application of chemotherapeutic agents bound to magnetic nanoparticles is a promising approach for site specific drug deposition . magnetic - drug - targeting ( mdt ) is intended to elevate the drug concentration in the region of interest , because the drug loaded particles are enriched by a focused external magnetic field to defined body compartments after application     [ 14 ] . this method can lead to both higher potency of antitumor treatments and reduction of negative side effects . in our model , drug loading was realized with mitoxantrone ( mto ) ( figure 1 ) , an anthracendion derivative which inhibits the dna and rna synthesis and causes dna strand breaks by intercalation     [ 5 , 6 ] . this agent is used extensively in clinical trials to treat fatal diseases including leukemia , lymphoma , cancers of the breast and prostate , and to treat multiple sclerosis     [ 8 , 9 ] . the applicability of this technique for the quantification of ferrofluids ( ff ) in tissue has been demonstrated previously for an extracted tumor slice model , a specific artery model , and is even in process for the whole body distribution . mto enrichment might be different , so we are interested in developing an easy multifunctional method to quantify the amount of mto , especially if it is bound to coated nanoparticles . various methods have been described to extract mto from blood plasma     [ 1216 ] and tissues   on its own . moreover there are established protocols to determine mto by hplc measurements in context of drug delivery systems like liposome carriers      and nanosphere vehicles     . protein precipitation with 0.5  m hydrochloric acid : acetonitrile ( 90  :  10 , v / v ) , as described by johnson et al .     , leads to a recovery of pure mto in porcine muscle tissue of more than 85% ( table 1 ) . nevertheless , measurement of mto bound to iron oxide nanoparticles is more difficult , and extracting whole organs of white new zealand rabbits this way was unfeasible . lu et al . developed a helpful method to determine mto     , which has been applied in the form of bovine serum albumin ( bsa ) nanospheres and revealed also promising results in our context . but in order to reliably capture even trace amounts of mto , an optimization of the extraction mode has been necessary , as well as the development of a subsequent solid phase extraction ( spe ) method . here we present a newly developed method to determine mto in different tissues and of different binding statuses . the analytical grade solvent , methanol , acetonitrile , chloroform were obtained from carl roth , germany and merck , germany . other chemicals were either delivered by merck , germany ( formic acid , sodium formate , phosphoric acid , potassium dihydrogen phosphate , and citric acid ) , carl roth , germany ( trichloroacetic acid and sodium citrate dihydrate ) , or sunlife , germany ( ascorbic acid ) . mto was delivered by neocorp pharma ( lot : 82056305 , expiration date : 05/2010 ) , germany ( test sample , a concentrate ( 2  mg / ml ) to produce an injection solution ) . the hplc analyses were performed by a waters alliance model consisting of a separation module ( 2695 series ) and a dual wavelength absorbance detector ( 2487 series ) . the separation was carried out using a 3.0  100  mm x - bridge phenyl column ( waters , germany ) with a particle diameter of 3.5  m ; the guard column consisted of the same material and was 3.0  20  mm of size . the column temperature was 55c , the mobile phase was made up of buffer ( 80  nm sodium formate and formic acid , ph 3.0 ) and methanol ( 80  :  20  v / v ) . flow was 1  ml / min , the injection volume was 50  l at room temperature . all measurements were performed after an hplc - method validation including selectivity , linearity , limit of quantification , precision , accuracy , recovery rate , and stability according to a validation protocol [ 21 , 22 ] . prior to the sample measurements , a calibration curve with mto - concentrations of 10  ng / ml20  000  ng / ml was done . for the extraction experiments , we used porcine muscle tissue and liver tissue which were doped with defined amounts of pure mto or mto bound to ff . this occurred by adding dissolved mto at defined values ( see tables 1 and 2 ) to tissue homogenates and incubating overnight at room temperature . due to the complexity of liver tissue and the successful determination here and moreover in muscle tissue , this method could be suitable to perform a complete biodistribution of mto in rabbits . liver or muscle tissue , doped with mto , was homogenized for 1 minute using an ultra turrax apparatus ( ika , staufen , germany ) to lyse the cell membranes prior to sonication . several extraction mixtures containing hydrochloric acid or phosphoric acid in combination with meoh were tested for extraction efficiency and their compatibility with the spe procedures ( see tables 1 and 3 ) . for the extraction , simple shaking for 4 or 24 hours was compared to vortex mixing and 1 to 4-fold ultrasonic treatment with a duration of 1 hour each using an ultrasonic bath ( us ) ( bandelin sonorex tk 52 , berlin , germany , without temperature control ) ( table 2 ) . an alternatively performed accelerated solvent extraction ( ase ) was done with ase 200 ( dionex , idstein , germany ) at temperatures of 40c and 80c according to a protocol of klejdus et al . . the cycle time was 10 minutes and just one cycle for each sample was performed . to sum up , each 0.5  g mto - containing tissue was treated 4 times for 1 hour with ultrasound in the extraction mixture ( 500  l water , 50  l ascorbic acid ( 20% ) in citrate buffer ( ph 3.0 ) 200  l methanole ( meoh ) , 200  l formic acid , 100  l , 20% trichloroacetic acid , 400  l chloroform ) followed by centrifugation for 10 minutes at 8000   g ( jouan mr 23 i , germany ) . the combined supernatant was concentrated via a bond elut plexa ( 200  mg , 6  ml , varian , darmstadt , germany ) cartridge after conditioning with acetonitrile ( mecn ) and 2% formic acid in water . the analyte was eluted with 5  ml 2% formic acid in mecn and dried under airstream . the water resolved residue was measured by hplc as described above to display the efficiency of the extraction . in terms of drug delivery with nanoparticles , only traces of mto are embedded in different tissues so that the whole organ has to be processed . the extraction of mto using 1  n hydrochloric acid ( hcl ) , meoh/1  n hcl ( 50  :  50  v / v ) , or 1  n hcl / mecn ( 90  :  10  v / v ) was efficient for extracting pure mto ( table 1 ) . when it was bound to ff , the yield decreased significantly . since a one - phase - extraction led to turbid , protein - rich supernatants , protein precipitation by trichloroacetic acid ( tca ) as well as carrez - clearing a new point of view offered the extraction mode of lu et al .      with a two - phase - extraction system to separate interfering lipophilic substances in organs , especially liver tissue . this method gave also the possibility to precipitate the protein fraction and save mto from decomposition . nevertheless , we modified this method because more tissue and less mto required an optimization . we used homogenization for 1 minute via ultra turrax ( ika ) to dehisce the cell membranes prior to sonication . simple vortexing and just one single extraction cycle obtained only 26% recovery ( table 2 ) . ultrasonic extraction was superior to simple vortexing , and multistep - extraction even increased the efficiency . a 4-fold extraction each for 1 hour was nearly quantitative ( 88  4% ) ( figure 2 ) . of course , the accelerated solvent extraction ( ase ) ( table 2 ) method led to high extraction rates in short time but this is very expensive . moreover , ase tends to require also a useful spe processing afterwards . to quantify the mto amount of a whole organ , a useful spe method was necessary . unfortunately all the extraction modes described in table 1 lead to turbid , protein - rich supernatants clogging the spe cartridge . using the rp-18 cartridge ( sep - pak - plus c18 , 360  mg , waters ) and a standard elution procedure with water and meoh , recovery of mto was not sufficient although pretesting with pure mto gave promising results ( > 90% ) ( table 3 ) . a recovery rate of 76% by extraction of mto in porcine liver tissue with phosphoric acid was adequate ( table 3 ) . it is also possible to extract , fixate , and elute mto bound to ff when using phosphoric acid or phosphate ; but in the presence of liver tissue , the recovery of mto connected to ff was scarce after the elution from the cartridge . in that case , the ff dissolved under these conditions and interestingly only traces of mto could be detected , no matter what kind of conditioning mode was used for the spe . most of the tested cartridges perform the fixation and elution of released mto bound to ff , but in presence of liver tissue only varian bond elut plexa gave appropriate yields with more than 80% recovery ( table 4 ) . to sum up , the whole method entails a 1-hour ultrasonic extraction repeated 3 of tissue with the 2-phase extraction - solution followed by concentration and purification via varian bond elut plexa cartridge and the consecutive hplc measurement ( figure 3 ) . chromatograms obtained from processed blank liver tissue did not show interfering peaks at the retention time of pure mto measured by hplc as seen in figure 4 . selectivity was proved from 6 blank animals using their liver , lung , kidneys and muscle tissue . kidney , lung , and muscle tissue show a baseline chromatographic resolution of at least 1.5 from all other sample components and can be declared as fully separated . this criterion can not be achieved for liver . the deficient baseline resolution could influence the analyte response . experiments were performed to evaluate to what extend the impurity peak will affect the final assay result . 6 extraction solutions doted with mto at the same amount , as the impurity peak is responding to ( 100  ng / ml ) , were concomitant analysed with identical amount of 100  ng / ml mto in mobile phase solution . t - test of these 2 different population means gave the result that the impurity peak influences the respond significantly . to meet the specific criteria that nonseparable impurity peaks influence the result at most for 0.5% mto , amounts more than 250  ng / ml can seriously be quantified . 5 independent sets of calibration standards including blank samples have been prepared comprising values from 10  ng / ml to 20000  ng / ml pure mto ( 0 , 10 , 20 , 50 , 100 , 1000 , 2000 , 5000 , 10000 , 20000  ng / ml ) ; the 10  ng / ml standard was detected as the lowest limit of quantification ( lloq ) . the maximum deviation of the back calculated values for the lloq was 11.8% and 13.4% for reading points unlike the lloq . the deviation of each reading point meets the fda specification for linearity of 15% ( lloq 20% ) . goodness of fit was not evaluated , because the correlation coefficient with more than 0.999 guarantees a sufficient linear correlation between mto concentration and analytical response over the whole range ( figure 5 ) . the lloq was determined according to the guidelines of fda for bioanalytical method validation and is defined as the 5-time response compared to blank samples . intraassay precision is determined for 3 concentrations of pure mto ( 5000 ; 10000 and 15000  ng / ml ) . the level for 5000  ng / ml gave 1.0% relative standard deviation , the according data for the 10000  ng / ml and 15000  ng / ml levels are 0.9% and 1.0% , respectively . interassay precision was performed on 5 different days for the same values mentioned for intraassay . the relative standard deviations were : 1.4% for 5000  ng / ml , 8.6% for 10000  ng / ml and 2.4% for 15000 ng / ml . intraassay accuracy is determined for 3 concentrations of unbound mto ( 5000 , 10000 , and 15000  ng / ml ) . the level for 5000  ng / ml gave a deviation of the mean from the true value of 1.4% ; for 10000  ng / ml measurements gave 1.7% and 1.8% for 15000  ng / ml , respectively and so meet the criteria which are 80120% for the target concentration . the accuracy values ( deviation of the mean from the true value ) were 2.1% for 5000  ng / ml , 5.6% for 10000  ng / ml , and 0.3% for 15000 liver tissue was used exemplarily to evaluate the recovery rate with variable tissue and mto amounts . the recovery rate of mto in 5  g liver tissue was evaluated for 3 different mto concentrations . series a was doped with 25  g , series b contained 2.5  g , and series c 0.25  g mto bound to ff . the total recovery rate ( extraction and spe ) for mto in liver tissue for series a ( 5000  ng / g ) was 76  2% for series b ( 500  ng / g ) 67  5% and for series c ( 50  ng / g ) 68  4% . the experiments were performed for 3 independent samples keeping in mind that the recovery can be different when using other tissues than liver . for biodistribution , recovery depends on the total weight of organs . the bigger the organs are the more mto can be extracted in comparison to smaller organs having the same drug concentration . processing the mto - containing samples , the extraction from tissue material and spe afterwards can lead to fragmentation of the analyte . we proved the stability of mto by treating mto and mto bound to ferrofluids in the extraction solution ( 12500  ng  mto / ml ) for 3 hours in an ultrasonic bath . reprocessing using bond elut plexa ( varian ) cartridges lead to recovery of 85  1% ( n = 3 ) in case of pure mto samples and 89  1% ( n = 3 ) in case of ferrofluid - bound mto samples . we used the method , described above with 4  1 hour ultrasonic extraction ( figure 3 ) , to measure the biodistribution of mto in rabbits ( new zealand white , charles river corp . new zealand white rabbits were used in accordance with the responsible authority ( regierung von mittelfranken , request : 54 - 2531.31 - 27/06 approved in april 2007 ) . the animals , weighing about 4.55.2  kg , were treated with ferrofluid - bound mto containing a drug amount of 10% to the regular systemic dose which requires 10  mg  mto / m body surface . complete organs of liver , lung , kidneys , and a muscle tissue sample were processed and treated in a 100  ml glass flask ( duran , germany ) with the respective amount of extraction solution increased to the amount for the weight of the entire organ as described above followed by spe . the application of this newly developed method has been proven suitable in a starting in vivo pilot study with new zealand white rabbits . first experiments with ferrofluid - bound mto showed that the highest enrichment could be found in the kidneys . the results exhibited the wide range of mto amounts in different tissues and proved the suitability of the method for different kinds of biological materials ( table 5 ) . to the best of our knowledge , none of the existing protocols , concerning the analysis of mitoxantrone in tissues , have been feasible for our approach of ferrofluid - bound mto . moreover , there is especially a lack of techniques to detect small amounts in large scaled tissues . with our method connecting efficient extraction procedures with enrichment strategies towards detectable extraction solutions , we are able to perform biodistribution studies . monitoring mto enrichment in different tissues in case of ff - bound drug delivery and overlapping the results with magnetorelaxometric measurements , complex biodistribution patterns after application of mto - bound iron oxide nanoparticles become low hanging fruits . current developments in chemotherapy increasingly base on nanoscaled delivery systems . nanoparticles , liposomes , encapsulations , micelles , and other nanostructures carrying chemotherapeutics require newly developed or modulated analytical methods . with this protocol in hand further detailed analytical instructions can be developed for adsorptive drug delivery systems using well - established and prevalent applied hplc . here we present an easy - to - perform and versatile applicable new method to determine even small amounts of mitoxantrone , regardless if it is in pure or ferrofluid - bound condition in tissues or other biological matrices . established procedures have not been useful in our context to detect mitoxantrone , especially in presence of ff and contained in tissue samples weighing more than 100  g which especially is necessary for medical application studies in cancer research . we applied this method to evaluate the biodistribution of mto in rabbit tissue . within first experiments , we could easily perform the measurements of whole organs and showed the applicability of this new method and its widespread possibilities and analytical applications .
in medicine , superparamagnetic nanoparticles bound to chemotherapeutics are currently investigated for their feasibility in local tumor therapy . 
 after intraarterial application , these particles can be accumulated in the targeted area by an external magnetic field to increase the drug concentration in the region of interest ( magnetic - drug - targeting ) . 
 we here present an analytical method ( hplc - uv ) , to detect pure or ferrofluid - bound mitoxantrone in a complex matrix even in trace amounts in order to perform biodistribution studies . 
 mitoxantrone could be extracted in high yields from different tissues . 
 recovery of mitoxantrone in liver tissue ( 5000  ng / g ) was 76  2% . 
 the limit of quantification of mitoxantrone standard was 10  ng / ml 12% . 
 validation criteria such as linearity , precision , and stability were evaluated in ranges achieving the fda requirements . as shown for pilot samples , biodistribution studies 
 can easily be performed after application of pure or ferrofluid - bound mitoxantrone .