compound were deposited on the NH surface using PPE as the additive.


be considered somehow negative for quantitative measure-ments.

Evaluation of linear responseAnalyses of irinotecan and irinotecan-spiked plasma sampleIn order to evaluate the possible use of LDI methods in quan-titative measurements of 1–3 concentrations in plasma, we undertook a preliminary investigation on 1 to determine the possible linear response of the above-described methods with respect to quantities of 1 deposited on different surfaces. Looking at the discussed data summarized in the histo-gram of Figure 9, the first attempts were performed by using NALDI method. It must be emphasized that the data obtained under the NALDI conditions are related to the deposition of 70 ng of 1 on the NALDI surface, i.e. one order of magni-tude lower than the quantity used under the MALDI condi-tions. To evaluate the possible linear response, quantities of 1 in the range 0.0085–0.0512 pmol (5–30 pg) were deposited (see the table in Figure 10). These values were chosen as the typical levels of 1 in the plasma of treated patients (500–3000 ng mL–1). Considering that the plasma sample depro-teinization procedure (necessary to obtain the release of 1 from plasma proteins) leads to a 1:5 dilution, the deposited quantities correspond to a volume of 1 µL. The [M + H]+ ion intensity data obtained for the concentrations of 1 that are of

interest are reported and plotted in Figure 10. As can be seen, the limit of detection (LOD) value is close to 0.008 pmol and the R2 value is particularly low (0.3544), showing that the NALDI method, because of the lack of linear response, is not effective for any quantitative analysis. Passing on to the spiked plasma sample, the situation is even worse: the NALDI spectra of the plasma samples spiked with 3000 ng mL–1 (i.e. for the highest concentration) of 1 do not show any signal related to 1, which indicates an ion-suppression effect.

Then it was thought of interest to undertake a series of measurements under MALDI conditions. This method showed, in the preliminary investigations, a [M + H]+ ion intensity for 1 lower than that obtained in NALDI (see Figure 9) but of the same order of magnitude. The evaluation of possibly linear responses was performed by depositing on the MALDI sample holder 1 µL of solutions of CHCA that contained 1 in different concentrations. Considering that the typical dose is in the order of 180 mg m–2, the quantities of 1 deposited were 0.085, 0.171, 0.256, 0.341, 0.427 and 0.512 pmol. Recorded spectra were acquired with the laser in a nine-point imaginary grid as described above (see Figure 4), in order to avoid the bias due to the operator’s search for a “sweet spot”, the resulting spectra were summed. This procedure was repeated for each sample spot. A peak integration of [M + H]+ was performed. MALDI MS measurements of 1, using CHCA as the matrix, were carried out in triplicate and the means of areas are plotted versus the irinotecan amount in Figure 11, together with the standard deviation plotted as error bars. The related regression line is y = 205,762x + 2222 with an R2 value of 0.9889.

Figure 9. Bar plot of the most intense ionic species for 1–3 versus a.i. under the different conditions tested. The plot corresponds to (a) the [M + H]+ ion for 1, (b) both [M + H]+ and [M + Na]+ for 2 and (c) [M + K]+ for 3.

Figure 10. Plot of six different irinotecan (1) amounts versus peak area. Data were obtained from three different NALDI measurements.


These results show that the instrumental response for the quantities of 1 of interest for pharmacological purposes exhibits a good linearity, and consequently seems to be promising for quantitative measurements in plasma

samples. However, in the case of plasma, quite severe limi-tations can originate from interfering species, as observed in the case of the NALDI measurements. For these reasons, further measurements were performed on plasma samples

Figure 11. Plot of six different irinotecan (1) amounts versus peak area. Data were obtained from three different MALDI measurements.

Figure 13. Plot of six different irinotecan (1) amounts versus peak area. Data were obtained from nine different MALDI measurements performed on three different days.

Figure 12. MALDI mass spectra of plasma spiked with irinotecan (1) at a concentration of 3000 ng mL–1 (a) before and (b) after depro-teinization with methanol containing 0.1% formic acid (v/v). Owing to a deproteinization process the amounts of irinotecan deposited were (a) 2.560 and (b) 0.512 pmol.


spiked with known amounts of 1. As can be seen in Figure 12(a), the MALDI spectrum of an untreated plasma sample spiked with the highest amount of 1 considered (2.560 pmol deposited ) does not show the presence of intense [M + H]+ ions of 1. Consequently, the same plasma-sample treatment as employed for LC-MS/MS measurements was performed, consisting of plasma deproteinization by methanol containing 0.1% formic acid (v/v). The spectrum obtained after deproteini-zation is reported in Figure 12(b): in this case a clearly detect-able [M + H]+ ion of 1 is present at m/z 587. Nine different measures were performed on three different days. The means of the areas are plotted versus drug amount in Figure 13 together with the standard deviation plotted as errors bars. The related regression line is y = 144,017x – 615.2 with an R2 value of 0.9766, also showing that in the case of plasma samples a good linear response can be achieved and, conse-quently, that the MALDI method can be employed for the quan-titative analysis of 1 in plasma samples.

ConclusionsThe possibility of evaluating the effective drug-plasma concen-tration in anticancer chemotherapy is a key issue to overcome the intra- and inter-individual variability in drug concentration, so as to achieve a real personalization of the drug treatment. For these reasons the development of analytical methods for TDM is of great interest. In the present investigation the capabilities of different LDI methods were tested. For this aim, MALDI and SALDI methods were employed for the characteri-zation of irinotecan (1), sunitinib (2) and 6-a-hydroxy paclitaxel (3) and the results so obtained are compared. Aside from

“classic” MALDI measurements, different SALDI methods were employed, in particular NALDI and carbon NH substrates; the latter were used either in the absence or the presence of liquid additives (glycerol, thioglycerol and PPE). Compounds 1 and 2 show easy formation of protonated molecular species under all the experimental conditions, but the highest a.i. was achieved under the NALDI conditions. [M + K]+ ionic species of low intensities are detected for 3. In order to evaluate the possibilities given by these approaches for the development of quantitative methods for TDM, the linear response for 1 was evaluated for both NALDI and MALDI on plasma samples spiked with known amounts of the compound, comparable with the levels present in the plasma of patients under drug treatment. Interestingly, even if the most intense [M + H]+ ions were generated by NALDI, a linear response was not obtained under these conditions (R2 = 0.3544). In the case of the plasma sample spiked with 1, no signal related to [M + H]+ was observed, indicating the presence of severe ion-suppression phenomena. The best results were obtained with MALDI. Under these conditions the linear response for pure compound 1 with R2 = 0.9889 was found. In the case of plasma-spiked samples a linear relationship between spiked quantities of 1 and signal intensity was found, with R2 = 0.9766. These preliminary results indicate the possibility of using

MALDI for the TDM of irinotecan and work is in progress in this direction.

AcknowledgmentsThis work was supported in part by grant (12214) “Application of Advanced Nanotechnology in the Development of Innovative Cancer Diagnostics Tools” from the Associazione Italiana Ricerca sul Cancro (AIRC) 5x1000 and in part by the “Grant Program for Young Investigator on Pediatric Research” 2013 from Fondazione CARIPARO.

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