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Use of pneumatic nebulization and laser ablation–inductively coupled plasma–mass spectrometry to study the distribution and bioavailability of an intraperitoneally administered Pt-containing chemotherapeutic drug

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Use of pneumatic nebulization and laser ablation–inductively coupled plasma–mass spectrometry to study the distribution and bioavailability of an intraperitoneally administered Pt-containing chemotherapeutic drug
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  ORIGINAL PAPER  Use of pneumatic nebulization and laser ablation  –  inductivelycoupled plasma  –  mass spectrometry to study the distributionand bioavailability of an intraperitoneally administeredPt-containing chemotherapeutic drug Deepti Gholap  &  Johanna Verhulst  &  Wim Ceelen  & Frank Vanhaecke Received: 5 October 2011 /Revised: 9 December 2011 /Accepted: 12 December 2011 /Published online: 15 January 2012 # Springer-Verlag 2012 Abstract  Quadrupole-based inductively coupled  –  massspectrometry (ICP-MS) with pneumatic nebulization as a means of sample introduction was employed for quantifica-tion of platinum in blood and tissue samples of rats with peritoneal carcinomatosis, receiving intraperitoneal treat-ment with the Pt-containing chemotherapeutic drug oxali- platin, and in the perfusate solution used for this purpose.The Pt levels were measured for various treatment condi-tions, i.e., with and without supporting treatment with thedrug bevacizumab and at two different temperatures. Limitsof detection obtained for platinum in blood and tissue sam- ples were 0.3 and 2.0 pg g , − 1 respectively. Evaluation of drug penetration into the tumor, under different conditionsof treatment, was carried out via laser ablation  –  ICP-MS.Quantitative mapping of the Pt distribution in tissue sectionsof rat was attempted relying on gelatin standards. Theresults show an influence of the temperature at which thetreatment is carried out, while supporting administration of the drug bevacizumab did not seem to affect the results. Keywords  Platinum.Laserablation  –  ICP  –  massspectrometry.Biologicalsamples.Quantitative mapping.Chemotherapeuticdrugs Introduction Cancers that occur within the gastrointestinal and gyneco-logical region disseminate through peritoneal spaces, lead-ing to an advanced form of metastasis, called peritonealcarcinomatosis (PC). Patients with PC are associated withshort survival and poor quality of life [1]. In cases where thedisease is limited to the peritoneal surface, systemic therapyalone is powerless [2] and, in many cases, complete surgicalremoval is impossible [3]. Cytoreductive surgery followed by hyperthermic (above body temperature) intraperitonealchemotherapy (HIPEC) with a Pt-based chemotherapeuticdrug is an aggressive approach that has been demonstrated toincrease the disease-specific survival rates in patients with PC[4  –  6].HIPECcombinesthe benefitsoflocal administrationof a chemotherapeutic drug (intraperitoneal chemotherapy) withdirect cytotoxic effects of heat on the tumor [7].HIPEC techniques are not yet standardized [8]. Effi-cacy of this approach in terms of, among others, tumor  penetration, blood levels, and synergism with other drugs, needs to be investigated in greater detail so as tostandardize this procedure to reduce morbidity and mor-tality. Availability of an analytical technique, allowingtrace-level determination of the chemotherapeutic drugin biological samples, combined with the possibility of visualizing drug penetration in the tumor mass, wouldundoubtedly prove to be beneficial in understanding andrefining treatment parameters. Electronic supplementary material  The online version of this article(doi:10.1007/s00216-011-5654-3) contains supplementary material,which is available to authorized users.Anal Bioanal Chem (2012) 402:2121  –  2129DOI 10.1007/s00216-011-5654-3 D. Gholap : F. Vanhaecke ( * )Department of Analytical Chemistry, Ghent University,Krijgslaan 281-S12,9000 Ghent, Belgiume-mail: Frank.Vanhaecke@UGent.beD. Gholape-mail: Deepti.Gholap@UGent.beJ. Verhulst  :  W. CeelenDepartment of Surgery, Ghent University Hospital,De Pintelaan 185,9000 Ghent, Belgium  Inductively coupled plasma   –  mass spectrometry (ICP-MS) is the most sensitive analytical technique available for trace element determination in biological samples. This, incombination with the limited amount of sample required for analysis and pronounced multi-element capability, has madeICP-MS the technique of choice for the determination of trace and ultra-trace elements in biological samples [9  –  11].When used in combination with laser ablation (LA) as a means of sample introduction, this technique is a powerfultool for mapping element distributions within thin biologicalsections [12  –  15]. The main advantages of LA-ICP-MS over other surface analytical tools, such as proton induced X-rayemission spectrometry, secondary ionization mass spec-trometry (SIMS), and synchrotron micro-XRF, are accessi- bility of the instrumentation, high sample throughput, a significantly lower rate of polyatomic ion formation (com- pared with SIMS), excellent limits of detection (down tosub-microgram-per-gram levels under optimum conditions),and a lateral resolution of a few micrometers [16  –  18]. Most of the mapping applications performed by LA-ICP-MSfocus on measurement of endogenous elements in soft andhard tissues [19  –  21]. Although mapping of derivatized pro-teins in electrophoresis gels has been demonstrated [22, 23] while a few publications focusing on clinical samples have been published [24  –  26], imaging of metallodrugs in tissuesstill remains a largely untapped area.The aim of the current work was to develop a reliablemethod for (a) quantitative determination of total Pt levels in blood and tissue samples and (b) visualizing the spatialdistribution of Pt in rat tumor sections after intraperitonealadministration of oxaliplatin. In vivo experiments involvedrats with PC, divided into various groups according to their mode of treatment. Rats belonging to groups 1 and 2 weretreated with intraperitoneal oxaliplatin at body temperature(37  –  38 °C, normotherm) and above body temperature(41  –  42 °C, hypertherm), respectively. Group 3 and 4 ratsreceived additional intravenous bevacizumab therapy beforeintraperitoneal oxaliplatin administration at normotherm andhypertherm conditions, respectively. Bevacizumab is a drugintended to prevent or slow down the growth of cancer cells by blocking the growth of blood vessels. It was postulatedthat addition of bevacizumab would reduce the intracellular  pressure and thereby increase penetration of oxaliplatin inthe tumor. Total platinum levels in blood and tissue samplesfor all the groups are determined following microwave-assisted acid digestion of the samples via quadrupole- based ICP-MS using pneumatic nebulization for sampleintroduction. Also, the Pt concentration in the perfusatesolution was monitored.In addition, we have utilized LA-ICP-MS to examine the penetration depth reached by Pt under various conditions,with and without additional bevacizumab treatment andunder normotherm and hypertherm conditions. To the best of our knowledge, this is the first attempt to quantitativelyvisualize Pt distribution by LA-ICP-MS in HIPEC-treatedrat tumors. We also demonstrate the utility of a calibrationstrategy based on gelatin standards for quantitative mappingof the target element distribution in biological thin sections.Preparation, characterization, and validation of the standardsand their application to the current area of interest arereported on. Experimental MaterialsOxaliplatin was purchased from Sanofi-Aventis, Diegem,Belgium. Bevacizumab was purchased from Genentech/ Roche, Anderlecht, Belgium. Hydrogen peroxide (30%,suprapur) was purchased from Sigma-Aldrich, Steinheim,Germany. Analytical grade 65 vol.% nitric acid (Chem-Lab NV, Zedelgem, Belgium) was used after additional purifica-tion via sub-boiling distillation in PFA equipment. Ultra-purewater obtained from a Direct-Q3 water purification unit (Millipore, Brussels, Belgium) was used for all dilution pur- poses. Gelatin purchased from VWR, Leuven, Belgium, wasused as the matrix material for preparing Pt-doped standards.Elemental standards of platinum and thallium (1 gL − 1 ) wereobtained from Inorganic Ventures, Christiansburg, VA, USA. Methods HIPEC procedure and samplingAdult athymic nude rats were housed under the followingconditions: ad libitum access to food and water, 12 hday/ night cycle, and 24 °C room temperature. The animalswere anesthetized with isoflurane. An incision was madein the abdomen. Inlet and outlet tubings were placed inthe peritoneal cavity for perfusion with the oxaliplatinsolution for 60 min. A roller pump circulated the perfus-ate at a flow rate of 50 ml min − 1 through a heat ex-changer, ensuring a temperature of 37  –  38 °C and 41.5  –  42.5 °C for normothermic and hyperthermic conditions,respectively. During perfusion, the body and perfusatetemperature were closely monitored by thermosensors placed in the abdomen. Samples of the perfusate werecollected at 0, 5, 15, 30, 45, and 60 min.For determination of the Pt concentration in blood duringHIPEC, 0.4 mL fractions of blood were collected in heparin-containing tubes from each animal via cardiac puncture at 5,15, 30, 45, and 60 min after the start of the treatment. At 60 min, the perfusate solution was removed, and the incisionwas sutured. 2122 D. Gholap et al.  At 75 min, 15 min after evacuation of the solution, a fixed volume of blood sample was withdrawn, and animalswere killed by means of intrapulmonal injection of 0.5 ml of T61 (a veterinary euthanasia drug).To determine the extent of drug penetration into thetumor mass, the tumor was carefully removed surgicallyfrom the rat  ’ s body and frozen until further sample prepara-tion was possible.Sample preparation  Blood   For determination of the total Pt concentration in the blood samples and tissue sections by pneumatic nebulization(PN)-ICP-MS, the samples first underwent a digestion step.Acid digestion was carried out in an MLS-1200 Mega, micro-wave digestion system (Milestone Inc, Sorisole, Italy). Inorder to eliminate variations arising from changes in thedensity of blood (viscosities of the blood samples were differ-ent), we decided to weigh the blood samples and calculate thePtconcentrationpergraminsteadofpermillliterofblood.Thefrozenbloodsampleswereallowedtoreachroomtemperature beforeapproximately 0.1 g sample was carefully weighed ina Teflon vessel. Subsequently, 2.0 ml of 23 vol.% HNO 3  wereadded, and the mixture thus obtained was subjected to a closedvessel microwave digestion step at 350 W for 30 min. After digestion, the clear pale yellow solution was transferred into a centrifuge tube, capped, and then kept refrigerated until analy-sis. About 500 μ  l of this digest was pipetted into a polypropyl-ene centrifuge tube, to which 150  μ  l of 1.0  μ  g g − 1 Tl solutionwas added as an internal standard. The final volume wasadjusted to 15 ml with 4.0 vol.% HNO 3 . Tissue  The entire tumor mass that was removed from the rat was embedded in a block of paraffin. From each of the tumor  blocks, two consecutive 20- μ  m thick sections were cut withthehelpofamicrotome,suchthattheentiredepthofthetumor was included in the section. Out of the two sections, onesection was placed on a glass support and kept refrigerateduntil further laser ablation  –  ICP-MS measurement. The other tumor section was accurately weighed and transferred into a Teflon vessel, into which 2.0 ml of 30.0 vol.% H 2 O 2  and1.0 ml of 65 vol.% HNO 3  were added. The mixture wassubjected to a microwave digestion program consisting of three steps. The first step started at 450 W for 5 min. In thesecond step, the power was raised to 650 Wand maintained at that value for 15 min. The last step during which the power was lowered to 500 W lasted for 5 min.To the resulting tissue digest, 100  μ  l of 1.0  μ  g g − 1 Tlsolution was added as an internal standard, and the finalvolume was adjusted to 10 ml with Milli-Q water.  Perfusate  The expected Pt concentration in the perfusatewas quite high. Hence, sample dilution was performed intwo steps. In the first step, 50  μ  l of the srcinal perfusatesolution was withdrawn and diluted to 15 ml with Milli-Qwater (solution A). In the next step, to 100  μ  l of solution A,150  μ  l of 1.0  μ  g g − 1 Tl solution was added, and the finalvolume was adjusted to 15 ml with 2 vol.% HNO 3 .Procedural blanks were prepared for all sample types.PN-ICP-MSLiquid sample analysis (digests) was performed using anXseries 2 quadrupole-based ICP-MS instrument (Thermo-Scientific, Bremen, Germany). Sample introduction was ac-complished via a concentric glass nebulizer mounted onto a Peltier-cooled conical spray chamber with impact bead. The peristaltic pump delivered sample at a flow rate of 500  μ  l min − 1 . A Cetac 500 autosampler (Cetac, Omaha, NE,USA) was deployed for automating sample introduction. Allthe isotopes of Pt were monitored, along with the major isotope of Tl ( 205 Tl). The instrument was operated at an RF power of 1,400 W. The nebulizer gas (argon) flow rate wasmaintained at0.85 Lmin − 1 . Five replicate measurementswere performed for every sample and standard solution.Calibration solutions containing 0.25, 0.5, 1, 5, 10, 15,and 20 ng g − 1 Pt were prepared and analyzed. Correlationcoefficients for the linear regression lines were always better than 0.99.Limit of detection and recoveryThe procedural limits of detection (LOD) were determinedseparately for blood, perfusate, and tissue samples. Thedetection limits were calculated as described by IUPAC(3s-criterion) [27].To determine loss of Pt during the analytical procedure,six different blood samples were spiked with a fixed amount of Pt prior to microwave digestion. For perfusates, matrixspike recovery was calculated in a similar manner after spiking six different perfusate samples just before measure-ment. The spike recovery was calculated as below:PercentRecovery ¼ SSR   SR SA   100Where SSR is the spiked sample result, SR is the sample(unspiked) result, and SA is the spike added.LA-ICP-MSA UP193HE 193 nm ArF* excimer-based laser ablationsystem (New Wave Research-ESI, Fremont, CA, USA)coupled to a quadrupole-based ICP-MS unit (ThermoXseries 2) was used to reveal the distribution of Pt withintumor sections of the of oxaliplatin-treated rats. The tumor sections were  “ rastered ”  by scanning a laser beam (with a  Bulk & spatially resolved Oxaliplatin determination in biological samples via ICP-MS 2123  diameter of 70  μ  m) over the entire surface. The rastering parameters were selected such as to provide square pixels inthe final elemental maps [28]. The ablated material wastransported into the ICP using helium as a carrier gas,subsequently admixed with argon post-ablation. The ICP-MS was set up in a time-resolved analysis mode, while onedata point was monitored per spectral peak. The laser abla-tion settings were optimized for ablation of tumor sectionsand standards. The main operating parameters are shown inTable 1.Basic image processing was carried out using IDL soft-ware (Interactive Data Language, IIT Visual informationsolutions, Boulder, CO, USA).Standards for LA-ICP-MS measurement A gelatin solution (10%  w/v  ) was prepared by mixing gel-atin powder with Milli-Q water and heating this mixture at 60 °C, until all the gelatin melts and the solution becomesclear and consistent. In a separate experiment, a stock solu-tion containing 250  μ  g g − 1 of Pt was prepared. Different volumes of the stock solution were taken and diluted to3.0 ml with the gelatin solution, so as to get 0.5, 1.0, 2.5,5.0, and 10 μ  g g − 1 Pt. From each of these standard solutions,100  μ  l was carefully withdrawn and placed on a flat glasssupport. Another 100  μ  l was introduced into a glass vial(20 ml). Both the glass vial and the glass support wereallowed to stand at room temperature overnight. The hard-ened gelatin film on the glass support was then used for LA-ICP-MS measurement as a   “ matrix-matched ”  Pt standard.The gelatin mass in the glass vial was dissolved in 10 ml1.0 vol.% HNO 3  by heating the solution at 40 °C. Different volumes from each of these dissolved gelatin standards werewithdrawn, spiked with 100  μ  l of 1.0  μ  g g − 1 Tl solution,and then adjusted to 10 ml with 2 vol.% HNO 3  so as toexpect a Pt concentration of 10 ng g − 1 in the final solution.The solutions were analyzed with PN-ICP-MS, and theactual Pt concentration in each of the srcinal (solid) gelatinstandards was back-calculated. Results and discussion Spectral interferencesWhile using a quadrupole-based ICP  –  mass spectrometer for Pt analysis, spectral interferences have to be carefully evalu-atedtoavoidsystematicerrors.ThesignalsofPtisotopes 194 Pt and  195 Pt are inseparable from those of oxide ions of Hf ( 179 Hf  16 O + ,  178 Hf  17 O + ,  177 Hf  18 O + [ 195 Pt] and  178 Hf  16 O + , 177 Hf  17 O + ,  176 Hf  18 O + [ 194 Pt]). However, since the back-ground concentration of Hf in biological samples is in thesame range as that of Pt, the MO + /M + ratio typically <2% andthe level of Pt added via the chemotherapeutic drug consider-ably higher, the risk of spectral interference is small. Never-theless,thisassumptionwasfurtherverifiedbymonitoringthe 195 Pt/  194 Pt isotope ratio in the samples and the standards. The 195 Pt/  194 Pt isotope ratio obtained for standards (1.0334) andsamples (1.0366), respectively showed no statistically mean-ingful difference.LOD and recoveryLimits of detection were calculated for all types of samples(blood, perfusate, tissue) according to the methodologydescribed above. The calculated LODs for Pt were muchlower than the Pt concentrations in the samples. LODs for  blood, perfusate, and tissue samples as measured byquadrupole-based ICP-MS were 0.3, 0.3, and 0.2 pg g − 1 ,respectively. Recovery values for blood and tissue sampleswere in the rage of 98±4% and 99±3%, respectively.BioavailabilityThe initial oxaliplatin concentration in the perfusate solutionused for performing IPEC was 0.18 mg mL − 1 . Figure 1shows the trend in Pt concentration in the perfusate solu-tions, monitored during a period of 60 min under varioustreatment conditions. After 60 min, the Pt concentration inthe perfusate was reduced to 79±8% and 75±13% of the Table 1  Optimized experimental parameters for LA-ICP-MS ICP-MS ThermoScientific Xseries 2 LA New Wave ResearchUP193HERF power (W) 1,400 Wavelength 193 nmCarrier gas flow rate (L min − 1 ) He, 0.5 Spot size ( μ  m) 70Make-up gas flow rate (L min − 1 ) Ar, 0.8 Lateral scanning speed ( μ  m s − 1 ) 70Sampling depth (mm) 60 Frequency (Hz) 10Total acquisition time (s) 1.0 Laser energy (J cm − 2 ) 0.85  –  1.0Analytes measured  13 C,  31 P,  190 Pt,  192 Pt,  194 Pt,  195 Pt,  196 Pt,  198 Pt Cones Nickel, Xt-type 2124 D. Gholap et al.  initial concentration when only oxaliplatin was used at nor-mothermic and hyperthermic conditions, respectively. In thecase where rats received bevacizumab as an additional treat-ment, the Pt concentration in the perfusate at 60 min was 77±7% and 73±7% of the initial concentration at normothermicand hyperthermic conditions, respectively.Blood concentrations of Pt were monitored during a  period of 75 min. Figure 2 shows the average blood concen-trations for the two different treatments at normothermic andhyperthermic conditions. Comparison of Pt concentrations for various treatments was carried out using SPSS v19 software.One-way ANOVA was used to compare concentrationsassociated to various treatments at each sampling point. A  P  <0.05 was considered significant. The investigation revealedthat temperature had a significant influence on both the treat-ments. In group 1 rats that received only oxaliplatin, the Pt concentrations increased significantly at higher temperature,already after 15 min (  P  0 0.002) of IP administration. Theincrease in Pt concentration at the 75 min end point was 32±4% higher at hyperthermic conditions than that at normother-mic conditions for group 1 rats.For group 2 rats, with additional bevacizumab treatment,a similar trend is observed upon using an increased temper-ature, although the difference becomes statistically signifi-cant at 75 min (  P  0 0.041) only. At the 75 min end point, theincrease in Pt concentration at hyperthermic conditions was32±9% higher than that at normothermic conditions.The above findings show that heat enhances systemicabsorption of the drug. The increase in Pt concentration in blood at higher temperature is undesirable, as it may lead tohigher systemic toxicity.Additional treatment with bevacizumab on the other handdid not exert any significant influence on the blood Pt concen-trations, neither under normothermic nor under hyperthermicconditions (  P   values >0.05 at all time points).Imaging for tissue penetrationLA-ICP-MS is a powerful technique for studying the spatialdistribution of metallodrugs in tissues. This technique pro-vides better sensitivity than most of the commonly available Fig. 1  Concentration (micrograms per gram) of oxaliplatin in perfus-ate solutions, sampled over a time span of 60 min in rats with andwithout bevacizumab treatment at normothermic and hyperthermicconditions Fig. 2  Platinum concentrationin the blood of rats with andwithout bevacizumabadministration at different temperaturesBulk & spatially resolved Oxaliplatin determination in biological samples via ICP-MS 2125
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