LC-MS quantification of drug metabolites is sometimes impeded by the availability of internal standards that often requires customized synthesis and/or extensive purification. Although isotopically labeled internal standards are considered ideal for LC-MS/MS based quantification, de novo synthesis using costly isotope-enriched starting materials makes it impractical for early stage of drug discovery. Therefore, quick access to these isotope-enriched compounds without chemical derivatization and purification will greatly facilitate LC-MS/MS based quantification. Herein, we report a novel 18O-labeling technique using metabolizing enzyme carboxylesterase (CES) and its potential application in metabolites quantification study. Substrates of CES typically undergo a two-step oxygen exchange with H218O in the presence of the enzyme, generating singly- and doubly-18O-labeled carboxylic acids; however, unexpected hydrolytic behavior was observed for three of the test compounds – indomethacin, piperacillin and clopidogrel. These unusual observations led to the discovery of several novel hydrolytic mechanisms. Finally, when used as internal standard for LC-MS/MS based quantification, these in situ labeled compounds generated accurate quantitation comparable to the conventional standard curve method. The preliminary results suggest that this method has potential to eliminate laborious chemical synthesis of isotope-labeled internal standards for carboxylic acid-containing compounds, and can be developed to facilitate quantitative analysis in early-stage drug discovery. 相似文献
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. 相似文献
An automated on-line method for simultaneous analysis of five phenothiazine drugs by high-performance liquid chromatography
(HPLC)/sonic spray ionization mass spectrometry (SSI-MS) has been established, using backflush column switching. A 400-μl
portion of serum sample diluted 81-fold with distilled water was subjected to the on-line system. In the system, an Oasis
HLB cartridge was used as the precolumn for extraction; large molecules such as proteins in serum were discarded by use of
distilled water containing 0.1% formic acid as a mobile phase. After switching a valve, the analytes trapped in the precolumn
were eluted in the backflush mode and separated by a Chromolith Performance RP-18e column, which is composed of C18-bonded monolithic silica. The column effluents were then introduced into the SSI-MS. The present method provided successful
separation and determination of six phenothiazines including an internal standard. Satisfactory linearities, reproducibility,
and sensitivity were obtained at concentration levels that matched the toxic levels of phenothiazines. All drug peaks appeared
within 18 min, and the system could be reequilibrated in only about 8 min for the next run. Because of the simplicity and
rapidness of the method, it is likely to be useful in the fields of emergency medicine and forensic toxicology. 相似文献
Tryptamine (TA) occurs in trace levels in the brain, but its role in the central nervous system is not clear. However, there is evidence that TA may be a neuromodulator since it binds to specific binding sites in the brain. TA was measured as a diheptafluorobutyryl derivative in rat whole brain by capillary gas chromatography—mass spectrometry using negative chemical ionization (NCI) and single ion monitoring (SIM). d4-TA was used as the internal standard. The ions m/z 532 and m/z 536 were monitored to identify TA and d4-TA, respectively and to calculate the concentration of TA in rat whole brain which was found to be 0.19 ± 0.08 ng g−1 (n = 8). The results confirm the earlier TA concentrations measured by GC—MS using positive electron impact ionization. However, NCI improved the signal/noise ratio of the method increasing its sensitivity for TA. 相似文献
Cyclic peptide disulfides of the general formula were synthesized from the corresponding peptide derivatives [Boc-Cys(Trt)(Gly)-n-Cys(Trt)-OBut] by oxidation with iodine in methanol and by subsequent removal of the terminal groups with trifluoroacetic acid. Acid ionization constants of the obtained peptides were determined by potentiometric titration in aqueous KCl (0.1 mol/L) medium. All compounds have two dissociable hydrogens, corresponding to carboxyl (pK1= 2.35–2.84) and to terminal amino group (pK2= 5.61–6.93); pK1, values show first an upward and then a downward trend with the increase in ring size; the opposite is true for pK2, values. These trends could be tentatively attributed to the intramolecular salt bridge (-COO——-NH+3-) formation. 相似文献
Group B Streptococcus (GBS) is an important invasive pathogen in neonates, pregnant women and the elderly. Serotype VI GBS, which has been rarely reported globally, has emerged as a significant pathogen in Asia. However, traditional serologic latex agglutination (LA) methods may fail to type isolates that lack of or low expression of CPS.
Methods
A total of 104 GBS strains were analyzed by MALDI-TOF MS. Multiplex PCR and multilocus sequence typing (MLST) were also performed to confirm their strains. The protein markers were purified with gel electrophoresis and LC-column, followed by identification with nanoLC–MS/MS analysis.
Results
Protein peak of 6251-Da was appeared in most (20/24, 92%) serotypes VI (94% ST-1 or single locus variant of ST-1), and protein peak of 6891-Da was appeared in most serotypes III (15/18, 83%) and Ib (19/23, 83%) strains. The protein peak of 6251-Da and 6891-Da were identified as CsbD family protein and UPF0337 protein gbs0600, respectively.
Conclusions
The protein peak of 6251 Da may play a role of emergence of ST-1 clone, serotype VI GBS in central Taiwan and could be useful in rapid identifying invasive serotype VI from III isolates, which is hardly achieved by LA. 相似文献
Summary: Homopolymers of a bis‐trifluorocarbinol substituted norbornene ( 1 ) (α,α‐bis(trifluoromethyl)bicyclo[2.2.1]hept‐5‐ene‐2‐ethanol or HFANB) and copolymers of 1 with t‐butyl ester of 5‐carboxylic acid ( 2 , t‐BuEsNB) were produced using palladium catalysts and olefinic chain transfer agents such as 1‐hexene and ethylene to control molecular weight. However, these low‐molecular‐weight polymers exhibited relatively low optical transparencies at 193 nm. In fact, the opacity (measured as optical densities in absorbance units per micron) of thin films of these homo‐ and co‐polymers was inversely proportional to their molecular weight. This relationship is consistent with an end group contribution to the film opacity. Spectroscopic analysis of these polymers by 1H NMR and MALDI‐TOF MS confirmed that 1‐hexene and ethylene chain transfer agents generated olefin‐terminated vinyl addition polymers. The olefinic end group contribution to optical density can be eliminated by appropriate chemical modification. Both epoxidation and hydrogenation of the polymer olefinic end groups generated very low optical density materials, independent of molecular weight, that are suitable as 193‐nm photoresist binder resins.
End group modification of vinyl and hexenyl‐terminated homopolymers of 1 by epoxidation or hydrogenation. 相似文献
Paenibacillus thiaminolyticus is a nonvirulent organism found in human and ruminant microbiota. However, P. thiaminolyticus can act as an opportunistic pathogen in humans. We describe a case of abdominal wall hematoma secondarily infected by P. thiaminolyticus. Our findings emphasize the risk for unusual Paenibacillus infections in otherwise healthy persons. 相似文献