Kayoko Minakata

Simple and selective determination of arsenite and arsenate by electrospray ionization mass spectrometry

Arsenic pollution of public water supplies has been reported in various regions of the world. Recently, some cancer patients are treated with arsenite (As(III)); most Japanese people consume seafoods containing large amounts of negligibly toxic arsenic compounds. Some of these arsenic species are metabolized, but some remain intact. For the determination of toxic As(III), a simple, rapid and sensitive method has been developed using electrospray ionization mass spectrometry (ESI-MS). As(III) was reacted with a chelating agent, pyrrolidinedithiocarbamate (PDC, C(4)H(8)NCSS(-)) and tripyrrolidinedithiocarbamate-arsine, As(PDC)(3), extracted with methyl isobutyl ketone (MIBK). A 1 microL aliquot of MIBK layer was directly injected into ESI-MS instrument without chromatographic separation, and was detected within 1 min. Arsenate (As(V)) was reduced to As(III) with thiosulfate, and then the total inorganic As was quantified as As(III). This method was validated for the analysis of urine samples. The limit of detection of As was 0.22 microg L(-1) using 10 microL of sample solution, and it is far below the permissible limit of As in drinking water, 10 microg L(-1), recommended by the WHO. Results were obtained in <10 min with a linear calibration range of 1-100 microg L(-1). Several organic arsenic compounds in urine did not interfere with As(III) detection, and the inorganic As in the reference materials SRM 2670a and 1643e were quantified after the reduction of As(V) to As(III).

Application of electrospray ionization tandem mass spectrometry for the rapid and sensitive determination of cobalt in urine

Recently, cobalt (Co) is reported to be taken as a supplement by athletes for improving anaerobic performance. For the diagnosis of abuse, the limit of detection (LOD) of Co in the analysis should be lower than the concentrations of Co in plasma and urine of normal persons. A simple, rapid and sensitive method has been developed for the determination of Co in urine. Co was complexed with diethyldithiocarbamate (DDC) and extracted with isoamyl alcohol in the presence of citric acid. The detection of Co was achieved by injecting a 1-microL aliquot of isoamyl alcohol containing Co-DDC complex directly into an electrospray ionization tandem mass spectrometric (ESI-MS-MS) instrument without chromatographic separation. The quantification was performed using selected reaction monitoring at m/z 291 of the product ion Co(C(4)H(10)NCS)(2)(+) which was produced by collision-induced dissociation from the precursor ion Co(DDC)(2)(+) at m/z 355. ESI-MS-MS data were obtained in less than 10 min with an LOD of 0.05 microg L(-1) and a linear calibration range of 0.1-100 microg L(-1) using 10 microL of urine. The procedure was validated with certified reference materials (SRM 2670a and SRM 1643e). This method is suitable for the analysis of Co in the laboratories already equipped with an ESI-MS-MS instrument.

Sensitive determination of arsenite and arsenate in plasma by electrospray ionization tandem mass spectrometry after chelate formation

Inorganic arsenite (As3+) and arsenate (As5+) are well-known poisons, and the toxicity of As3+ is about ten times that of As5+. In this study, a simple, rapid, and sensitive method was developed for As3+ in plasma using electrospray ionization (ESI) tandem mass spectrometry (MS-MS). After washing plasma with trichloroethylene (TCE), As3+ in the aqueous layer was reacted with pyrrolidinedithiocarbamate (PDC, C4H8NCSS-), and the produced As(PDC)3 was extracted with methyl isobutyl ketone (MIBK); a 1-µl aliquot of the MIBK layer containing As(PDC)3 was introduced into the MS-MS instrument in the direct-flow injection mode. Other arsenic compounds such as As5+, monomethyl arsonic acid, dimethyl arsinic acid, arsenobetaine, arsenocholine, and tetramethyl arsonium did not produce As(PDC)3. Therefore, without liquid chromatographic separation, As3+ alone could be detected after washing with TCE followed by solvent extraction of As(PDC)3 with MIBK. Thus, inorganic As5+ was reduced to As3+ with thiosulfate, and then the total inorganic As was quantifi ed as As3+; As5+could be calculated by subtracting As3+from the total inorganic As. The MS-MS quantification was performed by selected reaction monitoring using a peak at m/z 114 of a product ion (C4H8NCS)+ formed by collision-induced dissociation from the precursor ion As(PDC)2+ at m/z 367. The mass spectral identification on MS-MS spectrum was possible even at 1 ng As3+/ml plasma. The calibration curve for As3+ showed linearity from 0.5 to 100 ng/ml plasma. The limits of detection by selected reaction monitoring were 0.3 ng/ml in water and 0.2 ng/ml in plasma. The analysis could be completed in less than 15 min, because chromatographic separation was not necessary before the MS-MS detection.

Determination of Urine Luck in urine using electrospray ionization tandem mass spectrometry

A simple, rapid and sensitive method using tandem mass spectrometry (MS-MS) has been developed for the determination of chromate Cr6+ in urine. Cr6+ is a substantial component of Urine Luck, which is used to conceal the presence of drugs in urine. Cr6+ was complexed with diethyldithiocarbamate (DDC) and extracted with isoamyl alcohol in the presence of citric acid. Then a 1-μl aliquot of isoamyl alcohol containing Cr-DDC complex was directly injected into an MS-MS instrument without chromatographic separation. The quantification was performed using selected reaction monitoring at m/z 363.8 of product ion CrO(DDC)2+ obtained by collision-induced dissociation from the precursor ion, CrOH(DDC)3+ at m/z 513.1. This method was validated with the analysis urine samples obtained from volunteers. A linear calibration curve could be obtained in the range of 0.18–100 ng/ml. The limits of detection and quantification of Cr6+ were 0.05 and 0.18 ng/ml, respectively, using only 10 μl of urine. Results could be obtained in less than 10 min for a sample. After oxidation of Cr3+ to Cr6+, near 100% recovery was confirmed using standard reference materials such as SRM 2670a (low level and high level) and SRM 1643e. The most outstanding advantage of this ESI-MS-MS method is that it gives excellent product ion mass spectra for identification of Cr6+.

Platinum levels in various tissues of a patient who died 181 days after cisplatin overdosing determined by electrospray ionization mass spectrometry

Platinum (Pt) levels were determined in various tissues and body fluids obtained from a patient who died 181 days after cisplatin overdosing. The symptoms of cisplatin overdose, however, might have almost disappeared by day 40, and the patient’s death was ascribed to the recurrence of malignant lymphoma. Determination of Pt derived from cisplatin was performed by electrospray ionization mass spectrometry (ESI-MS) using silver (Ag) as internal standard. Pt and Ag complexed with diethyldithiocarbamate (DDC) in wetashed blood, and tissue solutions were extracted into isoamyl alcohol, and then acidified with oxalic acid. By injecting an aliquot of the isoamyl alcohol layer into a mass spectrometer in the direct flow injection mode, the quantitation was performed using the signals of Pt(DDC)3+ and Ag(DDC)2+ at m/z 639 and 403, respectively. The Pt levels ranged from 25ng/ml in blood to 2050ng/g wet weight in the liver of the patient, indicating that Pt remained at high levels in tissues, even after a period as long as 181 days after cisplatin overdosing.

Application of thermoresponsive HPLC to forensic toxicology: determination of barbiturates in human urine

A high-performance liquid chromatography (HPLC) method has been developed for assays of five barbiturates in human urine using a new thermoresponsive polymer separation column, which is composed of poly(N-isopropylacrylamide). By elevating the column temperature from 10°C to 50°C, the barbiturates metharbital, primidone, phenobarbital, mephobarbital, and pentobarbital became well separated by this method. The five barbiturates showed good linearity in the range of 0.2–10 μg/ml. Good accuracy, precision, and recoveries for these drugs were obtained at 1 and 5 μg/ml urine. The method with this new column type seems to have high potential for extensive use in forensic toxicology for analysis of many drugs and poisons by HPLC and HPLC-mass spectrometry.

Trace analysis of platinum in blood and urine by ESI-MS-MS

A simple, rapid, and sensitive method has been developed for determination of platinum (Pt) in blood and urine by tandem mass spectrometry (MS-MS). Pt4+ in wet-ashed blood or acid-treated urine was complexed with diethyldithiocarbamate (DDC), extracted with isoamyl alcohol, and acidified with oxalic acid; a 1-μl aliquot of the isoamyl alcohol layer containing the Pt-DDC complex was directly injected into the MS-MS instrument without chromatographic separation. The quantitation was performed using selected reaction monitoring at m/z 491 of the product ion Pt(DDC)2+, which was produced by collision-induced dissociation from the precursor ion Pt(DDC)3+ at m/z 639. This method was validated for the analysis of blood and urine samples; the limits of quantitation were about 0.3 and 0.1 ng/ml for blood and urine, respectively, using only 10 μl of sample. The calibration curves for Pt in urine and blood showed linearity from 0.1 to 30 ng/ml. Because chromatographic separation is not required before MS-MS detection, the analysis can be completed in less than 10 min.

Rapid and sensitive identification and determination of Urine Luck by ESI-MS after reduction of chromate

Urine Luck, the main component of which is pyridinium chlorochromate (PCC), is a popular adulterant used to conceal drugs present in urine samples. Electrospray ionization mass spectrometry with direct flow injection was employed to identify and quantitate PCC after complex formation of chromium (Cr6+) in PCC with diethyldithiocarbamate and its extraction with isoamyl alcohol. The quantitation was performed by selected ion monitoring at m/z 513, and the detection limit was 20 pg PCC (equivalent to 5pg Cr6+) in 1 µl of an injected volume. The integrated area of the mass chromatogram was proportional to the amount up to 2000 pg PCC with the limit of quantitation of 60 pg PCC in an injected volume. The recoveries of PCC from 50µl of urine spiked at 10−7 and 10−6M were 93% and 95%, respectively. The present method for analysis of Urine Luck is recommended for use in forensic analysis because of its speed, high sensitivity, and high specificity.

Electrospray ionization tandem mass spectrometric determination of monomethylarsonic acid and dimethylarsinic acid after adduct formation with citric acid

Inorganic arsenic species are metabolized to monomethylarsonic acid (MMA(V)) and dimethylarsinic acid (DMA(V)) and excreted into urine. A simple, rapid and sensitive method has been developed using electrospray ionization tandem mass spectrometry (ESI-MS-MS) for the simultaneous determination of MMA(V) and DMA(V). MMA(V) and DMA(V) in a sample were allowed to react with citric acid (CiA). Adduct compounds were extracted together with isoamyl alcohol (IAA). An aliquot (1-microL) of the IAA layer was directly injected into the ESI-MS-MS instrument, and was detected within 1 min. Quantification was done using selected reaction monitoring for MMA(V) and DMA(V) as follows: [MMAH + 2CiA - 3H(2)O](+) --> [MMAH + CiA - 2H(2)O](+) [DMAH + CiA + MeOH - 2H(2)O](+) --> [DMAH + MeOH - H(2)O](+) where MMAH and DMAH denote the protonated forms of MMA(V) and DMA(V), and MeOH denotes methanol (carrier liquid in ESI-MS-MS). This method was validated for the analysis of urine samples. The limit of detection of As was 0.3 microg L(-1) for MMA(V) and 0.6 microg L(-1) for DMA(V) using 10 microL of sample solution. Results were obtained in <10 min with a linear calibration range of 3-100 microg L(-1). Inorganic arsenic compounds (and other organic arsenic compounds) found in urine did not interfere with the detection of MMA(V) and DMA(V). Concentrations of MMA(V) and DMA(V) in the reference urine SRM 2670a were estimated after partial purification, and those in urine of a patient treated with As(2)O(3) were measured after dilution.

The restriction of all minerals in the diet enhancing paraquat toxicity is regarded primarily as the shortage of Mg

This study compares the effect of the restriction of Mg with that of all-minerals in the diet on the toxicity of paraquat. To compare the severity of the toxicity, several biological values were examined; kininogen in plasma, thiobarbituric acid reactive substances in liver, Ca level in kidney, and Mg levels in liver and kidney. Osteogenic disorder Shionogi rats that cannot synthesize vitamin C like humans did not display paraquat symptoms after receiving minute amounts of paraquat dichloride, i.e. 125 ppm in the diet for 8 days, and those biological values remained the same as those of the control. Rats fed with Mg at half of the recommended amounts also did not show any changes in those levels. The dosage of 125 ppm paraquat under the restriction of Mg, however, induced paraquat intoxication and increased those levels greatly. This result arises a question whether the intoxication is due to the imbalance of Ca and Mg or due to the shortage of Mg itself, because imbalance of Ca and Mg sometimes induces more serious effects than the shortage of Mg itself. Therefore, we fed rats an all-mineral restricted diet where the balance of Ca and Mg was maintained. The dosage of paraquat under all-mineral restriction, however, induced much more serious intoxication than that under Mg restriction. In conclusion, the shortage of Mg itself seems to be responsible for the induction of paraquat intoxication.