Investigation of the biotransformation of melarsoprol by electrochemistry coupled to complementary LC/ESI–MS and LC/ICP–MS analysis

Melarsoprol is the only currently available drug for treatment of the late stage of African trypanosomiasis (sleeping sickness). Unfortunately, the arsenic-containing drug causes serious side effects, for which the mechanisms have not been elucidated so far. This investigation describes the study of the melarsoprol biotransformation processes by electrochemical (EC) techniques. Based on EC, potential oxidation reactions of melarsoprol are examined. Moreover, the reactivity of melarsoprol, its metabolite melarsen oxide, and their oxidation products toward the tripeptide glutathione and the proteins hemoglobin and human serum albumin is evaluated. The combination of different analytical techniques allows the identification as well as the quantification of the biotransformation products. The hyphenation of liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI–MS) is applied for identification and structure elucidation, which implies the determination of exact masses and fragmentation patterns. For the selective detection of arsenic containing metabolites, LC coupled to inductively coupled plasma mass spectrometry is utilized. Based on the obtained data, the oxidative biotransformation of melarsoprol can be predicted, revealing novel species which have been suspected, but not been identified up to now. The results of the protein studies prove that melarsen oxide, the active derivative of melarsoprol, strongly binds to human hemoglobin and forms different adducts via the free cysteinyl groups of the hemoglobin α- and β-chain.     

Capillary HPLC–ICP MS mapping of selenocompounds in spots obtained from the 2-D gel electrophoresis of the water-soluble protein fraction of selenized yeast

A method based on ICP collision-cell MS detection in capillary HPLC was developed to gain an insight into the purity and identity of selenium-containing proteins separated by 1-D and 2-D electrophoresis. The bands and spots obtained after the separation of water-soluble proteins in selenized yeast were digested with trypsin prior to chromatography. Selenium could be detected down to the subpicogram level. The method, assisted by information obtained by MALDI TOF MS on the 5000 Da cut-off fraction, permitted the purity of bands and spots to be estimated and the efficiency of tryptic digestion and the quantity of selenium present in individual peptides to be evaluated. Owing to the high sensitivity and the lack of matrix suppression effects, the method provided chromatograms with signal-to-noise ratios of 10–1000 in conditions where the common ES Q–TOF MS detection failed.      

Investigation of Matrix Effects on the Determination of Carbadox and Olaquindox in Feed by LC–MS/MS

A comprehensive survey of matrix effects on the LC–MS/MS analysis of the banned antibiotic growth promoters carbadox and olaquindox in feed was carried out. Various factors of sample preparation procedure and measurement were systematically investigated by pre- and post-extraction addition and postcolumn infusion experiments. In general, strong signal suppression up to 70 % for carbadox and up to 90 % for olaquindox was observed when using different extraction solvents and techniques as well as different chromatographic conditions. Reduction of matrix effects was achieved by SPE clean-up and dilution of sample extracts. Nevertheless, matrix effect profiles determined by postcolumn infusion revealed, that reduction of signal suppression at a respective retention time cannot guarantee improvement of the methods performance. If high variability of matrix effects is present along the chromatographic run, accuracy might decrease despite reduced signal suppression. Besides method parameters, different feedingstuffs were investigated and showed similar matrix effects.

     

Investigation of Matrix Effects on the Determination of Carbadox and Olaquindox in Feed by LC–MS/MS

A comprehensive survey of matrix effects on the LC–MS/MS analysis of the banned antibiotic growth promoters carbadox and olaquindox in feed was carried out. Various factors of sample preparation procedure and measurement were systematically investigated by pre- and post-extraction addition and postcolumn infusion experiments. In general, strong signal suppression up to 70 % for carbadox and up to 90 % for olaquindox was observed when using different extraction solvents and techniques as well as different chromatographic conditions. Reduction of matrix effects was achieved by SPE clean-up and dilution of sample extracts. Nevertheless, matrix effect profiles determined by postcolumn infusion revealed, that reduction of signal suppression at a respective retention time cannot guarantee improvement of the methods performance. If high variability of matrix effects is present along the chromatographic run, accuracy might decrease despite reduced signal suppression. Besides method parameters, different feedingstuffs were investigated and showed similar matrix effects.     

Investigation of Euphrasia rostkoviana Hayne Using GC–MS and LC–MS

Gas and liquid chromatography coupled with mass spectrometry was applied to solve difficulties and reinvestigate the serious matrix problems affecting analysis of the active compounds in Euphrasia rostkoviana Hayne. The main groups of compounds were obtained by extracting the herb stepwise with n-hexane, chloroform, ethyl acetate and methanol. Polyamide column chromatography facilitated further separation. Phenolic/flavonoid- and terpenoid-type molecules were studied by GC–MS, HPLC and LC–MS–MS. The β-sitosterol content of the herb was determined by gas chromatography with flame ionisation detection (GC-FID). Caffeic acid, chlorogenic acid, coumaric acid and flavonoid glycosides of apigenin, luteolin, rhamnetine (hexoside), kaempferol (both hexoside and rutinoside) and quercetin (rutinoside) were identified in the fractions of the methanolic extract.     

Determination of the MRI contrast agent Gd-DTPA by SEC–ICP–MS

The simultaneous determination of Gd³⁺ and Gd-DTPA (DTPA: diethylenetriamino-pentaacetic acid), often used as contrast agent, is described. The proposed approach combines size-exclusion chromatography (SEC) and inductively coupled plasma–mass spectrometry (ICP–MS) for element-selective detection in order to determine also high-molecular Gd-complexes if present. This method was applied to the analysis of urine samples of a patient to whom Gd-DTPA was intravenously administered. The results showed that no conversion or adsorption of Gd-DTPA could be observed in any sample, even free Gd³⁺ could not be detected. Urine excretion behaviour was monitored and it was proved that Gd-DTPA was almost completely (>99%) excreted by urination within one day. Traces of Gd-DTPA could be measured in hair samples, but extraction with tetramethylammonium hydroxide (TMAH) resulted in degradation of Gd-DTPA.     

Determination of Pitavastatin in Human Plasma by LC–MS–MS

A simple and rapid LC–MS–MS assay was developed and validated for the quantitative determination of pitavastatin in human plasma. Sample pretreatment involved simple protein precipitation by addition of acetonitrile. Separation was on an Agilent 1.8 μm Zorbax SB-C18 column (150 mm × 4.6 mm) at 25 °C using isocratic elution with methanol–0.1% formic acid in water (85:15, v/v) at a flow rate of 0.4 mL min^?1. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the ion transitions m/z 422.0 → 290.1 for pitavastatin, and m/z 330.1 → 192.1 for paroxetine (IS). LC–MS–MS was found to improve the quantitation of pitavastatin in plasma and was successfully applied in pharmacokinetic studies.     

Determination of adrenolytic drugs by SPME–LC–MS

Five adrenolytic drugs have been analyzed by liquid chromatography–mass spectrometry (LC–MS). Samples were prepared by solid-phase microextraction (SPME) using polypyrrole fibers coated on stainless steel support as an adsorbent for the drugs. Adsorption efficiencies were 95% and were close for all the drugs investigated. Relative standard deviations (RSD), calculated for samples prepared in standard solutions, were in the range 2.5–13%, however RSD values for the drugs in human plasma were 2.5–4.5%. Using LC–MS the limit of detection (LOD) and the limit of quantification (LOQ) were in the ranges 0.11–0.18 and 0.39–0.54 ng mL⁻¹, respectively, for the five drugs.     

Advantages of LC–MS–MS compared to LC–MS for the determination of nitrofuran residues in honey

In the framework of developing analyses for exogenous contaminants in food matrices such as honey, we have compared data obtained by high-performance liquid chromatography coupled with mass spectrometry (LC–MS) to those provided by high-performance liquid chromatography and tandem mass spectrometry (LC–MS–MS). Initial results obtained with LC–MS showed that the technique lacked selectivity, which is why the method was validated by LC–MS–MS. This method involves a solid-phase extraction (SPE) of nitrofuran metabolites and nitrofuran parent drugs, a derivatization by 2-nitrobenzaldehyde for 17 h, and finally a clean-up by SPE. The data obtained show that the limits of detection varied between 0.2 and 0.6 μg kg⁻¹ for the metabolites and between 1 and 2 μg kg⁻¹ for nitrofuran parent drugs. The method was applied to different flower honeys. The results showed that nitrofurans (used as antibiotics) are consistently present in this matrix, the predominant compound being furazolidone. Figure Working bees     

Analysis of Nanafrocin in Foodstuffs of Animal Origin by LC–MS–MS

A new, rapid, and efficient method, multiple reaction monitoring liquid chromatography–tandem mass spectrometry, has been developed for analysis of nanafrocin in foodstuffs of animal origin. The researchers used a C₁₈ stationary phase coupled with triple-quadrupole tandem mass spectrometry in negative-electrospray mode. The limits of detection (LOD) and quantification (LOQ) were 0.005 and 0.01 mg kg^?1, respectively, in the matrixes. Detector response was found to be a linear function of concentration over the range 0.005–0.1 mg kg^?1 in each matrix. Mean overall recovery (n = 10) of nanafrocin varied from 71 to 101%. The results show that identification and quantification of nanafrocin residues in foodstuffs of animal origin can be successfully achieved by use of the proposed LC–MS–MS method.     

Sensitive redox speciation of neptunium by CE–ICP–MS

Capillary electrophoresis (CE) was used to separate the neptunium oxidation states Np(IV) and Np(V), which are the only oxidation states of Np that are stable under environmental conditions. The CE setup was coupled to an inductively coupled plasma mass spectrometer (Agilent 7500ce) using a Mira Mist CE nebulizer and a Scott-type spray chamber. The combination of the separation capacity of CE with the detection sensitivity of inductively coupled plasma mass spectrometry (ICP-MS) allows identification and quantification of Np(IV) and Np(V) at the trace levels expected in the far field of a nuclear waste repository. Limits of detection of 1?×?10^-9 and 5?×?10^-10 mol L^-1 for Np(IV) and Np(V), respectively, were achieved, with a linear range from 10^-9 to 10^-6 mol L^-1. The method was applied to study the redox speciation of the Np remaining in solution after interaction of 5?×?10^-7 mol L^-1 Np(V) with Opalinus Clay. Under mildly oxidizing conditions, a Np sorption of 31% was found, with all the Np remaining in solution being Np(V). A second sorption experiment performed in the presence of Fe²⁺ led to complete sorption of the Np onto the clay. After desorption with HClO₄, a mixture of Np(IV) and Np(V) was found in solution by CE–ICP–MS, indicating that some of the sorbed Np had been reduced to Np(IV) by Fe²⁺.     

Trace analysis of high-purity graphite by LA–ICP–MS

Laser-ablation inductively coupled plasma mass spectrometry (LA–ICP–MS) has been established as a very efficient and sensitive technique for the direct analysis of solids. In this work the capability of LA–ICP–MS was investigated for determination of trace elements in high-purity graphite. Synthetic laboratory standards with a graphite matrix were prepared for the purpose of quantifying the analytical results. Doped trace elements, concentration 0.5 μg g^–1, in a laboratory standard were determined with an accuracy of 1% to ± 7% and a relative standard deviation (RSD) of 2–13%. Solution-based calibration was also used for quantitative analysis of high-purity graphite. It was found that such calibration led to analytical results for trace-element determination in graphite with accuracy similar to that obtained by use of synthetic laboratory standards for quantification of analytical results. Results from quantitative determination of trace impurities in a real reactor-graphite sample, using both quantification approaches, were in good agreement. Detection limits for all elements of interest were determined in the low ng g^–1 concentration range. Improvement of detection limits by a factor of 10 was achieved for analyses of high-purity graphite with LA–ICP–MS under wet plasma conditions, because the lower background signal and increased element sensitivity. Received: 4 January 2001 / Revised: 27 March 2001 / Accepted: 28 March 2001     

Determination of Fenofibric Acid in Human Plasma by LC–MS/MS and LC–UV

A sensitive, high-throughput and economic liquid chromatographic method for determination of fenofibric acid in human plasma was developed and validated by ultraviolet detection and tandem mass spectrometry, then applied in pharmacokinetic study to investigate Lipanthyl™ 200 mg MC bioavailability under food and fasting conditions. Fenofibric acid with 2-chloro fenofibric acid-d6 (internal standard) was extracted from 100 µL of human plasma by acetonitrile in a single extraction step. 25 and 2 µL from supernatant were injected onto ACE column, 50 mm, 5 micron with 4.6 mm inner diameter for LC–UV and 2.1 mm for LC–MS/MS, and both systems were eluted isocratically by water:methanol:formic acid (35:65:0.1, v/v/v), with a constant flow rate of 1 mL min⁻¹. The established calibration curve was linear between 0.05–20 µg mL⁻¹, and the within- and between-day precisions were all below 13 % in both LC–MS/MS and LC–UV systems during validation, and accuracies ranged between 91 and 112 %. Twenty-eight healthy adult subjects participated in this clinical study, and the pharmacokinetic parameters including coefficient of variation were calculated and discussed. A dramatic decrease in C _max and AUC0-72 (3.63- and 1.85-fold, respectively) were observed for Lipanthyl™ MC under fasting conditions with more variable inter subject measurements comparing to the fed state.

     

A study of the extraction of plutonium from planchets by Aridus–ICP–SFMS

Two extraction processes of plutonium (Pu) on planchets from alpha spectrometry (AS) have been evaluated by inductively coupled plasma sector field mass spectrometry with a desolvator system (Aridus–ICP–SFMS). The samples were traced with known concentrations of ²³⁹Pu (1.2 × 10³ fg) and ²⁴²Pu (2 × 10³ fg) followed by an electrodeposition in planchets, according to the Hallstadius method. The processes of extraction were carried out with 50 mL of 0.36 mol L^?1 HNO₃ every 30 min up to 180 min in a glass beaker at 60 °C. The first process was on a hotplate and the second used an ultrasonic system. Finally, samples were evaporated to dryness, and resuspended in 10 mL of 0.72 mol L^?1 HNO₃ for evaluation. The results showed that at 120 min, a ~70 % recovery of ²³⁹Pu and a ~80 % recovery of ²⁴²Pu in both processes were obtained. The average recoveries of ²³⁹Pu and ²⁴²Pu at 180 min using the hotplate in plate were 93.4 ± 4.6 and 93.7 ± 4.2 % respectively, and with the ultrasonic system were 96.0 ± 4.3 and 98.2 ± 1.0 % respectively. In conclusion, both processes are suitable for Pu extraction, and Aridus–ICP–SFMS is an essential technique for the reassessments and quantification of Pu. In addition, procedural blanks spiked with 1 × 10² fg mL^?1 U were prepared for each process, in order to study the contribution of the ²³⁸U on the background signal at m/z = 239, which was 0.5 ± 0.2 cps, indicating that the contribution of ²³⁸U on the ²³⁹Pu signal was negligible. Furthermore, this methodology can be applied to sample planchets with environmental, food, biological and nuclear origin, and thereby to avoid repetitive analysis when Pu concentration determined by AS are under minimum detectable activities.     

Separation and identification of selenotrisulfides in epithelial cell homogenates by LC–ICP–MS and LC–ESI-MS after incubation with selenite

To elucidate how selenite is metabolised in the intestine after oral intake, it was incubated with homogenized epithelial cells from pigs. When the metabolites were analysed by LC–ICP–MS, two major selenium metabolites were separated in the supernatant from the homogenate. These metabolites were formed instantly but disappeared within 15 min. No other selenium-containing compounds appeared during this time. Hence, the secondary reaction products were either volatilised or precipitated. To verify the identity of the compounds, a larger amount of selenite was incubated with epithelial cells. The presence of Cys-Se-SG and GS-Se-SG was verified by LC–ESI-MS. Selenotrisulfides were synthesized by reaction of L-cysteine and L-glutathione with sodium selenite. The reaction mixture contained three main products: selenodicysteine (Cys-Se-Cys), selenocysteine glutathione (Cys-Se-SG), and selenodiglutathione (GS-Se-SG). The two transient selenium compounds in the epithelial cell incubation mixture co-eluted with the synthesized Cys-Se-SG and GS-Se-SG, respectively. The identities of these compounds were verified by LC–ESI-MS. Hence, these selenium metabolites have now been identified by ESI-MS after isolation from epithelial cells.     

Determination of Eleutherosides in Dietary Supplements by LC–MS/MS

A simple and specific method for the simultaneous determination of eleutherosides B and E in powdered rhizomes of Eleutherococcus senticosus extract and in solid and liquid dietary supplements was developed and validated. E. senticosus extracts, often mixed with other plants or herbal extracts, are widely used in food supplements because of the tonic and adaptogenic activities referred to the eleutherosides B and E. In this study, samples were analyzed by a liquid chromatography-electrospray tandem mass spectrometry (LC–ESI-MS/MS) method operated in single reaction monitoring (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values were fixed at 3 μg L^?1 and 10 μg L^?1, respectively, whereas linearity was established within 10–1,000 μg L^?1 range for both compounds. Good precision was obtained for both eleutherosides in terms of intra-day precision (RSD % lower than 4 %) and inter-day precision (RSD % lower than 6 %). Good percentage recoveries were obtained for both eleutherosides (91.5–103.6 %). Finally, the developed method was successfully applied to analyze a number of solid and liquid commercial dietary supplements containing E. senticosus extracts, also mixed with other herbal extracts.     

Determination of Amphotericin B in Human Cerebrospinal Fluid by LC–MS–MS

A simple, specific and sensitive column liquid chromatography–tandem mass spectrometry method has been developed for the determination of amphotericin B in human cerebrospinal fluid. Samples were prepared by dilution with methanol and quantitated by MS–MS detection in the positive mode. The determination was validated in the concentration range of 0.5–100 ng mL^?1 using 100 μL of human cerebrospinal fluid. The method was successfully used to support routine therapeutic drug monitoring.     

Development of analytical procedures for determination of total chromium by quadrupole ICP–MS and high-resolution ICP–MS,and hexavalent chromium by HPLC–ICP–MS,in different materials used in the automotive industry

A European directive was recently adopted limiting the use of hazardous substances such as Pb, Hg, Cd, and Cr(VI) in vehicle manufacturing. From July 2003 a maximum of 2 g Cr(VI) will be authorised per vehicle in corrosion-preventing coatings of key components. As no standardised procedures are available to check if produced vehicles are in agreement with this directive, the objective of this work was to develop analytical procedures for total chromium and Cr(VI) determination in these materials. The first step of this study was to optimise digestion procedures for total chromium determination in plastic and metallic materials by inductively coupled plasma mass spectrometry (ICP–MS). High resolution (HR) ICP–MS was used to examine the influence of polyatomic interferences on the detection of the ⁵²Cr⁺ and ⁵³Cr⁺ isotopes. If there was strong interference with m/z 52 for plastic materials, it was possible to use quadrupole ICP–MS for m/z 53 if digestions were performed with HNO₃+H₂O₂. This mixture was also necessary for digestion of chromium from metallic materials. Extraction procedures in alkaline medium (NH₄⁺/NH₃ buffer solution at pH 8.9) assisted by sonication were developed for determining Cr(VI) in four different corrosion-preventing coatings by HPLC–ICP–MS. After optimisation and validation with the only solid reference material certified for its Cr(VI) content (BCR 545; welding dusts), the efficiency of this extraction procedure for screw coatings was compared with that described in the EN ISO 3613 standard generally used in routine laboratories. For coatings comprising zinc and aluminium passivated in depth with chromium oxides the extraction procedure developed herein enabled determination of higher Cr(VI) concentrations. This was also observed for the screw covered with a chromium passivant layer on zinc–nickel. For coating comprising a chromium passivant layer on alkaline zinc the standardized extraction procedure was more efficient. In the case of painted metallic plate, because of a reactive matrix towards Cr(VI), its extraction without degradation was difficult to perform.