Manami Nishida

Direct extract derivatization for determination of amino acids in human urine by gas chromatography and mass spectrometry.

The purpose of this study was to develop a simple and accurate analytical method to determine amino acids in urine samples. The developed method involves the employment of an extract derivatization technique together with gas chromatography-mass spectrometry (GC-MS). Urine samples (300 microl) and an internal standard (10 microl) were placed in a screw tube. Ethylchloroformate (50 microl), methanol-pyridine (500 microl, 4:1, v/v) and chloroform (1 ml) were added to the tube. The organic layer (1 microl) was injected to a GC-MS system. In this proposed method, the amino acids in urine were derivatized during an extraction, and the analytes were then injected to GC-MS without an evaporation of the organic solvent extracted. Sample preparation was only required for ca. 5 min. The 15 amino acids (alanine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tyrosine, tryptophan, valine) quantitatively determined in this proposed method. However, threonine, serine, asparagine, glutamine, arginine were not derivatized using any tested derivatizing reagent. The calibration curves showed linearity in the range of 1.0-300 microg/ml for each amino acid in urine. The correlation coefficients of the calibration curves of the tested amino acids were from 0.966 to 0.998. The limit of detection in urine was 0.5 microg/ml except for aspartic acid. This proposed method demonstrated substantial accuracy for detection of normal levels. This proposed method was limited for the determination of 15 amino acids in urine. However, the sample preparation was simple and rapid, and this method is suitable for a routine analysis of amino acids in urine.

Extraction of amphetamines and methylenedioxyamphetamines from urine using a monolithic silica disk-packed spin column and high-performance liquid chromatography-diode array detection.

To overcome the limitations of solid-phase extraction, we developed a device comprising a spin column packed with octadecyl silane-bonded monolithic silica for extracting amphetamines and methylenedioxyamphetamines from urine. Urine (0.5mL), buffer (0.4mL), and methoxyphenamine (internal standard) were directly put into the preactivated column. The column was centrifuged (3000rpm, 5min) for sample loading and washed. The adsorbed analytes were eluted and analyzed by high-performance liquid chromatography, without evaporation. The results were as follows: linear curves (drug concentrations of 0.2-20microg/mL); correlation coefficients >0.99; detection limit, 0.1microg/mL. The proposed method is not only useful for drugs from biological materials but also highly reproducible for the analysis of these drugs in urine.

Simultaneous determination of amitraz and its metabolite in human serum by monolithic silica spin column extraction and liquid chromatography–mass spectrometry

A simple, rapid, sensitive, and specific liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the quantification of amitraz and its metabolite in human serum. Both the compounds were extracted using monolithic silica spin columns with acetonitrile. The chromatographic separation was performed on a reverse-phase C(18) column with a mobile phase of 10 mM ammonium formate-acetonitrile. The protonated analyte was quantitated in positive ionization by mass spectrometry. The method was validated over the concentration range of 25-1000 ng/ml for amitraz and its metabolite in human serum. For both compounds, the limit of detection was 5 ng/ml. The method was applied to serum samples taken from an attempted suicide patient, and only small volumes of serum were required for the simultaneous determination of these compounds.

Monolithic silica spin column extraction and simultaneous derivatization of amphetamines and 3,4-methylenedioxyamphetamines in human urine for gas chromatographic-mass spectrometric detection

A simple, sensitive, and specific method with gas chromatography-mass spectrometry was developed for simultaneous extraction and derivatization of amphetamines (APs) and 3,4-methylenedioxyamphetamines (MDAs) in human urine by using a monolithic silica spin column. All the procedures, such as sample loading, washing, and elution were performed by centrifugation. APs and MDAs in urine were adsorbed on the monolithic silica and derivatized with propyl chloroformate in the column. Methamphetamine-d(5) was used as an internal standard. The linear ranges were 0.01-5.0 microg mL(-1) for methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA) and 0.02-5.0 microg mL(-1) for amphetamine (AP) and 3,4-methylenedioxyamphetamine (MDA) (coefficient of correlation > or = 0.995). The recovery of APs and MDAs in urine was 84-94%, and the relative standard deviation of the intra- and interday reproducibility for urine samples containing 0.1, 1.0, and 4.0 microg mL(-1) of APs and MDAs ranged from 1.4% to 13.6%. The lowest detection limit (signal-to-noise ratio > or = 3) in urine was 5 ng mL(-1) for MA and MDMA and 10 ng mL(-1) for AP and MDA. The proposed method can be used to perform simultaneous extraction and derivatization on spin columns that have been loaded with a small quantity of solvent by using centrifugation.

Simultaneous determination of dibucaine and naphazoline in human serum by monolithic silica spin column extraction and liquid chromatography-mass spectrometry.

A simple, sensitive, and specific liquid chromatography-mass spectrometry (LC-MS) method for simultaneous determination of dibucaine and naphazoline from serum was developed and validated. The extraction procedure was performed using a monolithic silica spin column. Chromatographic separation of dibucaine and naphazoline was achieved on a C(18) reverse phase column with a mobile phase gradient (mobile phase A: 10 mM ammonium formate and mobile phase B: acetonitrile) at a flow rate of 0.2 mL/min. LC-MS was operated under the selective ion monitoring mode using the electrospray ionization technique in the positive mode. The retention times for naphazoline, dibucaine, and the internal standard (IS) were 6.7, 7.8, and 8.0 min, respectively. A linear graph was obtained for dibucaine and naphazoline with correlation coefficients >0.998 for all analytes by this method. The limit of quantification of dibucaine and naphazoline was 10 and 25 ng/mL, respectively. The mean recoveries were greater than 70%. Both compounds were stable under conditions of short-term storage, long-term storage as well as after freeze-thaw cycles. Monolithic spin column extraction and LC-MS analysis enabled the separation of dibucaine and naphazoline within 20 min.

On-column derivatization for determination of amphetamine and methamphetamine in human blood by gas chromatography-mass spectrometry.

A simple determination method of amphetamine (AP) and methamphetamine (MA) in human blood was developed using on-column derivatization and gas chromatography-mass spectrometry (GC-MS). AP and MA were adsorbed on the surface of Extrelut and then derivatized the N-propoxycarbonyl derivatives using propylchloroformate. Pentadeuterated MA was used as an internal standards. The recoveries of AP and MA from the spiked blood were 89.7 and 90.3%, respectively. The calibration curves showed linearity in the range of 12.5-2000 ng/g for AP and MA in blood. The coefficients of variation of intraday and interday were 0.42-4.58%. Furthermore, this proposed method was applied to some medico-legal cases of MA intoxication. MA and its metabolite AP were detected in the blood samples, and the correlation of the blood level of amphetamines and the behaviors of the victims was in good agreement with the criteria proposed by Nagata [Jpn. J. Legal Med. 37 (1983) 513].

Histological brain alterations following prenatal methamphetamine exposure in rats.

ABSTRACT  When pregnant women abuse methamphetamine, the foremost concern is the potential adverse effect of this substance on fetal development. Clinical studies in humans have found that exposure to methamphetamine during brain development can cause neurobehavioral abnormalities, such as aggressive behavior, learning problems, and poor social adaptation. In the present study, we examined the effects of prenatal methamphetamine exposure on brain development in rats. The first group of pregnant rats was administered methamphetamine at a dose of 5 mg/kg/day during gestational day (GD 10 to GD 20 [MA]. The second group of pregnant rats was injected with saline vehicle only [SAL]. On GD 21 their fetuses were removed and fetal brains were observed. We found various types of morphological damage in MA fetal brains, including microgyria, ectopia, and hemorrhage. In some cases, abnormal distribution of the leptomeninx, such as breach or accumulation, was observed in addition to these histological abnormalities. Therefore, we examined the expression of laminin, which is an important component of the pia mater, in the fetal brains. However, Western blot analysis revealed that there was no difference in expression amount of laminin in whole fetal brain between the MA and SAL groups. We concluded that methamphetamine use during pregnancy can cause histological brain alterations in fetuses. Morphological alterations of brain seen in the present study and previous human studies following prenatal exposure to methamphetamine might be related to the neurobehavioral abnormalities seen in patients who had been exposed to methamphetamine in utero.

In-matrix derivatization and automated headspace solid-phase microextraction for GC-MS determination of amphetamine-related drugs in human hair

A fully automated method for analysis of amphetamine-related drugs in human hair by gas chromatography-mass spectrometry (GC-MS) was developed using headspace solid-phase microextraction (SPME) and in-matrix derivatization. Amphetamines were extracted from hair under alkaline conditions, and were simultaneously derivatized to N-ethoxycarbonyl amphetamines with ethylchloroformate in a vial. An SPME fiber was then exposed to the headspace at 80°C for 10 min for extraction. The derivatives extracted into the stationary phase of the fiber were desorbed by exposing the fiber in an injection port of a GC-MS instrument. The calibration curves showed linearity up to 10ng/mg in hair. The detection limits ranged from 0.01 to 0.5 ng/mg according to the compound identity. No interferences were found, and the time required for analysis was about 30 min per sample. Furthermore, this proposed method was applied to diagnosis of methamphetamine intake in actual cases; methamphetamine and its metabolite amphetamine were able to be detected in hair of abuser patients admitted to a hospital.

Determination of dimethyl sulfoxide and dimethyl sulfone in urine by gas chromatography-mass spectrometry after preparation using 2,2-dimethoxypropane.

A method for routinely determination of dimethyl sulfoxide (DMSO) and dimethyl sulfone (DMSO(2)) in human urine was developed using gas chromatography-mass spectrometry. The urine sample was treated with 2,2-dimethoxypropane (DMP) and hydrochloric acid for efficient removal of water, which causes degradation of the vacuum level in mass spectrometer and shortens the life-time of the column. Experimental DMP reaction parameters, such as hydrochloric acid concentration, DMP-urine ratio, reaction temperature and reaction time, were optimized for urine. Hexadeuterated DMSO was used as an internal standard. The recoveries of DMSO and DMSO(2) from urine were 97-104 and 98-116%, respectively. The calibration curves showed linearity in the range of 0.15-54.45 mg/L for DMSO and 0.19-50.10 mg/L for DMSO(2). The limits of detection of DMSO and DMSO(2) were 0.04 and 0.06 mg/L, respectively. The relative standard deviations of intra-day and inter-day were 0.2-3.4% for DMSO and 0.4-2.4% for DMSO(2). The proposed method may be useful for the biological monitoring of workers exposed to DMSO in their occupational environment.

Monolithic silica capillary column extraction of methamphetamine and amphetamine in urine coupled with thin-layer chromatographic detection

A monolithic silica capillary column was first developed in Japan in 2001 as a new tool for better liquid chromatographic separation. The column is made of C18-bonded monolithic silica packed into a capillary glass tube (0.20 mm i.d.). In this study, we used this column for solid-phase extraction of methamphetamine (MA) and amphetamine (AP) in urine. Chromatographic separation was achieved by thin-layer chromatography (TLC) with double-spray detection of each spot. For extraction of amphetamines in urine, samples were mixed with phosphate buffer (pH 3.0, containing 20mM sodium octanesulfate), and the analytes were adsorbed to the column by passing the mixture through it. They were then eluted with a 10-μl volume of ethyl acetate and directly spotted onto a TLC plate. After development, the detection of MA was performed with Simon’s reagent. The plate was air-dried, and then over-sprayed with fluorescamine reagent for detection of AP. A fluorescent spot of AP was observed at 365 nm using an ultraviolet viewing system. The detection limits of MA and AP in urine were about 1.0 and 2.0μg/ml, respectively. To confirm the usefulness of this method, the eluent from the column was also analyzed by gas chromatography-mass spectrometry (GC-MS) after derivatization with heptafluorobutyric anhydride. In analysis of 30 actual urine samples, the results obtained by the monolith-TLC system were generally well in accordance with those obtained by GC-MS.