Determination of urinary iodine by inductively coupled plasma mass spectrometry.

BACKGROUND: Mild iodine deficiency is endemic in many countries of Europe including Belgium. Fast, accurate and specific methods for quantification of urinary iodine are needed. We describe in this report a specific ICP-MS method for the quantification of urinary iodine. METHOD: Samples and iodate calibrators were diluted 20 times into aqueous solution containing triton X-100, 1.5% HCl and (103)Rh as an internal standard. Prior digestion or oxidation was not necessary. Results were compared with those obtained by Sandell-Kolthoff (S-K) spectrophotometric method. RESULTS: Comparison of both methods showed good agreement. The Passing-Bablok regression between both methods was ICP-MS=0.986 (S-K)-7.51. The Bland-Altman difference plot showed a small but significant mean difference of -13.3 microg/L for ICP-MS. The between-day coefficient of variation (CV) was 13% at 89 microg/L. Limit of detection was 4 microg/L and limit of quantification was 20 microg/L. No carryover effect has been observed on series containing up to 50 samples. CONCLUSION: The ICP-MS method described here is fast, accurate and specific for the quantification of urinary iodine. Compared to the S-K method the urinary iodine concentrations measured by the ICP-MS method were slightly, but significantly lower. Consequently, the results of studies using S-K method should be compared with caution with those using the ICP-MS method.     

Manganese species from human serum,cerebrospinal fluid analyzed by size exclusion chromatography-, capillary electrophoresis coupled to inductively coupled plasma mass spectrometry

Manganese (Mn) at high concentrations can have adverse effects on health, mainly because of its toxicity to the central nervous system. Health impacts of Mn are known mostly from occupational health studies, but the exact mechanisms how Mn, being bound to transferrin (TF) in the blood, enters the brain – are unknown. Mn speciation at the neural barriers can help to obtain more information about the pathways and carriers. This paper summarizes investigations on the size distribution of Mn carriers (e.g. proteins, peptides, carbonic acids) in serum before the neural barriers and in cerebrospinal fluid (CSF) behind them as a first characterization step of the Mn carriers being involved in moving Mn across the neural barriers. Further identification of Mn-species in CSF was successfully achieved by CZE–inductively coupled plasma (ICP)–dynamic reaction cell (DRC)–mass spectrometry (MS). Serum samples showed Mn mean concentrations of 1.7±0.8 μg L⁻¹. The size distribution of Mn-carriers showed a main peak in the TF/albumin size fitting to the known physiological ligands. However, also an increasing Mn peak at 700 Da with increasing total Mn concentration was seen. Samples of CSF showed Mn mean concentrations of 2.6 μg L⁻¹=48 nM. In CSF Mn was found to be mostly bound to low-molecular-mass (LMM)-Mn carriers in the range of 640–680 Da. This is similar to the LMM compound in serum and to Mn–citrate complexes suggested to be present in body fluids. Citrate concentration was 573 μM, thus being in huge excess compared to Mn. CSF was further analyzed by CZE–ICP–DRC–MS. Several Mn-species were monitored and mostly identified. The most abundant Mn-species was Mn–citrate at a concentration of around 0.7 μg Mn L⁻¹.     

Quantification of 5-azacytidine in plasma by electrospray tandem mass spectrometry coupled with high-performance liquid chromatography

5-Azacytidine (5AC), a nucleoside analogue and hypomethylating agent, has anticancer properties and has been utilized in the treatment of various malignancies. 5AC is unstable and rapidly hydrolyzed to several by-products, including 5-azacytosine and 5-azauracil. A sensitive, reliable method was developed to quantitate 5AC using LC/MS/MS to perform pharmacokinetic and pharmacodynamic studies on 5AC combination therapy trials. Blood samples were collected in a heparinized tube and immediately processed for storage. To increase the stability of 5AC in plasma, 25 ng/mL tetrahydrouridine was added to the plasma and snap frozen. Plasma samples were extracted using acetonitrile then cleaned up by Oasis MCX ion exchange solid-phase extraction cartridges. 5AC was separated on an YMC Jsphr M80 C(18) column with gradient elution of ammonium acetate (2 mM) with 0.1% formic acid and methanol mobile phase. 5AC elutes at 5.0 +/- 0.2 min with a total run time of 30 min. Identification was through positive-ion mode and multiple reaction monitoring mode at m/z+ 244.9-->113.0 for 5AC and m/z+ 242.0-->126.0 for 5-methyl-2'-deoxycytidine, the internal standard. The lower limit of quantitation of 5AC was 5 ng/mL in human plasma, and linearity was observed from 5 to 500 ng/mL fitted by linear regression with 1/x weight. This method is 50 times more sensitive than previously published assays and successfully allows studies to characterize the pharmacokinetics and pharmacodynamics of 5AC.     

Speciation of trace elements in human serum by micro anion exchange chromatography coupled with inductively coupled plasma mass spectrometry

Speciation analysis of essential trace elements in human serum provides important information on nutritional status and homeostatic mechanisms regulating transport processes, acute phase reactions, and protection against oxidative damage. Anion exchange high-performance liquid chromatography (HPLC) combined with inductively coupled plasma mass spectrometry (ICP-MS) has proved to be a useful tool in speciation. Here we describe a fast method that can be applied to carry out the speciation of Fe, Cu, Zn, and Se in as little as 1 microl [corrected] of serum. The method employs monolithic anion exchange micro columns installed on a tandem HPLC system coupled on-line with an ICP-MS detector. The chromatographic separation is similar to those reported previously but with considerable gain in terms of time and sample requirement. Reproducibility is acceptable for most species. Using our method, we were able to find species-specific differences between different commercially available trace element reference materials. Because the method chosen to collect blood might interfere with speciation, the proposed methodology was used to compare heparinized plasma, ethylenediaminetetraacetic acid (EDTA) plasma, and serum from adult healthy volunteers. As expected, EDTA strongly affects speciation analysis (especially for Fe and Zn), whereas changes due to the use of lithium-heparin (Li-He) as anticoagulant appear to be minimized.     

Uranium quantification in semen by inductively coupled plasma mass spectrometry

In this study we report uranium analysis for human semen samples. Uranium quantification was performed by inductively coupled plasma mass spectrometry. No additives, such as chymotrypsin or bovine serum albumin, were used for semen liquefaction, as they showed significant uranium content. For method validation we spiked 2 g aliquots of pooled control semen at three different levels of uranium: low at 5 pg/g, medium at 50 pg/g, and high at 1000 pg/g. The detection limit was determined to be 0.8 pg/g uranium in human semen. The data reproduced within 1.4–7% RSD and spike recoveries were 97–100%. The uranium level of the unspiked, pooled control semen was 2.9 pg/g of semen (n = 10). In addition six semen samples from a cohort of Veterans exposed to depleted uranium (DU) in the 1991 Gulf War were analyzed with no knowledge of their exposure history. Uranium levels in the Veterans’ semen samples ranged from undetectable (<0.8 pg/g) to 3350 pg/g. This wide concentration range for uranium in semen is consistent with known differences in current DU body burdens in these individuals, some of whom have retained embedded DU fragments.     

Separation and determination of Se-compounds by liquid chromatography coupled with electrospray mass spectrometry

A method for Selenocystine and Selenomethionine determination by LC–ES–MS was developed in this work. The mass spectrometer was used in a positive mode and the m/z used for the identification of Selenomethionine and Selenocystine were 198.35 and 337.15, respectively.The selenium species were separated using a LC system. A silica chromatographic column (ZORBAX Eclipse XDB-C₈ of 50 mm length and 2.1 mm internal diameter (particle size 3.5 μm)) was used. The separation was realised in isocratic mode, using methanol:water (1:1) with 1% of acetic acid and a flow rate of 200 μL min⁻¹. The developed method was precise (RSD of 4.5% and 3.9% for Selenomethionine and Selenocystine, respectively) and sensible (limit of detection (LOD) 0.06 and 0.99 mg L⁻¹ for selenomethionine and selenocystine, respectively).     

Determination of ritodrine in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and sensitive HPLC/MS/MS method was developed and evaluated to determine the concentration of ritodrine (RTD) in human plasma. Liquid-liquid extraction with ethyl acetate was employed as the sample preparation method. The structural analogue salbutamol was selected as the internal standard (IS). The liquid chromatography was performed on a Hanbon Sci. & Tech. Lichrospher CN (150 mm x 4.6 mm, i.d., 5 microm) column (Hanbon, China) at 20 degrees C. A mixture of 0.03% acetic acid and methanol (50:50, v/v) was used as isocratic mobile phase to give the retention time 3.60 min for ritodrine and 2.94 min for salbutamol. Selected reaction monitoring (SRM) in positive ionization mode was employed for mass detection. The calibration functions were linear over the concentration range 0.39-100 ng mL(-1). The intra- and inter-day precision of the method were less than 15%. The lower limit of quantification was 0.39 ng mL(-1). The method had been found to be suitable for application to a pharmacokinetic study after oral administration of 20mg ritodrine hydrochloride tablet to 18 healthy female volunteers. The half-life is 2.54+/-0.67 h.     

Characterization of human transferrin glycoforms by capillary electrophoresis and electrospray ionization mass spectrometry

Carbohydrate Deficient Glycoprotein Syndrome (CDGS) is an inherited metabolic disease affecting all parts of the body. The biochemical diagnosis of this syndrome is based on the presence of a special marker in blood, Carbohydrate Deficient Transferrin (CDT), which is also a marker of chronic alcohol abuse. CDT is characterized by abnormal glycoforms of serum transferrin (Tf). In the present study, electrophoretic separation of human serum transferrin glycoforms was carried out using a bare fused-silica capillary and the glycoforms present in commercial Tf were baseline separated. The limit of detection (LOD) of human Tf was around the nmol concentration range. The LOD of the trisialo- and disialo-Tf, expressed as percentages of the tetrasialo-Tf peak area, were 0.5% for trisialo-Tf and 0.4% for disialo-Tf, and these values were appropriate for CDGS diagnosis. Moreover, Tf glycoforms were characterized using mass spectrometry (MS). The method was applied to the analysis of normal and pathological serum samples, after dilution. The results obtained suggest a way of making a rapid and simple CDGS diagnosis.     

Analysis of fluorescently labeled oligosaccharides by capillary electrophoresis and electrospray ionization mass spectrometry

Neutral oligosaccharides were fluorescently conjugated with 7-amino-1, 3-naphthalenedisulfonic acid. A mixture of fluorescently labeled chitobiose, chitotriose, and chitotetrose were successfully separated by preparative capillary electrophoresis (CE) and the individual components characterized by electrospray ionization-mass spectrometry (ESI-MS). By combining fluorescent labeling with CE, the use of highly specific exoglycosidases and ESI-MS, a more structurally complex N-linked glycan was analyzed.     

Profiling of Arabidopsis secondary metabolites by capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry

Large-scale metabolic profiling is expected to develop into an integral part of functional genomics and systems biology. The metabolome of a cell or an organism is chemically highly complex. Therefore, comprehensive biochemical phenotyping requires a multitude of analytical techniques. Here, we describe a profiling approach that combines separation by capillary liquid chromatography with the high resolution, high sensitivity, and high mass accuracy of quadrupole time-of-flight mass spectrometry. About 2000 different mass signals can be detected in extracts of Arabidopsis roots and leaves. Many of these originate from Arabidopsis secondary metabolites. Detection based on retention times and exact masses is robust and reproducible. The dynamic range is sufficient for the quantification of metabolites. Assessment of the reproducibility of the analysis showed that biological variability exceeds technical variability. Tools were optimized or established for the automatic data deconvolution and data processing. Subtle differences between samples can be detected as tested with the chalcone synthase deficient tt4 mutant. The accuracy of time-of-flight mass analysis allows to calculate elemental compositions and to tentatively identify metabolites. In-source fragmentation and tandem mass spectrometry can be used to gain structural information. This approach has the potential to significantly contribute to establishing the metabolome of Arabidopsis and other model systems. The principles of separation and mass analysis of this technique, together with its sensitivity and resolving power, greatly expand the range of metabolic profiling.     

Characterization and quantification of Bcl-2 antisense G3139 and metabolites in plasma and urine by ion-pair reversed phase HPLC coupled with electrospray ion-trap mass spectrometry

A novel ion-pair reversed phase electrospray ionization (IP-RP-ESI) liquid chromatography-mass spectrometry (LC-MS) method has been developed for identification and quantification of Bcl-2 antisense phosphorothioate oligonucleotides G3139 and metabolites in plasma. This method utilized solid phase extraction for desalting and matrix removal and detection by an ion trap mass spectrometer. Resolution was accomplished on a micro C18 column eluted with a mobile phase consisting of hexafluoro-2-propanol and triethylamine in methanol at 50 degrees C. Five G3139 metabolites were identified in plasma and urine from treated patients and rats. A cassette HPLC-MS/MS quantification method for G3139 and three metabolites was developed and validated with a limit of quantification (LOQ) of 17.6 nM in human and rat plasma with acceptable precision and accuracy. Plasma pharmacokinetics of G3139 and metabolites in these species were described.     

Large-scale identification of selenium metabolites by online size-exclusion-reversed phase liquid chromatography with combined inductively coupled plasma (ICP-MS) and electrospray ionization linear trap-Orbitrap mass spectrometry (ESI-MS(n))

A method was developed for the comprehensive cartography of the selenium metabolites synthesized in the process of conversion of selenite [Se(iv)] into organic compounds and enrichment of yeast with selenium. The number of compounds detected was considerably increased (49) owing to the optimization of the fractionation procedure and the use of UPLC. The increased purity of the minor selenoorganic amino acids and oligopeptides allowed successful on-line de-novo identification based on the exact mass (<1 ppm) and fragmentation (MS(2)/MS(3)) mass spectra obtained with high resolution and high mass accuracy. The quality of mass spectra allowed the re-interpretation of previously reported structures of some selenium species.     

Characterization of mannooligosaccharide caps in mycobacterial lipoarabinomannan by capillary electrophoresis/electrospray mass spectrometry

A new analytical approach based on capillary electrophoresis-electrospray mass spectrometry (CE/ESI-MS) has provided new insight into the characterization of mannooligosaccharide caps from lipoarabinomannans (LAMs), which are key molecules in the immunopathogenesis of tuberculosis. This analytical approach requires oligosaccharide labeling with the fluorophore 1-aminopyrene-3,6,8-trisulfonate (APTS) by reductive amination at the reducing termini. Optimization of the separation and ionization conditions, such as the choice of capillary electrophoresis (CE) electrolyte buffers, is presented and discussed. Anionic separation of the mono and oligosaccharide APTS derivatives was finally achieved with aqueous triethylammonium formate buffer. It was found that in contrast to the triethylammonium phosphate buffer, the triethylammonium formate buffer was appropriate for CE/ESI-MS coupling analysis of APTS-carbohydrate derivatives. In this case, negative ESI-mass spectra of APTS-carbohydrate adducts showed mainly (M-2H)2-pseudomolecular ions and some sequence fragment ions allowing their non-ambiguous structural characterization at the picomolar level. This analytical approach was successfully applied to more complex mixtures of carbohydrates released by mild acid hydrolysis of the lipoarabinomannans from Mycobacterium bovis BCG. The APTS-mannooligosaccharide cap adducts were separated by CE and their structural characterization achieved by CE/ESI-MS analyses. Mannooligosaccharide caps were routinely analyzed by capillary electrophoresis-laser induced fluorescence (CE-LIF) from 50 fmol of lipoarabinomannans with mannosyl capping (ManLAMs) but sensitivity was about 50 times lower using ESI-MS detection.     

Characterization of ricin heterogeneity by electrospray mass spectrometry, capillary electrophoresis, and resonant mirror

Electrospray mass spectrometry (ES/MS), capillary-zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and multianalyte resonant mirror are used to evaluate the heterogeneity of samples of ricin toxins extracted from five horticultural varieties of Ricinus communis seeds: R. communis zanzibariensis, carmencita, impala, sanguineus, and gibsonii. The investigation is also extended to the geographical provenance of the beans. Combining mass spectrometry, CE techniques, and resonant mirror results in a powerful analytical tool capable to characterize and differentiate between different varieties of ricin toxins. Each technique complements the others, adding another level of information. This study reveals a large extent of heterogeneity for each cultivar, demonstrating that ricin toxins consist of a series of glycosylated proteins most likely originating from a multigene family. By combining these techniques, it is possible to differentiate between zanzibariensis and the other four varieties, and that variations in the functional characteristics may be observed between the different cultivars. This study demonstrates that knowledge of the variety of R. communis beans used and their geographical provenance is essential before any type of investigation of ricin toxins is carried out. Consequently, any unusual behavior observed can only be attributed to that particular cultivar studied and not automatically extended to include all R. communis varieties.     

Quantitative analysis of PD 0332991 in mouse plasma using automated micro-sample processing and microbore liquid chromatography coupled with tandem mass spectrometry

In the oncology therapeutic area, the mouse is the primary animal model used for efficacy studies. Often with mouse pharmacokinetic (PK) and pharmacokinetic/pharmacodynamic (PK/PD) studies, less than 20 μL of total plasma sample volume is available for bioanalysis due to the small size of the animal and the need to split samples for other measurements such as biomarker analyses. The need to conduct automated "small volume" sample processing for quantitative bioanalysis has therefore increased. An automated fit for purpose protein precipitation (PPT) method using a Hamilton MicroLab Star (Reno, NV, USA) to support mouse PK and PK/PD studies for an oncology drug candidate PD 0332991, (a specific inhibitor of cyclin-dependent kinase 4 (CDK-4) currently in development) for processing "small volumes" was developed. The automated PPT method was achieved by extracting and processing 10 μL out of a minimum sample volume of 15 μL plasma utilizing the Hamilton MicroLab Star. A 96-conical shallow well plate by Agilent Technologies, Inc (Wilmington, DE, USA) was the labware of choice used in the automated Hamilton "small volume" method platform. Analyses of a 10 μL plasma aliquot from 15 μL of plasma study samples were conducted by both automated and manual PPT method. All plasma samples were quantitated using a Sciex API 4000 triple quadrupole mass spectrometer coupled with an Eksigent Express HT Ultra HPLC system. The chromatography was achieved using an Agilent microbore C(18) Extend, 1.0 × 50 mm, 3.5 μm column at a flow rate of 0.150 mL/min with a total run time of 1.8 min. Accuracy and precision of standard and QC concentration levels were within 90-107% and <14%, respectively. Calibration curves were linear over the dynamic range of 1.0-1000 ng/mL. PK studies for PD 0332991 were conducted in female C3H mice following intravenous administration at 1mg/kg and oral administration at 2mg/kg. PK values such as area under curve (AUC), volume of distribution (Vd), clearance (Cl), half life (T(1/2)) and bioavailability (F%) demonstrated less than 11% difference between the automated Hamilton and manual PPT methods. The results demonstrate that the automated Hamilton PPT method can accurately and precisely aliquot 10 μL of plasma from 15 μL or larger volume plasma samples. The fit for purpose Hamilton PPT method is suitable for routine analyses of plasma samples from micro-sampling PK and PK/PD samples to support discovery studies.     

Determination of procarbazine in human plasma by liquid chromatography with electrospray ionization mass spectrometry

Procarbazine is a cytotoxic chemotherapeutic agent used in the treatment of lymphomas and brain tumors. Its pharmacokinetic behavior remains poorly understood even though more than 30 years have elapsed since the drug was approved for clinical use. To characterize the pharmacokinetics of procarbazine in brain cancer patients during a phase I trial, a method for determining the drug in human plasma by reversed-phase high-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry (ESI-MS) was developed and thoroughly validated. Plasma samples were prepared for analysis by precipitating proteins with trichloroacetic acid and washing the protein-free supernatant with methyl tert-butyl ether to remove excess acid. The solution was separated on a Luna C-18 analytical column using methanol-25 mM ammonium acetate buffer, pH 5.1 (22:78, v/v) as the mobile phase at 1.0 ml/min. A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H](+) ions at m/z 222.2 for procarbazine and at m/z 192.1 for the internal standard (3-dimethylamino-2-methylpropiophenone). Procarbazine and the internal standard eluted as sharp, symmetrical peaks with retention times (mean+/-S.D.) of 6.3+/-0.1 and 9.9+/-0.3 min, respectively. Calibration curves of procarbazine hydrochloride in human plasma at concentrations ranging from 0.5 to 50 ng/ml exhibited excellent linearity. The mean absolute recovery of the drug from plasma was 102.9+/-1.0%. Using a sample volume of 150 microl, procarbazine was determined at the 0.5 ng/ml (1.9 nM) lower limit of quantitation with a mean accuracy of 105.2% and an interday precision of 3.60% R.S.D. on 11 different days over 5 weeks. During this same time interval, the between-day accuracy for determining quality control solutions of the drug in plasma at concentrations of 2.0, 15 and 40 ng/ml ranged from 97.5 to 98.2% (mean+/-S.D., 97.9+/-0.4%) and the precision was 3.8-6.2% (mean+/-S.D., 5.1+/-1.2%). Stability characteristics of the drug were thoroughly evaluated to establish appropriate conditions to process, store and prepare clinical specimens for chromatographic analysis without inducing significant chemical degradation. The sensitivity achieved with this assay permitted the plasma concentration-time profile of the parent drug to be accurately defined following oral administration of standard doses to brain cancer patients.     

Determination of dopamine and methoxycatecholamines in patient urine by liquid chromatography with electrochemical detection and by capillary electrophoresis coupled with spectrophotometry and mass spectrometry

The applicability of capillary electrophoresis (CE) with UV and mass spectrometric (MS) detection for the determination of dopamine and methoxycatecholamines in urine was evaluated in comparison with the liquid chromatography-electrochemical detection (LC-EC) method widely used in catecholamine analysis. The catecholamines in urine were deconjugated with acid or enzyme hydrolysis, purified by cation exchange (CEX) or solid-phase extraction (SPE) with a copolymer of N-divinylpyrrolidone and divinylbenzene and analyzed by LC-EC, CE-UV, and CE-MS. Acid hydrolysis was more effective in the deconjugation than enzymatic hydrolysis with Helix pomatia. However, the recoveries of HMBA, DA and NMN from spiked samples were less than 30% after acid hydrolysis and SPE purification. The CEX purification was more efficient than SPE in removing matrix compounds from the urine samples. The limits of detection were lower in LC-EC analysis than in CE-UV or CE-MS. Many factors in the analytical procedure caused deviations in the concentrations measured for urinary dopamine and methoxycatecholamines. The recovery of HMBA, which was used as the internal standard, was poor after acid hydrolysis and SPE purification. The purification methods were validated in conjunction with the analytical methods and therefore cross analysis was unsuccessful. The LC-EC method was the most sensitive, but CE-UV and CE-MS were sensitive enough for the determination of dopamine and methoxycatecholamines even in healthy patient urine. The EC and MS detections were superior to the UV detection in specificity since, after acid hydrolysis, some matrix compounds were migrating close to I.S., DA and 3MT.     

Determination of total arsenic concentrations in nails by inductively coupled plasma mass spectrometry

The analysis of trace elements in biological samples will extend our understanding of the impact that environmental exposure to these elements has on human health. Measuring arsenic content in nails has proven useful in studies evaluating the chronic body burden of arsenic. In this study, we developed methodology with inductively coupled plasma-mass spectrometry (ICP-MS) for the determination of total arsenic in nails. We assessed the utility of the washing procedures for removing surface contamination. Four types of preanalysis treatments (water bath, sonication, water bath plus sonication, and control) after sample decomposition by nitric acid were compared to evaluate the digestion efficiencies. In addition, we studied the stability of the solution over 1 wk and the effect of acidity on the arsenic signal. Arsenic content in the digested solution was analyzed by using Ar-N₂ plasma with Te as the internal standard. The results suggest that washing once with 1% Triton Χ-100 for 20 min for cleaning nail samples prior to ICP-MS analysis is satisfactory. Repeated measurement analysis of variance revealed that there was no significant difference among the various sample preparation techniques. Moreover, the measurements were reproducible within 1 wk, and acidity seemed to have no substantial influence on the arsenic signal. A limit of detection (on the basis of three times the standard deviation of the blank measurement) of 7 ng As/g toenail was achieved with this system, and arsenic recoveries from reference materials (human hair and nails) were in good agreement (95–106% recovery) with the certified/reference values of the standard reference materials. ICP-MS offers high accuracy and precision, as well as highthroughput capacity in the analysis of total arsenic in nail samples.     

Determination of platinum in human subcellular microsamples by inductively coupled plasma mass spectrometry

A fast and robust method for the determination of platinum in human subcellular microsamples by inductively coupled plasma mass spectrometry was developed, characterized, and validated. Samples of isolated DNA and exosome fractions from human ovarian (2008) and melanoma (T289) cancer cell lines were used. To keep the sample consumption to approximately 10 microl and obtain a high robustness of the system, a flow injection sample introduction system with a 4.6-microl sample loop was used in combination with a conventional pneumatic nebulizer and a spray chamber. The system was optimized with respect to signal/noise ratio using a multivariate experimental design. The system proved to be well suited for routine analysis of large sample series, and several hundreds of samples could be analyzed without maintenance or downtime. The detection limit of the method was 0.12 pg (26 pg/g) platinum. To avoid systematic errors from nonspectral interferences, it was necessary to use reagent matched calibration standards or isotope dilution analysis. An uncertainty budget was constructed to estimate the total expanded uncertainty of the method, giving a quantification limit of 2.3 pg (0.5 ng/g) platinum in DNA samples. The uncertainty was sufficiently low to study quantitative differences in the formation of Pt-DNA adducts after treatment with cisplatin using different exposure times and concentrations.