Compositional analysis of copoly (DL-lactic/glycolic acid) (PLGA) by pyrolysis-gas chromatography/mass spectrometry combined with one-step thermally assisted hydrolysis and methylation in the presence of tetramethylammonium hydroxide

Rapid and precise compositional analysis of copoly (DL-lactic/glycolic acid) (PLGA) was performed by pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) combined with one-step hydrolysis and methylation in the presence of tetramethylammonium hydroxide (TMAH). Pyrolysis of PLGA with TMAH gave two characteristic products, derivatives of DL-lactic acid and glycolic acid, which directly reflect the average molar composition of PLGA. The analytical results for PLGA samples with various compositional ratios were in good agreement with those obtained by 1H-NMR spectrometry, and the precision was satisfactory.     

Development of biodegradable co-poly( -lactic/glycolic acid) microspheres for the controlled release of 5-FU by the spray drying method

The water-soluble anti-cancer drug, 5-fluorouracil (5-fluoro-2,4-pyrimidinedione) (5-FU) is encapsulated into biodegradable co-poly ( -lactic/glycolic acid) (PLGA) using the spray drying method for the development of long-lasting controlled release systems. In this study, the effects of both polymeric composition and technological parameters on release profiles of 5-FU were investigated. The degradation of various microspheres was also investigated. The mixture of dichloromethane/chloroform/methanol (1:1:2 v/v) instead of dichloromethane/chloroform (1:1 v/v) resulted in the modification of morphology, while the physical structure of the microsphere varied from a porous PLGA microsphere to a dense PLGA microsphere. The results show that the average diameter was 2 μm and the anti-cancer drug loading of microspheres approached approximately 8% (w/w). In addition, the lactide/glycolide ratio of the polymer is an important parameter for controlling the release profile of the entrapped anticancer drug. Our results indicate that the mixture solvent using the spray drying method was more efficient than emulsification solvent diffusion.     

A study of some imidazo[l,5-a]benzodiazepin-6-ones by electron impact mass spectrometry. characterization by tandem mass spectrometry of a distonic fragment ion

The mass spectra of several imidazo[l,5-a]benzodiazepin-6-one derivatives of neuropharmacological significance were determined in order to establish their decomposition paths under electron impact. A novel distonic fragment ion was observed and it was possible to demonstrate by consideration of metastable ions and by tandem mass spectrometry that it undergoes isomerization to the corresponding classical structure.     

MALDI TOF/TOF tandem mass spectrometry as a new tool for amino acid analysis

This is the first report of an application of collisionally induced fragmentation of amino acids (AA) and their derivatives by MALDI TOF/TOF tandem mass spectrometry (MS). In this work, we collected the data on high-energy fragmentation reactions of a large group of protonated amino acids and their derivatives with the goal of determining which product ions are analyte specific and if yields of these fragment could be used for quantitative analysis. From 34 different amino acids (20 alpha-amino acids, beta-amino acids, homocysteine, GABA, and modified AA Met sulfone and sulfoxide, hydroxyproline, etc.) we observed that high yields of the target specific immonium ions and fragmentation patterns are most similar to EI or FAB CID on sector instruments. The major exceptions were two highly basic amino acids, Arg and Orn. It is noted that neither beta-, gamma-, nor delta-amino acids produce immonium ions. As might be predicted from high-energy CID work on peptides from the sectors and TOF/TOF, the presence of specific indicator ions in MALDI tandem MS allows distinguishing isomeric and isobaric amino acids. These indicator ions, in combination with careful control of data acquisition, ensure quantitative analysis of amino acids. We believe our data provide strong basis for the application of MALDI TOF/TOF MS/MS in qualitative and quantitative analysis of amino and organic acids, including application in clinical medicine.     

Determination of zearalenone by liquid chromatography/tandem mass spectrometry and application to a pharmacokinetic study

Zearalenone, a mycotoxin biosynthesized by various Fusarium fungi, is widely found as a contaminant in grains and animal feeds. This study describes a rapid and sensitive LC/MS/MS assay method for the quantification of zearalenone in rat serum. The assay was validated to demonstrate the specificity, linearity, recovery, lower limit of quantification (LLOQ), accuracy and precision. The multiple reaction monitoring was based on the transition of m/z 317.0 → 130.9 for zearalenone and 319.0 → 204.8 for zearalanone (internal standard). The assay utilized a single liquid–liquid extraction with t‐butyl methyl ether and isocratic elution, and the LLOQ was 0.5 ng/mL using 0.1 mL rat serum. The assay was linear over a concentration range from 0.5 to 200 ng/mL, with correlation coefficients >0.9996. The mean intra‐ and inter‐day assay accuracy was 101.2–112.9 and 96.3–108.0%, respectively. The mean intra‐ and inter‐day precision was between 1.3–7.6 and 3.6–10.6%, respectively. The developed assay was applied to a pharmacokinetic study after a bolus intravenous injection of zearalenone in rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural analysis of a glycoprotein by liquid chromatography-mass spectrometry and liquid chromatography with tandem mass spectrometry. Application to recombinant human thrombomodulin

Using recombinant human thrombomodulin (rhTM) expressed in Chinese hamster ovary (CHO) cells, we studied the structural analysis of a glycoprotein by liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS-MS). First, we analyzed the structure of both the O- and N-linked glycans in rhTM by oligosaccharide mapping using LC-MS equipped with a graphitized carbon column (GCC-LC-MS). Major O- and N-linked glycans were determined to be core 1 structure and fucosyl biantennary containing NeuAc(0-2) respectively. Next, the post-translational modifications and their heterogeneities, including the site-specific glycosylation, were analyzed by mass spectrometric peptide/glycopeptide mapping of trypsin-digested rhTM and precursor-ion scanning. Precursor-ion scanning was successful in the detection of five glycopeptides. Four N-glycosylation sites and their site-specific carbohydrate heterogeneity were determined by their mass spectra. O-Glycosylation could be estimated on the basis of its mass spectrum. We were able to identify partial beta-hydroxylation on Asn324 and Asn439, and O-linked glucose on Ser287 from the peptide/glycopeptide map and their mass spectra. We demonstrated that a sequential analysis of LC-MS and LC-MS-MS are very useful for the structural analysis of O- and N-linked glycans, polypeptides, and post-translational modifications and their heterogeneities, including site-specific glycosylation in a glycoprotein. Our method can be applied to a glycoprotein in biological samples.     

Measurement of bile acid CoA esters by high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS)

The novel and rapid assay presented here combines high-performance liquid chromatography and electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS) to directly measure and quantify the CoA esters of 3alpha,7alpha,12alpha-trihydroxy- and 3alpha,7alpha-dihydroxy-5beta-cholestan-26-oic acid (THCA and DHCA). The latter are converted inside peroxisomes to the primary bile acids, cholic and chenodeoxycholic acids, respectively. Prior to MS/MS, esters were separated by reversed-phase HPLC on a C(18) column using an isocratic mobile phase (acetonitrile/water/2-propanol) and subsequently detected by multiple reaction monitoring. For quantification, the CoA ester of deuterium-labelled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid (d(4)-CA) was used as internal standard. To complete an assay took less than 8 min.To verify the validity of the assay, the effect of peroxisomal proteins on the efficacy of extraction of the CoA esters was tested. To this end, variable amounts of the CoA esters were spiked with a fixed amount of either intact peroxisomes or peroxisomal matrix proteins and then extracted using a solid-phase extraction system. The CoA esters could be reproducibly recovered in the range of 0.1-4 micromol l(-1) (linear correlation coefficient R(2) > 0.99), with a detection limit of 0.1 micromol l(-1).In summary, electrospray ionization tandem mass spectrometry combined with HPLC as described here proved to be a rapid and versatile technique for the determination of bile acid CoA esters in a mixture with peroxisomal proteins. This suggests this technique to become a valuable tool in studies dealing with the multi-step biosynthesis of bile acids and its disturbances in disorders like the Zellweger syndrome.     

Identification of caffeic acid derivatives in Actea racemosa (Cimicifuga racemosa,black cohosh) by liquid chromatography/tandem mass spectrometry

Caffeic acid derivatives occurring in black cohosh [Cimicifuga racemosa (L.) Nutt., Actaea racemosa (Ranunculaceae)], some of which may have pharmacological activity, were analyzed using high-performance liquid chromatography (HPLC) electrospray ionization tandem mass spectrometry (ESI-MS/MS) with the aim of developing a methodology for their rapid identification in a complex plant matrix. Based on these studies, structurally characteristic product ions and neutral molecule losses were identified, which were then used during LC/MS/MS with product ion scanning, precursor scanning and constant neutral loss scanning to detect caffeic acid derivatives in a crude extract of black cohosh. Several caffeic acid derivatives were detected, and the identification of six of them were confirmed by comparison with authentic standards including caffeic acid, ferulic acid, isoferulic acid, fukinolic acid, cimicifugic acid A, and cimicifugic acid B. Four other compounds were detected that appeared to be caffeic acid derivatives based on LC/MS/MS retention times, molecular weights, and fragmentation patterns during MS/MS. Since standards were unavailable for these four compounds, they were tentatively identified using LC/MS/MS as cimicifugic acid E, cimicifugic acid F, dehydrocimicifugic acid A, and dehydrocimicifugic acid B. Dehydrocimicifugic acid A and dehydrocimicifugic acid B have not been reported previously to be constituents of black cohosh.     

Determination of long-acting release octreotide, an octapeptide analogue of somatostation, in human plasma by liquid chromatography/tandem mass spectrometry

A sensitive and selective method for the determination of long-acting released octreotide in human plasma has been developed based on liquid chromatography/tandem mass spectrometry (LC/MS/MS). Octreotide and the internal standard, triptorelin, were precipitated from the matrix, washed with dichloromethane and subsequently separated by reversed-phase high-performance liquid chromatography (HPLC) employing a 1% formic acid/methanol gradient system. Detection was by electrospray ionization mass spectrometry in the positive ion mode using multiple-reaction monitoring. The assay was linear in the concentration range 0.0500-50.0 ng/mL with intra- and inter-day precision (as relative standard deviation) of <2.95% and <8.37%, respectively. The limit of detection was 0.0200 ng/mL. The method was applied to a pharmacokinetic study of long-acting released octreotide in healthy volunteers given an intramuscular injection containing 20 mg octreotide.     

Determination of diazepam and its metabolites in human urine by liquid chromatography/tandem mass spectrometry using a hydrophilic polymer column

Diazepam and its major metabolites, nordazepam, temazepam and oxazepam, in human urine samples, were analyzed by liquid chromatography (LC)/tandem mass spectrometry (MS/MS) using a hydrophilic polymer column (MSpak GF-310 4B), which enables direct injection of crude biological samples. Matrix compounds in urine were eluted first from the column, while the target compounds were retained on the polymer stationary phase. The analytes retained on the column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. All compounds showed base-peak ions due to [M+H]+ ions on LC/MS with positive ion electrospray ionization, and product ions were produced from each [M+H]+ ion by LC/MS/MS. Quantification was performed by selected reaction monitoring. All compounds spiked into urine showed method recoveries of 50.1-82.0%. The regression equations for all compounds showed excellent linearity in the range of 0.5-500 ng/mL of urine. The limits of detection and quantification for each compound were 0.1 and 0.5 ng/mL of urine, respectively. The intra- and inter-day coefficients of variation for all compounds in urine were not greater than 9.6%. The data obtained from actual determination of diazepam and its three metabolites, oxazepam, nordazepam and temazepam, in human urine after oral administration of diazepam, are also presented.     

Isomer differentiation in 7, 12-dimethylbenz[a]anthracene-pyridine adducts by fast atom bombardment tandem mass spectrometry

Three isomeric 7,12-dL-nethylbenz[α]anthracene-pyridine adduct salts, namely.. the 5-N-pyridinium-7,12-dimethylbenz[α]anthracene perchlorate, the 7-N-pyridiniummethylene-12methylbenz[ α]anthracene picrate, and the 7-methyl-12-N-pyridiniummethylenebenz[ α]anthracene picrate, were studied by fast atom bombardment tandem mass spectrometry using high energy collisional-activated dissociation (CAD). The CAD mass spectra of the molecular cations and the (M – pyridine)⁺ ions allow one to distinguish positional isomers on the basis of daughter ion peak height ratios. The differences in the CAD mass spectra of the (M – pyridine)⁺ ions are probably due in part to formation of isomer-specific fused-ring tropyliumions.     

Quantification of benzo[a]pyrene diol epoxide DNA-adducts by stable isotope dilution liquid chromatography/tandem mass spectrometry

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants found in car exhausts, charbroiled food, and tobacco smoke. Three pathways for the metabolic activation of B[a]P to ultimate carcinogens have been proposed. The most widely accepted pathway involves cytochrome-P450 (CYP) 1A1- and/or 1B1-mediated formation of B[a]P-7,8-oxide, which undergoes epoxide hydrolase-mediated metabolism to the proximate carcinogen B[a]P-7,8-dihydro-7,8-diol. Further CYP1A1- and/or CYP1B1-mediated activation of the dihydrodiol results in the formation of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE), the ultimate carcinogen. In previous studies, it was demonstrated that (+)-anti-B[a]PDE was the most potent tumorigen of the CYP-derived B[a]PDE diastereomers. We have developed a stable isotope dilution, liquid chromatography multiple reaction monitoring/mass spectrometry (LC-MRM/MS) assay for all eight (+/-)-anti-B[a]PDE-derived dGuo and dAdo DNA-adducts. The LC-MRM/MS assay was rigorously validated and used to show that (+)-anti-trans-B[a]PDE-dGuo was the major adduct formed when naked DNA and human bronchoalveolar adenocarcinoma H358 cells were treated with (+/-)-anti-B[a]PDE. The preference for DNA-adducts derived from (+)-anti-B[a]PDE was even more apparent in cellular DNA. Thus, the increased potency of (+)-anti-B[a]PDE as a tumorigen is most likely due its ability to preferentially form DNA-adducts when compared with (-)-anti-B[a]PDE. Also, the adduct profile suggests that this occurs by binding of (+)-anti-B[a]PDE to DNA in a manner that facilitates covalent binding to dGuo rather than dAdo residues.     

Simultaneous determination of magnesium lithospermate B, rosmarinic acid, and lithospermic acid in beagle dog serum by liquid chromatography/tandem mass spectrometry

A rapid, sensitive and specific isocratic liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous quantitative determination of magnesium lithospermate B (MLB), rosmarinic acid (RA), and lithospermic acid (LA) in beagle dog serum, with silibinin as internal standard. The serum samples were treated by liquid-liquid extraction and analyzed using LC/MS/MS with a TurboIonSpray source. A short run-time (3 min) fulfilled the need for monitoring serum levels of MLB, RA, and LA in large-scale studies. The calibration curves for MLB, RA, and LA were linear over the ranges 8-2048, 4-1024, and 4-1024 ng/mL, respectively, with coefficients of correlation >0.999. The intra- and inter-day precision (CV) of analysis was <10%, and accuracy ranged from 90-104%. This quantitation method was successfully applied to a pharmacokinetic study of salvianolate administrated by intravenous infusion with dosage of 6 mg/kg in beagle dogs.     

A new approach for measuring protein adducts from benzo[a]pyrene diolepoxide by high performance liquid chromatography/tandem mass spectrometry

The long-range goal of the present study is the development of a general approach for in vivo dosimetry of reactive metabolites of polycyclic aromatic hydrocarbons (PAHs), to be used as a tool in cancer risk assessment. With benzo[a]pyrene (BaP) chosen as indicator and a model of PAHs this study aims at the development of a method for the determination of adducts to histidine (His) in hemoglobin (Hb) and serum albumin (SA) of reactive metabolites of BaP. The predominantly mutagenic metabolite of BaP has been shown to be a diolepoxide isomer, +(anti)r-7, t-8-dihydroxy-t-9,10-epoxy-7,8, 9,10-tetrahydrobenzo[a]pyrene (+BPDE). In comparison with other methods for protein degradation, hydrazinolysis was found to be sufficiently effective and mild. The His adduct isolated after protein hydrazinolysis, with protection by tert-butyloxycarbonyl (Boc) of the hydrazide and alpha-amino groups, was shown to be N(im)- +/- (r-7, t-8, t-9-trihydroxy-7, 8, 9, 10-tetrahydrobenzo[a]pyren-c-10-yl)-N(alpha), N(2)-bis(tert-butyloxycarbonyl)-L-histidinehydrazide. Isomers of this compound, used as references, were synthesized and characterized by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Adducts in Hb and SA from in vitro treatment with BPDE were characterized after hydrazinolysis by HPLC-UV/MS, muHPLC/MS/MS and gas chromatography/mass spectrometry (GC/MS). Approximately 70 and 10% of the isolated BPDE adducts from SA and Hb, respectively, were His adducts. Other products were released as BaP tetrols and BaP triols. For the purpose of enrichment/purification of BPDE-His adducts, C(18) and cation exchange solid phase extraction (SPE) were utilized. The sensitivity obtained by this new approach, based on hydrazinolysis of protein, enrichment by SPE and analysis with muHPLC/MS/MS (APCI), is in the low-fmole range.     

Analysis of pesticide residues in fruits, vegetables, and milk by gas chromatography/tandem mass spectrometry.

A method for detection, quantitation, and confirmation of more than 100 pesticides by gas chromatography (GC) with ion trap mass spectrometry (MS/MS) has been developed. The sensitivity of this method for many analytes is equal to or lower than those of selective GC detectors such as flame photometric detectors and electrolytic conductivity detectors. Using MS/MS, very low detection limits and good confirmation (1 precursor ion and 2 or more product ions) are achieved simultaneously. The entire list of pesticides is screened with 2 injections per sample. Samples are introduced onto the column by a temperature-programmed cold injection to maximize response. Each pesticide is run with its own unique set of parameters, which fragment the compound, retaining only the precursor ion. This ion is then refragmented to create a product spectrum. The selectivity of MS/MS gives a very clean spectrum, making compound identification and confirmation clear, even with a relatively dirty food matrix. If care is taken to maintain the injection port and guard column, this method can reliably identify and confirm more than 100 pesticides at the low parts-per-billion range.     

Highly sensitive assay for tiotropium, a quaternary ammonium, in human plasma by high-performance liquid chromatography/tandem mass spectrometry

Tiotropium bromide, a long-acting inhaled bronchodilator analogous to ipratropium bromide, is currently undergoing development for the treatment of chronic obstructive pulmonary disease. To evaluate its systemic absorption in humans, we have developed a rapid and sensitive method for its determination in human plasma based on high-performance liquid chromatography with tandem mass spectrometric detection (HPLC/MS/MS). Reversed-phase chromatography of tiotropium and the internal standard clenbuterol was carried out using acetonitrile/10 mM ammonium acetate (1% formic acid) 40:60 as mobile phase in a run time of 3.0 min. The sample preparation involved deproteination with acetonitrile, extraction into dichloromethane and back-extraction into hydrochloric acid. The assay was linear over the concentration range 0.500-50.0 pg/mL with intra- and inter-day precision (as relative standard deviation) both 相似文献   

Structural alterations of humic acid fractions in a steel slag-compost fertilizer during fertilization. Analysis by pyrolysis/methylation-gas chromatography/mass spectrometry

Alterations in the structural features of humic acids (HAs) in a steel slag-compost fertilizer for supplying soluble Fe(II) to barren ground in coastal areas were monitored during a 6-month period of fertilization. HAs, the major organic fractions in the fertilizer, were extracted at different periods of time (2, 4 and 6 months) in the absence and presence of steel slag. To focus on the polymeric structure of the HA-backbone and the organic fractions, which are sorbed onto the HA-polymeric matrix (e.g., lipids), the extracted HA samples were analyzed by pyrolysis-gas chromatography/mass spectrometry under conditions where the sample is methylated with tetramethylammonium hydroxide (TMAH-py-GC/MS). The structural features analyzable by TMAH-py-GC/MS for the HA were dramatically altered in the presence of steel slag during the fertilization period. The major modification involved a decrease in phenolic moieties and a significant increase in the sulfur-containing pyrolysate compounds. These results indicate that the steel slag has a significant effect in altering the structure of HA in the fertilizer. TMAH-py-GC/MS analysis of the HAs indicated that the sulfur in the HAs was present in an organic form and not as elemental sulfur. Significant increases in the levels of biomarker fatty acids (iso- and anteiso-C_15:0 and C_17:0 carboxylic acids), which are related to the activity of sulfate reducing bacteria, suggests that the significant alterations in the HA structures in the presence of steel slag can be attributed to the sulfurization of organic matter in the fertilizer to form hydrogen polysulfides by the reduction of sulfate ions in seawater and/or steel slag via microbial processes.     

Validation of a screening method for corticosteroids in doping analysis by liquid chromatography/tandem mass spectrometry

A selective and sensitive method for the screening of nine corticosteroids in human urine has been validated. Analyses were performed using an ion trap instrument equipped with an electrospray ionisation (ESI) interface. All corticosteroids were separated in less than 20 min after liquid/liquid extraction with diethyl ether. The limit of detection for all substances was 4 ng/mL or lower. The method was applied to detect betamethasone after the intramuscular injection of Diprophos. Betamethasone could be detected for up to 12 days after administration. Validation of the chromatographic separation and mass spectrometric identification of mixtures of betamethasone and dexamethasone are also presented.     

Quantitation and metabolism of mitoquinone, a mitochondria-targeted antioxidant, in rat by liquid chromatography/tandem mass spectrometry

Mitoquinone (MitoQ10 mesylate) is a mitochondria-targeted antioxidant undergoing development for the treatment of neurodegenerative diseases. The aim of this study was to develop and validate an assay based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) to determine mitoquinone and to detect and identify the metabolites of MitoQ10 in rat plasma after an oral dose. After a simple protein precipitation step, plasma samples were analyzed by reversed-phase liquid chromatography using gradient elution with acetonitrile/water/formic acid. Electrospray ionization in the positive ion mode with multiple reaction monitoring (MRM) was used to analyze mitoquinone employing the deuterated compound (d3-MitoQ10 mesylate) as internal standard. The calibration curve for mitoquinone was linear over the concentration range 0.5-250 ng/mL with a correlation coefficient>0.995. The method was sensitive (limit of quantitation 0.5 ng/mL) and had acceptable accuracy (relative error<8.7%) and precision (intra- and inter-day coefficient of variation<12.4%). Recoveries of mitoquinone at concentrations of 1.5, 20 and 200 ng/mL were in the range 87-114%. The method was successfully applied to a pharmacokinetic study in rat after a single oral dose in which four metabolites of MitoQ10 were tentatively identified as hydroxylated MitoQ10, desmethyl MitoQ10 and the glucuronide and sulfate conjugates of the quinol form of MitoQ10.