• Mass Spectrometry Letters
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• v.2 no.2
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• pp.33-36
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• 2011

The misuse of anabolic androgenic steroids is of particular concern in sports and society. Thus, it is of great importance to discriminate endogenous steroids such as testosterone or testosterone prohormones from their chemically identical synthetic copies. In this study, gas chromatography-combustion/isotope ratio mass spectrometric (GC-C/IRMS) method has been developed and validated for discriminating the origin of anabolic androgenic steroids. The method involves the solid-phase extraction, enzymatic hydrolysis with ${\beta}$-glucuronidase, HPLC-fractionation for the cleanup and analysis by GC-C/IRMS. The difference(${\Delta}^{13}C$) of urinary ${\delta}^{13}C$ values between synthetic analogues and endogenous reference compounds (ERC) by GC-C/IRMS was used to elucidate the origin of steroids, and intra- and inter-day precision, specificity and isotope fractionation were evaluated. The present GC-C/IRMS method combined with HPLC cleanup was accurate and reproducible enough to be successfully applied to the test of urine sample from suspected anabolic steroid abusers.

• Journal of Food Hygiene and Safety
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• v.5 no.3
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• pp.159-164
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• 1990

To detect and quantitation residual antibiotics and antibacterial agents in meats, we performed a biological assay employing the three microorganisms Bacillus subtilis ATCC 6633, Micrococcus luteus ATCC 9341, and Bacillus cereus var. mycoides ATCC 11778 for the screening purpose and developed a Gas Chromatography-mass Spectrometry(GC/MS) analysis for the confirmation and quantiation. In the biological assay (paper disk method), three test solution are used depending on the character of the residual antibiotics and antibacterial agents, follow by a simple clean up procedure which includes homogenization with Mcilvaine buffer, defatting with includes homogenization with Mcilvaine buffer, defatting with hexane, extraction with chloroform, clean-up by Sep-Pak $C_{18}$ and Bakerbond SPE carboxylic acid column. The chloroform layer is used for the analysis of sulfa agents. macrolides antibiotics and antibacterial agents, Adsorbed materials in the Sep-Pak $C_{18}$ were also employed for th analysis of penicillins and tetracyclines. Effluents from the Sep-Pak $C_{18}$ were cleaned-up one more by Bakerbond 10 SPE COOH column and employed for the analysis of aminoglycosides. In the instrumental analysis by using the GC/MSD, residual antibiotics and antibacterial agent were quantitated by selected ion monitoring (SIM) mode after derivatization. A simultaneous analysis of six residual antibiotic and antibacterial agent such as oxytetracycline, penicillin, ampicillin, choliraphenicol and thiamphenicol was developed with simple cleanup procedures revealing good recovery and reproducibility. Also, simultaneous detection of macrolides antibiotics such as erythromycin, spiramycin, and oleandomycin was developed after acid hydrolysis due to their large molecular structures. Because of the high reproducibility and selectivity of these two methods, it is very desirable that the combination of the two methods be used in the bioassay for the screening of residual antibiotics and antibacterial agent and that GC/MSD analysis be used for the confirmation and quantitation.

• Analytical Science and Technology
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• v.7 no.1
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• pp.65-78
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• 1994

The herbicide mixture of 7 triazines and 9 phenoxyalkanoic acid esters was simultaneously separated and determinated with the selected ion monitoring by using gas chromatography/mass spectrometry. The extraction recoveries of those herbicides from the reagent water were studied for the organic solvent extraction(LLE) with methylene chloride. The calibration curves of them showed good linearity over the concentration range of 0.2~0.5ng/ml and the detection limits were 0.2~0.5ng/ml for 100ml of water. This analytical method could be applied to the drinking water and biological sample.

• Analytical Science and Technology
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• v.8 no.2
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• pp.139-159
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• 1995

Positive ion mass spectra of some corticosteroids were obtained by using liquid chromatography-mass spectrometry(LC-MS). The base peak of each compound showed the protonated molecular ion [$MH^+$], ammonium adduct ion [${MNH_4}^+$] or [$MH^+-60$] ion according to its chemical structure and other characteristic mass ions were [$MH^+-18$], [${MNH_4}^+-18$] and so on. Several rat urinary metabolites of methylprednisolone acetate after the oral administration were detected by the thermospray LC-MS. The identified major metabolites were 20-hydroxymethylprednisolone(20-HMP), methylprednisolone(MP) and methylprednisone(11-KMP), which were supposed to be formed by deacetylation at the position of C-21, reduction at C-20, oxidation at C-11, or due to the bond cleavage between C-17 and C-20.

• Bulletin of the Korean Chemical Society
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• v.32 no.4
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• pp.1158-1164
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• 2011

5-Methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT), a psychoactive tryptamine derivative, is a hallucinogenic drug of abuse. In this study, 5-OH-DIPT and its metabolites were identified and the quantitative method was developed and validated by using hydrophilic interaction chromatography-tandem mass spectrometry (HILICMS/MS). Chromatographic separation was achieved on an Atlantis HILIC silica column ($5{\mu}m$, $100{\times}2.1\;mm$). The metabolites of 5-MeO-DIPT in rat urine were characterized via Q1 scanning and product ion scanning. As a consequence, 5-MeO-IPT, 5-OH-DIPT, 6-OH-5-MeO-DIPT and their glucuronide conjugates were detected and identified as the metabolites of 5-MeO-DIPT. Subsequently, a quantitative method for 5-MeO-DIPT and its major metabolites, 5-MeO-IPT and 5-OH-DIPT, was developed in multiple reactions monitoring (MRM) mode. The calibration curves for all analytes evidenced good linearity over the concentration range of 1-1000 ng/mL with linear correlation co-efficients ($r^2$) in excess of 0.99. The intra- and inter-day accuracy and precision were 92.2-110.2% and 1.5-9.9%, respectively.

• YAKHAK HOEJI
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• v.36 no.1
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• pp.26-36
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• 1992

The metabolic profile of triprolidine, 2-[1-(4-methylphenyl)-3-(1-pyrrolidinyl-1-propenyl)] pyridine, was determined in rat urine and bile. The free fractions of urinary and biliary extracts were obtained without hydrolysis, and the conjugated fractions of extracts were obtained with enzyme hydrolysis using ${\beta}-glucuronidase$ from Escherichia coli. The mixture of N-methyl-N-trimethylsilyltrifluoroacetamide/trimethylsilyl chloride (100 : 1, v/v) was used to derivatize the extracts and then analyzed by gas chromatography/mass spectrometry. Hydroxymethyltriprolidine, hydroxytriprolidine, triprolidine carboxylic acid, dihydroxytriprolidine 1, dihydroxytriprolidine 2, oxotriprolidine carboxylic acid and unchanged triprolidine were detected in rat urine and bile, which were obtained after oral treatment with triprolidine hydrochloride. The maximum urinary excretion rate of triprolidine and hydroxymethyltriprolidine which were extracted from free fraction was at 1 to 2 hours after drug administration. Hydroxymethyltriprolidine was detected in conjugated fraction, and the maximum urinary excretion rate of that metabolite was at 2 to 3 hours in rat. In rat bile analysis, triprolidine was detected only in free fraction and its biliary excretion rate showed the maximum within 30 minutes after drug administration and decreased continuously thereafter. The excretion percentage of triprolidine and hydroxymethyltriprolidine to the initial dose of the parent drug in bile and urine of rats were all low.

• Analytical Science and Technology
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• v.8 no.3
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• pp.229-236
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• 1995

Multiple-step gradient method was used for the analysis of free amino acids in physiological fluids by high-performance liquid chromatography with diode array detector on the Amino Quant $C_{18}$ column under the condition of pH 7.2 of buffer solutions. Plasma samples of normal Korean people and abnormal Korean people who have schizophrenia were subjected to derivatization with o-phthalaldehyde in the presence of 3-mercaptopropionic acid. Quantitative analysis of amino acids in physiological fluids by internal standard method gave highly reproducible results within a relative standard deviation of less than 2~6%. And amino acids amounts of physiological fluids of Korean people gave some different results from those of foreigners. There was large differences in tyrosine amount between normal and abnormal man.

• Bulletin of the Korean Chemical Society
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• v.31 no.9
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• pp.2493-2496
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• 2010

Erythropoietin (EPO) is mainly produced in kidney and stimulates erythropoiesis. The use of recombinant EPOs for doping is prohibited because of its performance enhancing effect. This study investigated whether biosimilar EPOs could be differentiated from endogenous one by iso-electro-focusing plus double blotting and SDS-PAGE for antidoping analysis. The established method was validated with positive control urine. The band patterns were reproducible and meet the criteria, which was made by world anti doping agency (WADA). Isoelectric focusing was conducted in pH range 2 to 6. Recormon (La Roche), Aropotin (Kunwha), Epokine (CJ Pharm Co.), Eporon (Dong-A), Espogen (LG Life Sciences), and Dynepo (Shire Pharmaceuticals) were detected in basic region. All biosimilars showed discriminative isoelectric profiles from endogenous EPO profiles, but they showed different band patterns with the reference one except Epokine (CJ Pharm Co.). Next, SDS-PAGE of biosimilar EPOs resulted in different molecular weight patterns which were distributed higher than endogenous EPO. Commercial immune assay kit as an immune affinity purification tool and immobilized antibody coated magnetic bead were tested for the purification and concentration of EPO from urinary matrix. The antibody-coated magnetic bead gave better purification yield. The IEF plus double blotting and SDS-PAGE with immunoaffinity purification method established can be used to discriminate biosimilar EPOs from endogenous EPO.

• Mass Spectrometry Letters
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• v.9 no.1
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• pp.30-36
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• 2018

A quantitation method for free amino acids in human serum was developed using a stepwise-dilution method and a bimodal cation exchange (CEX)/hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry system equipped with an electrospray ionization source (ESI/MS/MS). This method, which was validated using quality control samples, was optimized for enhanced selectivity and sensitivity. Dithiothreitol (DTT) was used as a reducing agent to prevent the oxidation of a serum sample ($50{\mu}L$), which was then subjected to stepwise dilution using 3, 30, and 90 volumes of acetonitrile containing 0.1% formic acid. Chromatographic separation was performed on an Imtakt Intrada Amino Acid column ($50mm{\times}3mm$, $3{\mu}m$) in mixed mode packed with CEX and HILIC ligands embedded in the stationary phase. Underivatized free amino acids were eluted and separated within 10 min. As a result of the validation, the precision and accuracy for the inter- and intraday assays were determined as 2.11-11.51% and 92.82-109.40%, respectively. The lowest limit of quantification (LLOQ) was $0.5-4.0{\mu}g/mL$ and the matrix effect was 80.22-115.93%. The proposed method was successfully applied to the quantitative analysis of free amino acids in human serum.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 1996.12a
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• pp.64-64
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• 1996

Transgenic animal and cell line models which are recently developed in toxicology field combined with molecular biological technique, are powerful tools for studying of mutation in vivo and in vitro, respectively. The Big Blue mutagenesis assay system is one of the most widely used transgenic systems. Especially, for the study of direct acting mutagens, Big Blue cell line is very useful and powerful to evaluate mutagenicity because the mutation frequency and mutationspectrlun showed no distinct differences between cell line and animal. The Big Blue cell lines carry stably integrated copies of lambda shuttle vector containing lac I gene as a mutational target. These lambda shuttle vectors are useful for various mutagenesis related studies in eukaryotic system due to their ability to be exposed mutagen and then transfer a suitable target DNA sequence to it convenient organism for analysis. We tried to assess the mutagenic effect of 4-NQO with Big Blue cell line. After the treatment of 4-NQO, genomic DNA was isolated and lambda shuttle vector was packaged by in Vitro packaging and then these were plated on bacterial host in the presence of X-gal to screen mutation in the lac I. We determined MF as a ratio of blue plaques versus colorless plaques and now undergoing the mutation spectrum of 4-NQO in lac J gene sequence.