Analysis of mono-phosphate nucleotides as a potential method for quantification of DNA using high performance liquid chromatography–inductively coupled plasma-mass spectrometry

The determination of total deoxyribonucleic acid (DNA) concentration is of great importance in many biological and bio-medical analyses. The quantification of DNA is traditionally performed by UV spectroscopy; however the results can be affected greatly by the sample matrix. The proposed method quantifies phosphorus in digested calf thymus DNA and human DNA by high performance liquid chromatography (HPLC) combined with inductively coupled plasma mass spectrometry (ICP-MS). The method presented showed excellent baseline separation between all four DNA mono-nucleotides and 5′UMP. The ability of LC-ICP-MS to provide an internal check that only DNA derived phosphorus was counted in the assay was demonstrated by establishing a mass balance between the total phosphorous signal from undigested DNA and that from the speciated DNA. Column recoveries ranging from 95% to 99% for phosphorus resulted in a mass balance of 95% ± 0.5% for standard nucleotides, determined by LC-ICP-MS, compared to total DNA determined by flow injection coupled to ICP-MS (FI-ICP-MS). The method for quantification was validated by analysis of NIST SRM 2,372; a total speciated DNA recovery of 52.1 ng/μL, compared with an expected value of 53.6 ng/μL, was determined by external calibration. From repeat measurements, a mass balance of 97% ± 0.5% for NIST DNA was achieved. The method limits of detection for individual nucleotides were determined between 0.8 and 1.7 μg L⁻¹ (³¹P) for individual nucleotides by LC-ICP-MS, and 360 ng L⁻¹ for 5′AMP by direct nebulisation.     

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

Quadrupole-based inductively coupled–mass spectrometry (ICP-MS) with pneumatic nebulization as a means of sample introduction was employed for quantification of platinum in blood and tissue samples of rats with peritoneal carcinomatosis, receiving intraperitoneal treatment with the Pt-containing chemotherapeutic drug oxaliplatin, and in the perfusate solution used for this purpose. The Pt levels were measured for various treatment conditions, i.e., with and without supporting treatment with the drug bevacizumab and at two different temperatures. Limits of detection obtained for platinum in blood and tissue samples were 0.3 and 2.0 pg g_,⁻¹ respectively. Evaluation of drug penetration into the tumor, under different conditions of treatment, was carried out via laser ablation–ICP-MS. Quantitative mapping of the Pt distribution in tissue sections of rat was attempted relying on gelatin standards. The results show an influence of the temperature at which the treatment is carried out, while supporting administration of the drug bevacizumab did not seem to affect the results.     

One-milliliter wet-digestion for inductively coupled plasma mass spectrometry (ICP-MS): determination of platinum-DNA adducts in cells treated with platinum(II) complexes

Platinum (Pt)–DNA adducts formed by the anti-tumor agent cisplatin are recognized by the DNA mismatch repair (MMR) system. To investigate the involvement of MMR proteins including hMLH1 in the removal of these adducts, we developed a mL-scale wet-digestion method for inductively coupled plasma mass spectrometry (ICP-MS). The detection limit was 0.01 ng mL^–1 Pt, which corresponded to 2 pg Pt/g DNA when 10 g of DNA was used. The mean relative errors were 5.4% or better for a dynamic range of 0.01–10 ng mL^–1 Pt. DNA (~500 g) had no matrix effect. To improve the accuracy, DNA preparations were treated with ribonuclease and the apparent reduction in the concentration of Pt was corrected using cellular DNA levels, which were determined with Hoechst 33258. No significant differences were observed, in terms of the formation of Pt–DNA adducts or their removal over 6 h, between hMLH1-deficient HCT116 cells, a human colorectal cancer cell line, and hMLH1-complemented HCT116+ch3 cells (n=5; P>0.05), indicating that the hMLH1-dependent DNA repair systems contribute to neither the formation nor the removal of the adducts at detectable levels. In addition, approximately 19% of the adducts were removed within 6 h in both cell lines. A time course analysis (~24 h) suggested that the removal of cisplatin-generated Pt–DNA adducts follows first-order kinetics (t_1/2=32 h). The amount of Pt–DNA adduct formed by oxaliplatin in 1 h was 56% (ratio of means) of that generated by an equimolar concentration of cisplatin in HCT116. The proposed procedure could be useful for determining Pt–DNA adducts formed by Pt(II) complexes.     

Determination of cyanide in blood by electrospray ionization tandem mass spectrometry after direct injection of dicyanogold

An electrospray ionization tandem mass spectrometric (ESI-MS-MS) method has been developed for the determination of cyanide (CN^–) in blood. Five microliters of blood was hemolyzed with 50 μL of water, then 5 μL of 1 M tetramethylammonium hydroxide solution was added to raise the pH of the hemolysate and to liberate CN^– from methemoglobin. CN^– was then reacted with NaAuCl₄ to produce dicyanogold, Au(CN)₂^–, that was extracted with 75 μL of methyl isobutyl ketone. Ten microliters of the extract was injected directly into an ESI-MS-MS instrument and quantification of CN^– was performed by selected reaction monitoring of the product ion CN^– at m/z 26, derived from the precursor ion Au(CN)₂^– at m/z 249. CN^– could be measured in the quantification range of 2.60 to 260 μg/L with the limit of detection at 0.56 μg/L in blood. This method was applied to the analysis of clinical samples and the concentrations of CN^– in the blood were as follows: 7.13 ± 2.41 μg/L for six healthy non-smokers, 3.08 ± 1.12 μg/L for six CO gas victims, 730 ± 867 μg for 21 house fire victims, and 3,030 ± 97 μg/L for a victim who ingested NaCN. The increase of CN^– in the blood of a victim who ingested NaN₃ was confirmed using MS-MS for the first time, and the concentrations of CN^– in the blood, gastric content and urine were 78.5 ± 5.5, 11.8 ± 0.5, and 11.4 ± 0.8 μg/L, respectively.     

Methods for the separation and quantification of arsenic species in SRM 2669: arsenic species in frozen human urine

Two independent liquid chromatography inductively coupled plasma-mass spectrometry (LC/ICP-MS) methods for the separation of arsenic species in urine have been developed with quantification by standard additions. Seven arsenic species have been quantified in a new NIST frozen human urine Standard Reference Material (SRM) 2669 Arsenic Species in Frozen Human Urine, Levels 1 and 2. The species measured were: arsenite (As(III)), arsenate (As(V)), monomethylarsonate (MMA), dimethylarsinate (DMA), arsenobetaine (AB), arsenocholine (AC), and trimethylarsine oxide (TMAO). The purity of each arsenic standard used for quantification was measured as well as the arsenic species impurities determined in each standard. Analytical method limits of detection (L _D) for the various species in both methods ranged from 0.2 to 0.8 μg L⁻¹ as arsenic. The results demonstrate that LC/ICP-MS is a sensitive, reproducible, and accurate technique for the determination of low-level arsenic species in urine. Measurements of the arsenic species 3 years after initial production of the SRM demonstrate the stability of the arsenic species in the urine reference material.     

Simple, Sensitive, and Rapid LC–ESI-MS Method for Quantification of Mitiglinide in Human Urine

A sensitive and specific liquid chromatographic method with electrospray ionization mass spectrometry (LC–ESI-MS) has been developed and validated for identification and quantification of mitiglinide in human urine. A simple liquid–liquid extraction procedure was followed by separation on a C₁₈ column with gradient elution, and detection using a single-quadrupole mass spectrometer in selected-ion-monitoring (SIM) mode. The method was tested using six different batches of urine. Linearity was established for the mitiglinide concentrations in the range 0.005–1.0 μg mL⁻¹, with a coefficient of determination (r) of 0.9998 and good back-calculated accuracy and precision. Intra- and inter-day precision (as RSD, %) was below 10% and accuracy for mitiglinide ranged from 85 to 115%. The lower limit of quantification was reproducible at 0.002 μg mL⁻¹ for 500 μL urine. The proposed method enables unambiguous identification and quantification of mitiglinide in pre-clinical and clinical studies.     

Determination of phenazopyridine in human plasma by high performance liquid chromatography

Summary A simple, low-cost, sensitive and selective HPLC method was developed for the determination of phenazopyridine in human plasma. The method employs UV detection of phenazopyridine and of the internal Standard at 2 different wavelengths. Calibration curves were linear over a large dynamic range, i.e., within 0.05–10.0 μg mL⁻¹ with limit of quantification of 0.05 μg mL⁻¹, and a limit of detection of 0.01 μg mL⁻¹.     

Determination of thorium and uranium in activated concrete by inductively coupled plasma mass spectrometry after anion-exchange separation

A sensitive analytical method was established for the determination of Th and U in activated concrete samples. The method combines an anion-exchange separation step with an ICP-MS determination technique. In the ICP-MS measurement, a few μg mL^–1 of Al and Ca, a few ng mL^–1 of Mn, La, Ce, Nd and Pb and pg mL^–1 amounts of Li, Zr, Nb and Ba coexisting in the anion-exchange fraction of Th and U did not interfere. No adverse interference effects were observed in real sample analyses. The obtained detection limits (3σ, n = 10) of Th and U were 2.3 and 1.8 pg mL^–1, respectively. The analytical precisions for ca. 5 μg g^–1 Th and ca. 1 μg g^–1 U in real activated concrete samples were equally less than 7% RSD. The accuracies obtained by the analysis of GSJ rock standard samples were –18.1 to 0.4% for the Th determination and –14.0 to –5.7% for the U determination. The method uses the conventional absolute calibration curve. The internal standard calibration is unnecessary. Received: 14 March 1999 / Revised: 13 July 1999 / Accepted: 15 July 1999     

Size characterization and quantification of silver nanoparticles by asymmetric flow field-flow fractionation coupled with inductively coupled plasma mass spectrometry

A method for determining the size of silver nanoparticles and their quantification by asymmetric flow field-flow fractionation coupled with inductively coupled plasma mass spectrometry (ICP-MS) is proposed and was tested in consumer products. Experimental conditions were studied in detail to avoid aggregation processes or alteration of the original size distributions. Additionally, losses from sorption processes onto the channel membrane were minimized for correct quantification of the nanoparticles. Mobile phase composition, injection/focusing, and fractionation conditions were evaluated in terms of their influence on both separation resolution and recovery. The ionic strength, pH, and the presence of ionic and nonionic surfactants had a strong influence on both separation and recovery of the nanoparticles. In general, better results were obtained under those conditions that favored charge repulsions with the membrane. Recovery values of 83 ± 8% and 93 ± 4% with respect to the content of silver nanoparticles were achieved for the consumer products studied. Silver nanoparticle standards were used for size calibration of the channel. The results were compared with those obtained by photon correlation spectroscopy and images taken by transmission electron microscopy. The quantification of silver nanoparticles was performed by direct injection of ionic silver standard solutions into the ICP-MS system, integration of the corresponding peaks, and interpolation of the fractogram area. A limit of detection of 5.6 μg L^-1 silver, which corresponds to a number concentration of 1×10¹² L^-1 for nanoparticles of 10 nm, was achieved for an injection volume of 20 μL.     

GC determination of nicotine in subcutaneous adipose tissue obtained by minimal trauma tissue biopsy

Summary Nicotine has been analyzed by gas chromatography nitrogen-phosphorus detection in tissue samples obtained by repeated minimal trauma tissue biopsies from human subcutaneous adipose tissue. For sample preparation, a single extraction step of the tissue samples with chloroform was performed at 30–45°C. Calibration curves generated with spiked porcine subcutaneous adipose tissue were linear over a concentration range from 0.20 to 100 μg g⁻¹, therefore, the limit of quantification was fixed at 0.20 μg g⁻¹. The limit of detection was found to be 0.05 μg g⁻¹ adipose tissue. The recovery of nicotinespiked porcine subcutaneous adipose tissue by chloroform extraction was 100±8%. The performance of the assay was not affected by the complex lipid matrix. The method was employed for analysis in a clinical study on nicotine penetration from a transdermal delivery system through the skin of moderate smokers. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999.     

Chromatographic and spectral behaviour and detection of hepatotoxic nodularin in fish, clam, mussel and mouse tissues using HPLC analysis

Summary High-performance liquid chromatography (HPLC) was applied in the analysis of nodularin (NODLN), a potent, bioaccumulable hepatotoxin. The behaviour of NODLN in biological matrices and possibility to analyse biota samples for NODLN content was examined using a conventional HPLC/diode array detector method that uses C₁₈ solid-phase cartridge clean up. Tissues of European flounder, blue mussel (spiked and naturally contaminated), clam (exposed to NODLN in an aquariuml and mouse (subjected to i. p. administration of NODLN) were analysed. UV detection was 5 times more sensitive than electrochemical detection. Recovery of NODLN from spiked tissues was 59% for mussel, 53% for flounder, and 44–75% for mouse tissues. NODLN was detected in clams exposed with NODLN, but not in naturally contaminated mussels where NODLN conjugation occurs. Through the use of spectral processing, free NODLN was unambiguously identified from tissue samples. The HPLC method showed limits of quantification between 90 and 150 μg NODLN kg⁻¹ dw. The method proved applicable for routine tissue analysis and can be used in the monitoring of acutely toxic NODLN levels.     

In vivo detection of DNA adducts induced by cisplatin using capillary HPLC–ICP-MS and their correlation with genotoxic damage in Drosophila melanogaster

The antitumoral effect of cisplatin [cis-diamminodichloroplatinum(II)] in mammals is related to its binding to DNA components. However, there is a lack of specific chemical methods to selectively detect those adducts formed in vivo at low concentrations. In this work, a new sensitive and selective method of determining cisplatin–DNA adducts based on the use of element-selective mass spectrometry is proposed, and the method is then applied to detect cisplatin adducts induced in vivo in somatic cells of Drosophila melanogaster. The bioanalytical strategy proposed here allows the determination of the most important DNA adduct formed between adjacent guanine units of the same DNA strand with cisplatin, and it is based on the coupling of capillary liquid chromatography (cap-LC) to inductively coupled plasma mass spectrometry (ICP-MS). This set-up allows the simultaneous monitoring of the Pt (from the drug) and P (from the DNA components) present in these adducts, once they have been cleaved by enzymatic hydrolysis of the DNA samples. Using this instrumental set-up, the adducts of cisplatin formed in vivo when D. melanogaster flies are exposed to different cisplatin concentrations can be detected and their concentration determined. The results obtained show a direct correlation between the concentration of cisplatin adducts, the induced genotoxic damage (measured as DNA strand breaks using the Comet assay) and the cisplatin concentration. Figure The work illustrates the complementary use of bioanalytical and biological information to study cisplatin interactions with DNA is vivo at biologically relevant concentrations of the drug     

MEKC Determination and Pharmacokinetic Study of Danshensu in Rabbits after Intragastric Administration of the Aqueous Extract from Danshen

A micelle eletrokinetic capillary chromatography (MEKC) method was used to determine Danshensu in rabbit blood plasma and tissues (liver, lung, kidney, brain, heart and spleen). The separation was achieved with a buffer consisting of 30 mmol L⁻¹ borax and 50 mmol L⁻¹ SDS (pH 9.0), and with an applied voltage of 7.0 kV. Validation of the method showed good sensitivity, reproducibility and precision. The calibration curve for Danshensu was linear over the concentration range of 0.4–400 μg mL⁻¹. The limit of detection (LOD) was 0.08 μg mL⁻¹ (S/N = 3). The validated method has been successfully applied for the pharmacokinetic and the tissue distribution studies of Danshensu after intragastric administration of the aqueous extract from traditional Chinese medicine Danshen.     

Determination of gallium originated from a gallium-based anticancer drug in human urine using ICP-MS

Urine analysis gives an insight into the excretion of the administered drug which is related to its reactivity and toxicity. In this work, the capability of inductively coupled plasma mass spectrometry (ICP-MS) to measure ultratrace metal levels was utilized for rapid assaying of gallium originating from the novel gallium anticancer drug, tris(8-quinolinolato)gallium(III) (GaQ₃), in human urine. Sample dilution with 1% (v/v) HNO₃ as the only required pre-treatment was shown to prevent contamination of the sample introduction system and to reduce polyatomic interferences from sample components. The origin of the blank signal at masses of gallium isotopes, 71 and 69, was investigated using high-resolution ICP-MS and attributed, respectively, to the formation of ³⁶Ar³⁵Cl⁺ and ⁴⁰Ar³¹P⁺ ions and, tentatively, to a triplet of doubly charged ions of Ba, La, and Ce. The accuracy and precision performance was tested by evaluating a set of parameters for analytical method validation. The developed assay has been applied for the determination of gallium in urine samples spiked with GaQ₃. The achieved recoveries (95–102%) and quantification limit of 0.2 μg L⁻¹ emphasize the practical applicability of the presented analytical approach to monitor renal elimination of GaQ₃ at all dose levels in clinical trials that are currently in progress.     

Gas chromatographic-mass spectrometric assay for 2-methoxyethanol and 2-methoxyacetic acid in human plasma and its application to pharmacokinetic studies

Summary A new assay has been developed for simultaneous measurement of 2-methoxyethanol (ME) and its major toxic metabolite, 2-methoxyacetic acid (MAA) in human blood by gas chromatography-mass spectrometry. Over the concentration range 0.3 to 200 μgml⁻¹, ME and MAA could be determined with a pooled coefficient of variation (eleven concentrations, six replicate samples) of 6.5% and 8.7%, respectively. The limits of quantification of ME and MAA were 0.19 and 0.05μg mL⁻¹, respectively. The respective mean (SD) recoveries of ME and MAA were 101.3 (2.6)% and 98.2 (4.9)% (n=3). A study of the effects of human contact with ME liquid showed that this new method is a specific, sensitive, simple, and reliable method both for the pharmacokinetic studies and for the biological monitoring of occupational exposure to ME.     

Platinum concentration in silicone breast implant material and capsular tissue by ICP-MS

Inductively coupled plasma-mass spectrometry (ICP-MS) was used to determine the concentration of platinum (Pt) in silicone breast implant gel (range, 0.26–48.90 g g^–1 Pt; n=15), elastomer (range, 3.05–28.78 g g^–1 Pt; n=7), double lumen (range, 5.79–125.27 g g^–1 Pt; n=7), foam (range, 5.79–8.36 g g^–1 Pt; n=2), and capsular tissue (range, 0.003–0.272 g g^–1 Pt; n=15). The results show that very high levels of Pt are present in the encasing elastomer, double lumen, and foam envelope materials. Silicone breast implants can be a source of significant Pt exposure for individuals with these implants.     

Simultaneous Determination of Baicalin, Baicalein, Wogonin, Oxysophocarpine, Oxymatrine and Matrine in the Chinese Herbal Preparation of Sanwu-Huangqin-Tang by Ion-Paired HPLC

A sensitive and reliable ion-paired high-performance liquid chromatographic method has been established for the simultaneous quantification of six major active ingredients, namely baicalin, baicalein, wogonin, oxysophocarpine, oxymatrine and matrine in the Chinese herbal preparation, Sanwu-Huangqin-Tang. HPLC analyses were performed on a Phenomenex luna C₁₈ column with mobile phase of methanol–acetonitrile–aqueous phosphoric acid at a flow rate of 0.9 mL min⁻¹. The complete separation was achieved within 35 min for the six target constituents. A good linear regression relationship between peak-areas and concentrations was obtained over the range of 12.10–242.0 μg*mL⁻¹ for baicalin, 5.05–101.0 μg*mL⁻¹ for baicalein, 0.95–19.0 μg*mL⁻¹ for wogonin, 2.75–55.0 μg*mL⁻¹ for oxysophocarpin, 2.75–55.0 μg*mL⁻¹ for oxymatrine and 4.90–98.0 μg*mL⁻¹ for matrine, respectively. The repeatability was evaluated by intra- and inter-day assays with relative standard deviation (RSD) being less than 5.1%. The recoveries, measured at three concentration levels, varied from 93.8 to 102.1%. The assay was successfully applied for determination of six bioactive compounds in Sanwu-Huangqin-Tang. The interaction of chemical constituents was observed when the herbs were used in compatibility. The results indicated that the developed assay method was rapid, accurate and could be readily utilized as a quality control method for Sanwu-Huangqin-Tang.     

Determination of total and non-water soluble iodine in atmospheric aerosols by thermal extraction and spectrometric detection (TESI)

Iodine has recently been of interest in atmospheric chemistry due to its role in tropospheric ozone depletion, modification of the HO/HO₂ ratio and aerosol nucleation. Gas-phase iodine chemistry is tightly coupled to the aerosol phase through heterogeneous reactions, which are dependent on iodine concentrations and speciation in the aerosol. To date, the only method available for total iodine determination in aerosols is collection on filters by impaction and quantification by neutron activation analysis (NAA). NAA is not widely available to all working groups and is costly to commission. Here, we present a method to determine total iodine concentrations in aerosol impact filter samples by combustion of filter sub-samples (∼5 cm²) at 1,000 °C, trapping in deionised water and quantification by UV/Vis spectroscopy. Both quartz and cellulose filters were analysed from four separate sampling campaigns. The method proved to be sensitive (3σ = 6 ng absolute iodine ≈ 3 pmol m⁻³) precise (RSD ∼ 5%) and accurate, as determined by external and standard addition calibrations. Total iodine concentrations ranged from 10 pmol m⁻³ over the Southern Ocean to 100 pmol m⁻³ over the tropical Atlantic, in agreement with previous estimates. The soluble iodine concentration (extracted with water and measured by ICP-MS) was then subtracted from the total iodine to yield non-water-soluble iodine (NSI). The NSI fraction ranged from 20% to 53% of total iodine, and thus can be significant in some cases.     

Quantitative trace analysis of a broad range of antiviral drugs in poultry muscle using column-switch liquid chromatography coupled to tandem mass spectrometry

A liquid chromatography–tandem mass spectrometry method for the analysis of seven antiviral drugs, zanamivir, ribavirin, oseltamivir, oseltamivir carboxylate, amantadine, rimantadine and arbidol, in poultry muscle is reported. The antiviral drugs were extracted from the homogenized poultry muscle sample using methanol. The extract was purified using tandem solid-phase extraction combining a cation exchange cartridge and a phenylboronic acid cartridge. To prevent excessive matrix effects, the analytes were separated from the matrix constituents using a column-switch liquid chromatography system combining a reversed-phase and a Hypercarb analytical column. Detection was carried out using tandem mass spectrometry. The method was fully validated according to 2002/657/EC [1] and proved to be adequate for quantification and confirmation of zanamivir and ribavirin at 10 μg kg⁻¹, oseltamivir, oseltamivir carboxylate, amantadine and rimantadine at levels below 1.0 μg kg⁻¹ and for qualitative confirmatory analysis of arbidol at levels below 1 μg kg⁻¹.     

Determination of bisphenol-a and related compounds in human saliva by gas chromatography—mass spectrometry

Summary A simple and sensitive method for the determination of trace amounts of bisphenol-A (BPA), bisphenol-A diglycidyl dimethacrylate (bis-GMA), bisphenol-A dimethacrylate (bis-DMA) and triethyleneglycol dimethacrylate (TEGDMA) in human saliva is proposed. These materials are used in dental restorations, as composites and sealants, and are sometimes detected in human saliva after dental treatment. The proposed method involves protein precipitation using acetonitrile followed by acidification, evaporation of the solvent and dissolution with dichloromethane prior to injection into a GC-MS. Thermal derivatization in the injection system was used for the identification and quantification of bis-GMA. Clean-up is not necessary using SIM mode. Bisphenol-F (BPF) was used as internal standard. The linear range was 15 to 1000 μg·L⁻¹ for BPA, 50 to 10 000 μg·L⁻¹ for bis-GMA, 50 to 1000 μg·L⁻¹ for bis-DMA and 1 to 100 μg·L⁻¹ for TEGDMA. The detection limits were 3,15,10 and 0.3 μg·L⁻¹ for BPA, bis-GMA, bis-DMA and TEGD-MA, respectively. Validation of the proposed method was carried out by using the standard addition methodology. Samples of 10 mL of human saliva collected 1 h after dental treatment were analysed in order to assess the applicability of the method to detect and quantify such compounds originated from methacrylic resins used in odontological treatment.