Acidic metabolite profiling analysis of catecholamine and serotonin as O-ethoxycarbonyl/tert-butyldimethylsilyl derivatives by gas chromatography-mass spectrometry.

A novel derivatization method was developed for the simultaneous determination of six acidic metabolites of catecholamine and serotonin by gas chromatography-mass spectrometry (GC-MS). The metabolites were converted to O-ethoxycarbonyl/tert-butyldimethylsilyl (EOC/TBDMS) derivatives for the direct GC-MS analysis in selected ion monitoring mode. Their mass spectral pattern as EOC/TBDMS derivatives showed characteristic fragment ions of [M - 15](+) and [M - 57](+), which permitted rapid and accurate structural confirmation of acidic metabolites. The present method was linear (r ≥ 0.998), reproducible (percentage relative standard deviation = 1.0-10.0) and accurate (% relative error = -9.7-9.8) with detection limits of 0.001-4.7 ng/mL. When applied to human urine samples, the method allowed simultaneous determination of six acidic metabolites of catecholamine and serotonin.

Chiral separation of N-methyl-DL-aspartic acid in rat brain tissue as N-ethoxycarbonylated (S)-(+)-2-octyl ester derivatives by GC-MS.

A selective and sensitive analytical method was developed for enantiomeric separation and determination of N-methyl-DL-aspartic acid (NMA). The method involved the conversion of each enantiomer into N-ethoxycarbonylated (S)-(+)-2-octyl ester derivative for the direct separation by gas chromatography-mass spectrometry (GC-MS). The diastereomeric derivatives showed characteristic mass spectral properties for analysis by selected ion monitoring mode (SIM) and enabling enantioseparation on an achiral capillary column. Two enantiomers were baseline separated, and the detection limits for N-methyl-L-aspartic acid (NMLA) and N-methyl-D-aspartic acid (NMDA) were 0.07 and 0.03 ng/g, respectively. When applied to rat brain tissues for absolute configuration of NMA, only NMDA was determined, while NMLA was monitored as lower than the limit of detection.

Nitrogen fixation by a marine non-heterocystous cyanobacterium requires a heterotrophic bacterial consort.

Cultures of the non-heterocystous cyanobacterium, Leptolyngbya nodulosa, could be grown indefinitely in media devoid of combined nitrogen. Acetylene reduction assays showed that these cultures fixed nitrogen in the dark period of a diurnal cycle under micro-oxygenic or anaerobic conditions. Addition of DCMU to cultures induced much higher rates of nitrogenase activity, most of which occurred in the light. Measurements of activity in the presence of chloramphenicol indicated that nitrogenase is synthesized in darkness and probably destroyed in the subsequent light period. Neither the dark-mediated nitrogenase in the absence of DCMU nor light-mediated activity in the presence of DCMU could be sustained for more than 3 days without a photoperiodic light/dark cycle. Axenic cultures could not be grown in the absence of combined nitrogen and did not demonstrate any acetylene reduction activity. An identical nifH gene sequence was found in axenic and non-axenic cultures of L. nodulosa. RT-PCR demonstrated that this gene was expressed only in non-axenic cultures. Western blotting showed that the Fe-protein of nitrogenase is absent in cultures that are incapable of acetylene reduction, indicating that the lack of nitrogenase activity is likely due to the absence of the enzyme. These observations strongly indicate that L. nodulosa contains a functional nitrogenase which is not expressed in the absence of heterotrophic bacteria.

Plasma free fatty acid level patterns according to cardiovascular risk status in postmenopausal women.

BACKGROUND: The present study examines changes in the pattern of plasma free polyunsaturated fatty acids (PUFA) according to menopausal status and the existence of diabetes mellitus (DM) or coronary heart disease (CHD) in Korean women. METHODS: The participants were as follows; premenopausal women (PRE, n=20) and postmenopausal women without any known chronic disease (POST, n=35), with DM (DM, n=35), or with angiographically proven CHD (CHD, n=30). Plasma free fatty acids were measured in all participants. RESULTS: Healthy premenopausal women had a higher ratio of omega-3/omega-6 PUFA than postmenopausal women (p=0.001). As expected, the PRE group had higher docsapentaenoic acid (DPA) and docosahexaenoic acid (DHA) levels (p<0.05) and lower arachidonic acid levels (p<0.05) than the POST group. In turn, the healthy POST group had higher levels of DPA and DHA compared to the DM or CHD groups (p<0.05). There were significant shifts of increased omega-6 and decreased omega-3 among the women, according to each disease. CONCLUSIONS: There was a significant relationship between omega-3 and omega-6 PUFA profiles and risk for CHD in women. This metabolic profile of PUFA might be an important surrogate marker in postmenopausal women.

Gas chromatographic-mass spectrometric analyses of cholesterol and its precursors in rat plasma as tert-butyldimethylsilyl derivatives.

BACKGROUND: Cholesterol and its metabolic precursors occurring in metabolic pathways are important biochemical indicators in pathological conditions. METHODS: A method for the simultaneous determination of cholesterol and its metabolic precursors, such as lanosterol and 7-dehydrocholesterol, in rat plasma is demonstrated. It involves their extraction after saponification, followed by conversion to tert-butyldimethylsilyl (TBDMS) derivatives for analysis by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode (GC-SIM-MS). RESULTS: The characteristic fragment ions of [M-57], m/z 443, 483, and 441 permitted the accurate and selective detection of cholesterol and its precursors in rat plasma. The whole procedure of TBDMS derivatization, with subsequent GC-SIM-MS analysis, was linear (r>or=0.9994), reproducible (% relative standard deviation=2.2 to 7.5), and accurate (% relative error=-5.6 to 7.7), with detection limits of 0.02 to 0.07 ng/ml. Recoveries were measured to be ranged from 89.5 to 95.4%. CONCLUSION: The present method was useful for the quantification of cholesterol and its precursors in rat plasma samples of 1 microl.

N-ethoxycarbonylation combined with (S)-1-phenylethylamidation for enantioseparation of amino acids by achiral gas chromatography and gas chromatography-mass spectrometry.

N-ethoxycarbonylation was combined with (S)-1-phenylethylamidation for enantioseparation of amino acids by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) on achiral capillary columns. The method provided complete enantioseparations of 12 amino acids as diastereomeric N-ethoxycarbonyl/(S)-1-phenylethylamides with exceptional resolutions for proline (R(s) > or = 9.9) and pipecolic acid (R(s) > or = 10.2). GC-MS analysis in selected ion monitoring mode employing standard addition method, facilitated quantitation of D-pipecolic acid in kidney bean (0.95 microg/10 mg) and adzuki bean (0.14 microg/10 mg). The peak area ratios indicated that they had the identical chiral composition at 2.5% for D-pipecolic acid and 97.5% for L-pipecolic acid.

Patterns of plasma fatty acids in rat models with adenovirus infection.

Adenoviral vectors are among the most promising vectors available for human gene therapy. However, the use of recombinant adenoviral vectors, including replicationcompetent adenovirus (RCA), raises a variety of safety concerns in relation to the development of new therapies based on gene therapy. To examine how organic compounds change in rat plasma following the injection of adenovirus, beta-galactosidase expressing recombinant adenovirus (designated rAdLacZ) or RCA, we investigated the content of fatty acids (FAs), which are important biochemical indicators in pathological conditions. Pattern recognition analysis on the level of FAs in rat plasma is described for the visual discrimination of adenovirus infection groups from normal controls. Plasma FAs from four control rats (normal group), and from four rats with rAdLacZ infection and six rats with RCA infection (the two abnormal groups), were examined by gas chromatography-mass spectrometry in selected ion monitoring modes as their tert-butyldimethylsilyl derivatives. In total, 20 FAs were positively detected and quantified. The results of the Studentos t-test on the normal mean of two abnormal groups, the levels of three FAs (p< 0.05) from rAdLacZ group and eleven FAs (p < 0.05) from RCA group were significantly different. When star symbol plotting was applied to the group mean values of 20 FAs after normalization to the corresponding normal mean values, the resulting eicosagonal star patterns of the two infected groups were distorted into similar shapes, but were distinguishable from each other. Thus, these approaches will be useful for screening and monitoring of diagnostic markers for the effects of infection following the use of adenoviral vectors in gene therapy.

N-Menthoxycarbonylation combined with trimethylsilylation for enantioseparation of beta-blockers by achiral dual-column gas chromatography.

Solvent extractive two-phase menthoxycarbonyl (MnOC) derivatization was combined with trimethylsilyl (TMS) reaction for enantioseparation of beta-blockers by gas chromatography employing achiral DB-5 and DB-17 dual-columns of different polarity. beta-Blockers in alkaline solution were vortex-mixed with menthyl chloroformate present in dichloromethane to be extracted as diastereomeric N-MnOC derivatives. The subsequent O(N)-TMS reaction allowed complete enantioseparations of two beta-blockers and partial separations of five as N-MnOC/O(N)-TMS derivatives in a single analysis. The temperature-programmed retention index sets were characteristic of each derivative, facilitating chiral discrimination of each enantiomer.

Simultaneous retention index analysis of urinary amino acids and carboxylic acids for graphic recognition of abnormal state.

Simultaneous profiling analysis of urinary amino acids (AAs) and carboxylic acids (CAs) was combined with retention index (I) analysis for graphic recognition of abnormal metabolic state. The temperature-programmed I values of the AA and CA standards measured as ethoxycarbonyl (EOC)/methoxime (MO)/tert-butyldimethylsilyl (TBDMS) derivatives were used as the reference I values. Urine samples were subjected to the sequential EOC, MO and TBDMS reactions for the analysis by gas chromatography (GC) and GC-mass spectrometry. The complex GC profiles were then transformed into their respective I patterns in bar graphic forms by plotting the normalized peak area ratios (%) of the identified AAs and CAs against their reference I values as the identification numbers. When the present method was applied to infant urine specimens from normal controls and patients with inherited metabolic diseases such as phenylketonuria, maple syrup urine disease, methylmalonic aciduria or isovaleric aciduria, each I pattern of bar graph more distinctly displayed quantitative abundances of urinary AAs and CAs in qualitative I scale, thus allowing graphic discrimination between normal and abnormal states.

Sequential ethoxycarbonylation, methoximation and tert-butyldimethylsilylation for simultaneous determination of amino acids and carboxylic acids by dual-column gas chromatography.

Amino acids (AAs) in alkaline solution were first ethoxycarbonylated with subsequent methoximation of keto acids (KAs). After acidification and solid-phase extraction, tert-butyldimethylsilylation was performed for direct analysis by gas chromatography (GC) on dual-columns with different polarities, which provided simultaneous separation of multiple amino acids, carboxylic acids (CAs) and keto acids, facilitating accurate peak confirmation based on matching with retention index sets characteristic of each analyte. The present method was linear (r2 > or = 0.9955) with good precision (0.1-9.4%) and accuracy (-8.6 to 9.9%), allowing simultaneous screening for diagnostic amino acids along with carboxylic acids and keto acids in urine from a phenylketonuria patient.

Chiral discrimination of multiple profens as diastereomeric (R)-(+)-1-phenylethylamides by achiral dual-column gas chromatography.

Profens were converted into diastereomeric (R)-(+)-1-phenylethylamides using ethyl chloroformate and triethylamine in dichloromethane. Gas chromatographic analysis on dual-columns with different polarities provided complete enantioresolution of eight profens, facilitating chiral discrimination based on matching with retention index sets characteristic of each enantiomer. The present method was linear (r >/= 0.9992) with good precision (0.8-6.0%) and accuracy (-9.3 to 0.003%), allowing detection of trace (R)-profens in optical purity test on four (S)-profen mixture in a single run. And the method allowed simultaneous enantiomeric screening for ibuprofen enantiomers and their chiral metabolites excreted in urine following administration of racemic ibuprofen.

Optical purity determination of (S)-ibuprofen in tablets by achiral gas chromatography.

An optical purity test was indirectly performed on (S)-ibuprofen as its diastereomeric (R)-(+)-1-phenylethylamide derivative using achiral gas chromatography (GC). The method for the determination of trace (R)-ibuprofen (optical impurity), within the range 1.0 to 50 ng, from a racemic ibuprofen standard was linear (r = 0.9997) with acceptable precision (% RSD < or = 5.3) and accuracy (% RE = 0.7 - -3.9). Similar results were obtained with the method validation for the quantification of (S)-ibuprofen within the range 0.1 to 2.0 microg using a (S)-ibuprofen standard. When applied to seven different commercial (S)-ibuprofen products, their optical purities (98.7 - 99.1%) were determined with good precision (% RSD < or = 4.0).

Solid-phase extraction of L-muscone from aqueous samples with Amberlite XAD-4 for gas chromatographic assay.

An efficient analytical method was devised for the accurate L-muscone assay in aqueous samples. It involves solid-phase extraction of L-muscone in adsorption mode using XAD-4 as the sorbent and dichloromethane modified with 10% (v/v) methanol as the eluting solvent. The gas chromatographic analysis of the eluate residue dissolved in toluene on a DB-5MS capillary column provided complete resolution of L-muscone from the co-extracted interferences. The overall method showed excellent linearity (r2 > or = 0.9994) in the range of 0.1 to 2.0 microg/mL with good intra- and inter-day precisions (% RSD = 2.5 to approximately 7.3) and with high extraction recovery rates (> or = 98.1%). When the present method was applied to a L-muscone herbal drink product, the within-batch RE (%) in the labeled concentration (1.5 microg/mL) for the three randomly chosen bottles were -2.4, -1.3 and -3.3 with high precision (% RSD < or = 3.1). The present method is considered to be suitable for quality control evaluation on liquid drinks and other complex formulations fortified with L-muscone.

Combined isobutoxycarbonylation and tert-butyldimethylsilylation for the GC/MS-SIM detection of alkylphenols, chlorophenols and bisphenol A in mackerel samples.

The alkylphenols, chlorophenols, and bisphenol A were determined by gas chromatography/mass spectrometry-selected ion monitoring (GC/MS-SIM) followed by two work-up methods for comparison: isobutoxycarbonyl (isoBOC) derivatization and tert-butyldimethylsilyl (TBDMS) derivatization. Eleven endocrine disrupting chemicals (EDCs) of phenols in biological samples were extracted with acetonitrile and then the acetonitrile layer underwent freezing filtration 60 degrees C for 2 hours. Solid-phase extraction (SPE) was used with XAD-4 and subsequent conversion to isoBOC or TBDMS derivatives for sensitivity analysis with the GC/MS-SIM mode. For isoBOC derivatization and TBDMS derivatization the recoveries were 92.3 approximately 150.6% and 93.8-108.3%, the method detection limits (MDLs) of bisphenol A for SIM were 0.062 microg/kg and 0.010 microg/kg, and the SIM responses were linear with the correlation coefficient varying by 0.9755-0.9981 and 0.9908-0.9996, respectively. When these methods were applied to mackerel samples, the concentrations of the 11 phenol EDCs were below the MDL.

Enantioseparation of flurbiprofen and ketoprofen in patches and in urine excretions by achiral gas chromatography.

The enantiomeric composition tests on flurbiprofen and ketoprofen present in patch products and in urine excretions following patch applications were performed as diastereomeric (R)-(+)-1-phenylethylamides by achiral gas chromatography and by gas chromatography-mass spectrometry in selected ion monitoring mode. The method for determination of (R)- and (S)-enantiomers in the range from 0.1 to 5.0 microg was linear (r > or = 0.9996) with acceptable precision (% RSD < or = 5.2) and accuracy (% RE = 0.6 approximately -2.4). The enantiomeric compositions of flurbiprofen in one patch product and of ketoprofen in five different products were identified to be racemic with relatively good precision (< or = 6.4%). The urinary excretion level of (R)-flurbiprofen was two times higher than its antipode, while the comparable excretion levels of (R)- and (S)-enantiomers for ketoprofen were observed.

Retinal damage in chloroquine maculopathy, revealed by high resolution imaging: a case report utilizing adaptive optics scanning laser ophthalmoscopy.

A 53-year-old Asian woman was treated with hydroxychloroquine and chloroquine for lupus erythematosus. Within a few years, she noticed circle-shaped shadows in her central vision. Upon examination, the patient's visual acuity was 20 / 25 in both eyes. Humphrey visual field (HVF) testing revealed a central visual defect, and fundoscopy showed a ring-shaped area of parafoveal retinal pigment epithelium depigmentation. Fundus autofluorescence imaging showed a hypofluorescent lesion consistent with bull's eye retinopathy. Adaptive optics scanning laser ophthalmoscope (AO-SLO) revealed patch cone mosaic lesions, in which cones were missing or lost. In addition, the remaining cones consisted of asymmetrical shapes and sizes that varied in brightness. Unlike previous studies employing deformable mirrors for wavefront aberration correction, our AO-SLO approach utilized dual liquid crystal on silicon spatial light modulators. Thus, by using AO-SLO, we were able to create a photographic montage consisting of high quality images. Disrupted cone AO-SLO images were matched with visual field test results and functional deficits were associated with a precise location on the montage, which allowed correlation of histological findings with functional changes determined by HVF. We also investigated whether adaptive optics imaging was more sensitive to anatomical changes compared with spectral-domain optical coherence tomography.

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.

The free fatty acid metabolome in cerebral ischemia following human mesenchymal stem cell transplantation in rats.

BACKGROUND: Mesenchymal stem cells (MSCs) have the potential to promote brain repair and improve recovery following stroke. We investigated changes in free fatty acids (FFAs) following intravenous human MSC (hMSC) transplantation into rats that had undergone transient middle cerebral artery occlusion (MCAo). METHODS: Rats were subjected to 2-hours MCAo, followed by intravenous transplantation of hMSC or phosphate-buffered saline (PBS) at one day after MCAo. All rats were sacrificed 5 days after MCAo. Metabolic profiling of free fatty acids (FFAs) level was assessed in plasma and brain from control rats (n=8), PBS-treated MCAo rats (n=6), and hMSC-treated MCAo rats (MCAo+hMSC, n=6). RESULTS: The levels of some FFAs in plasma and brain samples of the MCAo and MCAo+hMSC groups were significantly different from those of the control group. The percentage composition of myristic acid in plasma and those of myristic acid, linoleic acid, and eicosenoic acid in brain tissues of the MCAo+hMSC group were significantly reduced compared to those in the untransplanted MCAo group. CONCLUSION: Our metabolic approach has provided insights into understanding the complexity of biochemical and physiological events that occur in ischemic brain injury and the transplantation effects of MSCs in stroke.

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.