Kyoung-Rae Kim

Capillary electrophoretic profiling and pattern recognition analysis of urinary nucleosides from thyroid cancer patients

Metabolic profiling analysis by capillary electrophoresis (CE) was combined with pattern recognition methods to see some correlation between urinary nucleoside levels and thyroid cancer. A total of 15 nucleosides were identified in urines from 12 female thyroid cancer patients and 12 healthy females studied. From the scatter plot evaluation, inosine showed the highest estimated diagnostic power with ca. 97.725% confidence level, followed by N2-methylguanosine. Star symbol graphs showed differences in levels of both minor and major nucleosides between cancer and normal groups more efficiently, compared with histogram. The stepwise discriminant analysis (SDA) selected N2-methylguanosine, N2,N2-dimethylguanosine and 1-methylguanosine as the most discriminating variables between thyroid cancer and normal groups. The canonical discriminant analysis (CDA) correctly classified all urine specimens studied into two separate clusters of cancer and normal groups in a canonical plot. The principal component analysis (PCA) distinguished cancer patients from normal controls in a principal component plot. The cluster analysis (CA) yielded a dendrogram displaying group separation without any single wrong linkage.

Gas chromatographic profiling and screening for phenols as isobutoxycarbonyl derivatives in aqueous samples

An efficient method is described for the simultaneous determination of phenol and 49 substituted phenols present in aqueous samples. The method is based on the extractive two-phase isobutoxycarbonyl (isoBOC) derivatization with subsequent solid-phase extraction (SPE) for the direct analysis by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Phenolic hydroxyl groups in acidic aqueous solutions were allowed to react with isobutyl chloroformate present in the dichloromethane phase containing triethylamine. The resulting isoBOC derivatives were then recovered by SPE using Chromosorb P in normal-phase partition mode, followed by direct GC and GC-MS analysis. Using this combined procedure, linear detector responses were obtained in the concentration range of 0.5-8 microg ml(-1), with correlation coefficients varying from 0.925 to 0.999 for most of the phenols studied except for 2,4-dinitorphenol (0.789). The temperature-programmed retention index (I) sets as measured on DB-5 and DB-17 dual-capillary columns of different polarity were characteristic of each isoBOC phenol derivative and thus, useful in the screening for isomeric phenols by I matching only. The mass spectral patterns, exhibiting characteristic [M-100]+, [M-200]+ and [M-300]+ ions for the mono-, di- and trihydroxybezenes, respectively with common ions at m/z 57, facilitated their rapid structural confirmation. The present method allowed rapid screening for phenols when applied to water samples spiked with phenols.

Determination of hair polyamines as N-ethoxycarbonyl-N-pentafluoropropionyl derivatives by gas chromatography-mass spectrometry

An efficient method is described for the simultaneous determination of hair polyamines, such as 1,3-diaminopropane, putrescine, cadaverine, spermidine and spermine, by gas chromatography-mass spectrometry (GC-MS) with the selected ion-monitoring (SIM) mode. The method is based on the extractive two-phase ethoxycarbonyl (EOC) reaction of amino functions in aqueous solutions combined with subsequent pentafluoropropionyl (PFP) derivatization of the remaining active hydrogen atoms for the direct analysis by GC-SIM-MS. The detection limits for SIM of the polyamines as N-EOC-N-PFP derivatives ranged from 1 to 10 ng/g hair, while their recovery rates varied in the range of 76.42-93.38%. This method demonstrated a good overall accuracy (% bias) and precision (% C.V.) as 3.32-11.05% and 5.88-14.71%, respectively. When applied to 0.6 M HCl extracts of hair samples from 11 healthy men and 19 healthy women, all five polyamines were positively detected at the concentrations of 8.82-871.87 ng/g. Both in median and mean concentrations, the most abundant hair polyamine was spermidine, followed by spermine, putrescine, 1,3-diaminopropane and cadaverine in the male group, while the order of 1,3-diaminopropane and cadaverine was reversed in the female group. The levels of polyamines, except for cadaverine, in hair specimens studied were found to be higher in men than in women.

Capillary electrophoretic profiling and pattern recognition analysis of urinary nucleosides from uterine myoma and cervical cancer patients.

Capillary electrophoretic (CE) profiling analysis combined with pattern recognition methods is described for the correlation between urinary nucleoside profiles and uterine cervical cancer. Nucleosides were extracted from urine specimens by solid-phase extraction in affinity mode using phenylboronic acid gel. CE separation was carried out with an uncoated fused-silica capillary (570 mm x 50 microm I.D.) maintained at 20 degrees C, using 25 mM borate-42.5 mM phosphate buffer (pH 6.7) containing 200 mM sodium dodecyl sulfate as the run buffer under the applied voltage of 20 kV. A total of 15 nucleosides were positively identified in urine samples (2 ml) from eight uterine myoma (benign tumor group), 10 uterine cervical cancer (malignant tumor group) patients and 10 healthy females (normal group) studied. The star symbol plots drawn based on each mean concentration of nucleosides normalized to that in normal group enabled one to discriminate malignant and benign groups from normal group. In addition, canonical discriminant analysis performed on the nucleoside data of 28 individual urine specimens correctly classified into three separate clusters according to groups in the canonical plot.

Rapid screening for acidic non-steroidal anti-inflammatory drugs in urine by gas chromatography-mass spectrometry in the selected-ion monitoring mode

A rapid screening procedure is described for the simultaneous determination of various acidic non-steroidal anti-inflammatory drugs (NSAIDs) at sub-nanogram levels. The procedure involves solid-phase extraction (SPE) of NSAIDs using Chromosorb P as the adsorbent in partition mode, with subsequent single-step conversion to tert-butyldimethylsilyl (TBDMS) derivatives, followed by direct analysis by gas chromatography-mass spectrometry (GC-MS). The characteristic [M-57]+ high-mass ions constituting the base peaks in the electron-impact mass spectra of most TBDMS derivatives permitted sensitive detection of NSAIDs by GC-MS in selected-ion monitoring (SIM) mode, even in the presence of higher levels of coextracted urinary organic acids. The detection limit for SIM of each drug was in the range 0.03-0.9 pg. When applied to urine samples (250 microliters) spiked with NSAIDs, the present GC-SIM-MS method allowed simultaneous screening for various NSAIDs with good overall precision and accuracy in the range of 10-40 ng.

Capillary electrophoresis of nonprotein and protein amino acids without derivatization.

An efficient separation of eleven nonprotein amino acids (NPAAs) and three protein amino acids containing aromatic moieties was achieved by capillary electrophoresis without derivatization. The fourteen amino acids were well separated with a 100 mM sodium phosphate run buffer (pH 2.0) using a 57 cm fused‐silica capillary (50 μm ID, 50 cm effective length) at 20°C. With an electric field of 351 V/cm, the time needed for the separation was less than 20 min. Under optimum conditions, excellent linear responses were obtained in the concentration range of 5—100 μM, with the linear correlation coefficient ranging from 0.9785 or greater. The relative standard deviations of the migration times and the corrected peak areas were found to be 1.5—3.9% and 8.0—11.5%, respectively. In order to improve the limit of detection (LOD), simple stacking and large volume stacking using an EOF pump (LVSEP) methods were used. Improved LODs were about 300 nM in stacking and below 15 nM for five small NPAAs in LVSEP.

Separation of triacylglycerols and free fatty acids in microalgal lipids by solid-phase extraction for separate fatty acid profiling analysis by gas chromatography

Microalgal lipids were separated into two fractions, triacylglycerols (TAGs) and free fatty acids (FFAs), by solid-phase extraction employing sodium carbonate as the sorbent and dichloromethane (20% by volume) in n-hexane as the extracting solvent. The TAG fraction was then saponified, followed by acidification, extraction and tert-butyldimethylsilyl esterification. The FFA fraction was directly acidified, extracted and derivatized. From the lipid extracts of eight microalgal species examined, a total of 13 fatty acids were detected in the TAG fractions and nine were found in the FFA fractions, with at much higher total TAG content in all microalgae. Oleic acid was the most prominent fatty acid in three species, alpha-linolenic acid was more abundant in two others, and palmitic acid was present in highest concentration in the remaining three species.

Enantioseparation of chiral amino acids as the N(O,S)-ethoxycarbonylated diastereomeric esters by achiral dual-capillary column gas chromatography

The enantioseparation of 30 racemic amino acids in a single analysis is described for the determination of their absolute configurations. Two-phase extractive ethoxycarbonyl (EOC) reaction with ethyl chloroformate present in the dichloromethane phase was performed to recover amino acids from alkaline aqueous solutions. The resulting N(O,S)-EOC amino acids extracted into an organic solvent after acidification were reacted with a chiral alcohol such as (S)-(+)-3-methylbutan-2-ol, (S)-(+)-butan-2-ol and (S)-(+)-octan-2-ol for gas chromatographic analysis on achiral dual-capillary DB-5 and DB-17 columns of different polarities. Among the chiral reagents examined, (S)-(+)-3-methylbutan-2-ol provided the best diastereomeric structures in resolving all the racemic amino acids into their enantiomeric pairs with high resolution factors (1.2-8.0). Moreover, the temperature-programmed retention index (I) values measured on the two columns were characteristic of each enantiomer. Hence simple I matching with the reference values was useful in cross-checking for chemical identification and also chiral discrimination. When the present method was applied to a fermented dairy product (Yakult), D-alanine, D-aspartic acid, D-glutamic acid and D-proline were positively detected along with their respective L-forms in addition to glycine.

Gas chromatographic profiling and pattern recognition analysis of urinary organic acids from uterine myoma patients and cervical cancer patients

An efficient organic acid profiling and pattern recognition method is described for the correlation between urinary organic acid profiles and uterine cervical cancer. After methoximation of keto acids in alkalinized urine samples, all free organic acids were recovered by a dual solid-phase extraction procedure, followed by conversion to tert.-butyldimethylsilyl derivatives for the profiling analysis by dual-capillary column gas chromatography (GC) with subsequent screening for acids by retention index (I) library matching. A total of 50 organic acids were positively identified in urine samples (0.25 ml) from 12 uterine myoma (benign tumor group) and 14 uterine cervical cancer (malignant tumor group) patients studied. When the GC profiles were simplified to their corresponding organic acid I spectra in bar graphical form, characteristic patterns were obtained for each average of benign and malignant tumor groups. Stepwise discriminant analysis performed on the GC data selected 16 acids as the variables discriminating between the two groups. Canonical discriminant analysis applied to these 16 variables correctly classified 26 urine samples into two separate clusters according to tumor types in the canonical plot.

Simultaneous clinical monitoring of lactic acid, pyruvic acid and ketone bodies in plasma as methoxime/tert-butyldimethylsilyl derivatives by gas chromatography–mass spectrometry in selected ion monitoring mode

Simultaneous determination of lactic acid, pyruvic acid, 3-hydroxybutyric acid and acetoacetic acid for clinical monitoring of lactic acidosis and ketone body formation in human plasma (20 microL) was performed by gas chromatography-mass spectrometry in selected ion monitoring (SIM) mode after generating methoxime/tert-butyldimethylsilyl derivatives. All of the targeted carboxylic acids were detected by characteristic fragment ions, which permitted sensitive and selective identification in the presence of co-extracted free fatty acids and other acidic metabolites at much higher levels. The method was linear (r>or=0.9991), reproducible (% relative standard deviation=1.2-5.8), and accurate (% relative error=-7.2-7.6), with detection limits of 0.05-1.7 ng/mL. This rapid, accurate and selective method using minimal plasma samples (20 microL) is useful in the clinical monitoring of lactic acidosis and ketone body formation in plasma.