Fractionation Chromatography Research Articles

Laser scattering centrifugal liquid sedimentation method for the accurate quantitative analysis of mass-based size distributions of colloidal silica.

This paper proposes a laser scattering centrifugal liquid sedimentation (LS-CLS) method for the accurate quantitative analysis of the mass-based size distributions of colloidal silica. The optics comprised a laser diode light source and multi-pixel photon-counting detector for detecting scattered light intensity. The unique optics can only detect the light scattered by a sample through the interception of irradiated light. The developed centrifugal liquid sedimentation (CLS) method comprised a light-emitting diode and silicon photodiode detector for detecting transmittance light attenuation. The CLS apparatus could not accurately measure quantitative volume- or mass-based size distribution of poly-dispersed suspensions, such as colloidal silica, because the detecting signal includes both transmitted and scattered light. The LS-CLS method exhibited improved quantitative performance. Moreover, the LS-CLS system allowed the injection of samples with concentrations higher than that permitted by other particle size distribution measurement systems with particle size classification units using size-exclusion chromatography or centrifugal field-flow fractionation. The proposed LS-CLS method achieved an accurate quantitative analysis of the mass-based size distribution using both centrifugal classification and laser scattering optics. In particular, the system could measure the mass-based size distribution of approximately 20mgmL-1 poly-dispersed colloidal silica samples, such as in a mixture of the four mono-dispersed colloidal silica, with high resolution and precision, thereby demonstrating high quantitative performance. The measured size distributions were compared with those observed through transmission electron microscopy. The proposed system can be used in practical setups to achieve a reasonable degree of consistency for determining particle size distribution in industrial applications.

A Sesquiterpene Enol Carboxylic Acid from Pleurotus tuber regium Sclerotium

As a follow-up to an earlier report on the antihyperlipidaemic activity guided fractionation study on the chloroform extract from the sclerotia of the edible mushroom Pleurotus tuber regium, this present study is reporting the isolation of a sesquiterpene enol carboxylic from the active chromatography fraction of the chloroform extract of this edible mushroom. The purity of the compound was confirmed from TLC and Melting point determination. The chemical structure was elucidated using spectroscopic techniques (Mass spectrometry, 1&2D Nuclear magnetic resonance and Infra red spectroscopy). The isolated compound coded as compound P, exhibited molecular mass of 256 with molecular formular of C14H24O4 and from the 1 &2D NMR and IR analyses, the compound P was identified as a sesquiterpene enol carboxylic acid with a sytemic name of 2,3-dihydroxy-10-propylcyclodec-2-ene-1-carboxylic acid.

Identification of 2,4-Di-tert-butylphenol (2,4-DTBP) as the Major Contributor of Anti-colon cancer Activity of Active Chromatographic Fraction of Parkia javanica (Lamk.) Merr. Bark Extract

Parkia javanica (Lamk.) Merr. is an ethnomedicinal leguminous plant species from northeastern India with a long history of medicinal use among various tribes of this region to treat cholera, dysentery, stomach aches, diarrhea and food poisoning, having antibacterial, wound-healing, anticancer and anti-inflammatory properties. Therefore, in this current study, the methanolic bark extract was carried out and fractionated by using flash chromatography, examined the cytotoxicity of the respective fractions on colon cancer cell lines, and evaluated the major phytochemical compounds present in the fractions using Gas Chromatography-Mass Spectrometry (GC-MS) chemical profiling. Chemical profiling of the fractions by GC-MS revealed in fraction-1 and -2, 2,4-Di-tert-butylphenol was the major compound (50.740% in fraction-1, 21.277% in fraction-2, and 7.859% in fraction-3) having reported anticancer activity. The gradation of the presence of this compound in the fractions was corroborated by the gradation of anti-colon cancer activity of the respective fractions on both the colon carcinoma cell lines. However, the presence of D-Allose in a substantial amount (20.870%) in only fraction-3 could not increase the anticancer activity of fraction-3 over the other two fractions. An in vitro cytotoxic assay guided evaluation of three flash chromatographic fractions (fraction-1, -2, and -3) of methanolic extract of Parkia javanica bark showed significant anticancer properties on two human colon carcinoma cell lines (HCT116 and SW480). The order of efficacy of the fractions was fraction-1> fraction-2 > fraction-3. In a time and dose-dependent experiment, fraction-1, being the most active one, showed an IC50 value of 16.25 µgml-1 (24 hrs), 9.94 µgml-1 (48 hrs), and 9.38 µgml-1 (72 hrs) on HCT116 and 35 µgml-1 (24 hrs), 20.14 µgml-1 (48 hrs), and 19.71 µgml-1 (72 hrs) on the SW480 cell line. Parkia javanica bark extract is bestowed with the potential of anti-colon cancer property and upon chemical profiling of different chromatographic fractions of the extract, 2,4-Di-tert-butylphenol has been identified as the primary anticancer component of the extract.

Investigation on selective separation of oxygen-containing compounds in lignite and sub-bitumite

To realize the high value-added utilization of low-rank coal and elucidate the selectivity of solvent structure and oxygen-containing compounds in low-rank coal, Zhaotong (ZT) lignite and Naomaohu (NMH) sub-bitumite were subjected to thermal dissolution in three types of oxygen-containing solvents with different properties, namely ethanol (ET), tetrahydrofuran (THF) and ethyl acetate (EA). The soluble portions (SPs) obtained were separated by column chromatography into three types of fractions: hydrocarbon (HY), non-hydrocarbon (NHY) and asphaltene (ASP). The results indicated that the yields of SPs in all three solvents for each coal was ET > THF > EA, and the ratios of column chromatographic fractions of SPs of the two coals were significantly different, with lower asphaltene content in the SPs of NMH. The thermal dissolution process of the three solvents significantly reduced the oxygen content of low-rank coal, and the deoxygenation capacity was in the order of ET > THF > EA. More medium or low polarity compounds could be obtained from NMH than from ZT through thermal dissolution process. ET showed excellent selectivity for the generation of both phenols and esters, where the phenols were mainly alkylphenols. THF could release highly condensed polycyclic compounds from coal thus more naphthalenols containing substituent groups were detected in phenols. And it was more selective to COO bonds in NMH than ZT from the results of XPS. More esters were detected in the SPs of NMH compared with ZT. EA had a strong selectivity for CO in low-rank coals, and the thermal dissolution process released a large number of esters, which could be divided into alkanoates and aromatic acid esters. This study can provide a theoretical basis for extracting high value-added chemicals from low-rank coal under mild conditions.

Bioassay-guided Antidiabetic Study of Chromatographic Fractions of Boswellia Dalzielii Hutch. Leaf Extract

Background: Boswellia dalzielli Hutch. (Burseraceae) is a medicinal plant, which is used locally by the local dwellers for the management and treatment of microbial-related diseases, neurological conditions, stomach spasms, diabetes, etc. Objectives: This study aimed at isolating a phytochemical of anti-diabetic potentials from the leaf of Boswellia dalzielii in alloxan-induced diabetic rats. Methods: The n-butanol fraction of the leaf of B. dalzielii was fractionated using column chromatography. Fractions obtained were screened phytochemically and by antidiabetic study. Results: Encoded column fraction B4 (150 mg/kg) produced a maximum reduction (72.45%) in fasting blood glucose (FBG) of animals after 7 hours, which was significantly (P<0.05) different from the controls (alloxan-induced diabetic rats) and was better than glibenclamide (52.67%). The re-column fractions obtained from fraction B4 were pooled based on similar Rf values and encoded B41-B48, and subjected to further antidiabetic evaluation on alloxan-induced mice. Eight sub-fraction with doses of 50 mg/kg each were administered to all the groups. Fraction B44 had the highest reduction of FBG by 65.63%, whose effect was significantly higher than the non-treated diabetic mice (negative control) and glibenclamide (52.68%) at 2.0 mg. Further purification of sub-fraction B44 with Sephadex LH-20 yielded encoded fractions A, B, and C. Isolate C showed the highest inhibition of glycemia (22.85%) when the dose of 10 mg/kg was administered (p.o). Conclusion: The antidiabetic effect of the plant in laboratory animals (rats and mice) may be due to the presence of the isolated phenolic compounds.

Biotin Accounts for Only Half of the Total Avidin-Binding Substances in Human Serum

In studies using an avidin-binding assay to measure the serum or plasma concentration of biotin, biotin is sometimes assumed to be equal to the avidinbinding substances detected. To provide a range of values for serum concentrations of biotin, bisnorbiotin, and biotin sulfoxide, HPLC was used to separate avidinbinding substances in human serum, and the chromatographic fractions were assayed for avidin-binding substances (biotin and biotin metabolites). In sera from 15 normal fasting adults, substantial concentrations of avidin-binding substances other than biotin were detected. Two of the principal substances were identified as bisnorbiotin and biotin sulfoxide based on their chromatographic properties. The serum concentrations of bisnorbiotin and biotin sulfoxide varied widely among the individuals. In three subjects, the concentration of bisnorbiotin exceeded that of biotin. The presence of avidinbinding substances in addition to biotin may have confounded previous measurements of the concentration of biotin in serum, plasma, and blood when avidin-binding assays were used. Because bioassay methods for biotin sometimes use organisms for which one or more of these biotin metabolites are growth factors, measurements of biotin in blood using some bioassays are likely to overestimate the concentrations of biotin.

Sustainable chromatographic purification of milbemectin: Application of high-speed countercurrent chromatography coupled with off-line atmospheric pressure solid analysis probe-high resolution mass spectrometry

Isolation of valuable chemicals is an important process in reagent manufacturing for the pharmaceutical and food science industries. This process is traditionally time-consuming, expensive, and consumes vast amounts of organic solvents. Considering green chemistry and sustainability concerns, we sought to develop a sustainable chromatographic purification methodology for obtaining antibiotics by focusing on the reduction of organic solvent waste generation. Milbemectin (mixture of milbemycin A3 and milbemycin A4) was successfully purified using high-speed countercurrent chromatography (HSCCC) and pure fractions (>98% purity, HPLC) could be identified using the organic solvent fee atmospheric pressure solid analysis probe mass spectrometry (ASAP-MS). The organic solvents required for HSCCC could be redistilled and recycled for continued HSCCC purification, thus reducing the consumption of organic solvent (n-hexane/ethyl acetate) by 80+%. Optimization of the two-phase solvent system (n-hexane/ethyl acetate/methanol/water, 9/1/7/3, v/v/v/v) for HSCCC was assisted computationally, thereby reducing solvent waste from an experimental determination. Our proposal application of HSCCC and offline ASAP-MS provides proof of concept for a sustainable, preparative scale, chromatographic purification methodology for obtaining antibiotics in high purity.

Column Chromatography Fractionation and Antioxidant Activity of Passiflora foetida Leaves

Available synthetic antioxidants have been reported to have mutagenic and toxic effects. On the other hand, natural antioxidants show their superiority as they are not or less toxic. Passiflora foetida has the potential as an antioxidant, but the investigation of the antioxidant activity of the P. foetida chromatography column fraction has not been reported. This studied aims to investigate the antioxidant activity of the column chromatographic fractions of P. foetida leaves. An antioxidant assay using the DPPH and FRAP methods. The extraction was carried out by graded maceration, then fractionation using column chromatography. The antioxidant activity test was carried out using the DPPH and FRAP methods. Thin Layer Chromatography analysis was performed to determine the chromatogram pattern. The EC50 using DPPH method from n-hexane extract: 129.035 µg/mL, ethyl acetate extract: 206.398 µg/mL, methanol extract: 97.453 µg/mL, while the EC50 using FRAP method from n-hexane extract: 67.851 µg/mL, ethyl acetate extract : 68.981 µg/mL, and methanol extract: 58.787 µg/mL. Column chromatography fractions have antioxidant activity, with FMetPF6 as the fraction with the best activity, with percent inhibition 41.85±1.96 at concentration 25 µg/mL (DPPH), and with percent antioxidant activity 26.03±0.84 at concentration 9 µg/mL (FRAP). Passiflora foetida leaves have great potential as an antioxidant; both the extract and its fractions have antioxidant activity. The FMetPF6 has the best activity compare to other extracts and fractions. Further analysis to determine the various compounds in FMetPF6 using LC-MS/MS will facilitate the active compound's isolation.

Euphorbia Graminea Jacq. (Euphorbiaceae): A Comparative Antimicrobial Evaluations of Stem and Root Extracts

Antimicrobial resistance is a global issue. Euphorbia plants are used locally to treat microbial infections. This study examined the antimicrobial potential of Euphorbia graminea stem and root extracts. The stems and roots extracts of E .graminea were extracted using 80% methanol and tested for antimicrobial activity at concentrations between 4.69-300mg/mL against non-clinical isolates (S. aureus, E. coli, P. aeruginosa, C. albican, A. niger). The active roots extract was fractionated using vacuum liquid chromatographic fractionations (VLC) and the resulting fractions bulked and tested against the organisms at 6.25-100mg/mL. The MIC of extracts and vlc bulked fractions were tested at 0.39-6.25 mg/mL. The root extract recorded higher antimicrobial activities over the stem extract especially against S. aureus and E.coli, hence was fractionated. Among the vlc sub-fractions of the roots extract, fractions A (2) recorded no activity against the test organisms while fractions C (9-10) recorded 7.50 and 3.50 mm against S. aureus and E. coli only at the maximum concentration of 100mg/mL. However, fractions B (3-8) conspicuously gave zones of inhibitions far higher than the other fractions. This study has shown that the roots extract of E. graminea has higher antimicrobial activities more than the stems, further justifying the ethno-botanical potentials of the plant in treating skin infections.

Old Meets New: Mass Spectrometry-Based Untargeted Metabolomics Reveals Unusual Larvicidal Nitropropanoyl Glycosides from the Leaves of Heteropterys umbellata.

The Aedes aegypti (Diptera: Culicidae) mosquito is the vector of several arboviruses in tropical and subtropical areas of the globe, and synthetic pesticides remain the most widely used combat strategy. This study describes the investigation of secondary metabolites with larvicidal activity from the Malpighiaceae taxon using a metabolomic and bioactivity-based approach. The workflow initially consisted of a larvicidal screening of 394 extracts from the leaves of 197 Malpighiaceae samples, which were extracted using solvents of different polarity, leading to the selection of Heteropterys umbellata for the identification of active compounds. By employing untargeted mass spectrometry-based metabolomics and multivariate analyses (PCA and PLS-DA), it was possible to determine that the metabolic profiles of different plant organs and collection sites differed significantly. A bioguided approach led to the isolation of isochlorogenic acid A (1) and the nitropropanoyl glucosides karakin (2) and 1,2,3,6-tetrakis-O-[3-nitropropanoyl]-beta-glucopyranose (3). These nitro compounds exhibited larvicidal activity, possibly potentialized by synergistic effects of their isomers in chromatographic fractions. Additionally, targeted quantification of the isolated compounds in different extracts corroborated the untargeted results from the statistical analyses. These results support a metabolomic-guided approach in combination with classical phytochemical techniques to search for natural larvicidal compounds for arboviral vector control.

Antioxidant Guided Fractionation of Blackberry Polyphenols Show Synergistic Role of Catechins and Ellagitannins

In the present study, blackberry extract was prepared using a previously optimized solid-liquid extraction method in 70% aqueous acetone aimed at the recovery of its principal phenolics. Subsequently, 0.5 g of freeze-dried extract was subjected to flash chromatography fractionation, which was conducted on a C18 column using a binary solvent system of water and methanol at 10 mL/min. The total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) activities of the obtained 42 flash fractions were determined, and a strong positive correlation (r ≥ 0.986) was exhibited among them. Furthermore, the graph of the antioxidant indices of the flash fractions resembled the flash chromatogram, suggesting a good correlation among the compounds within the chromatographic peaks and the antioxidant indices. LC-MS/MS identified as many 28 phenolics, including cinnamtannin A2 reported for the first time in blackberries. This study further established the role of dominant anthocyanins (cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside), but uniquely those of ellagitannins and catechins on the antioxidant capacity of blackberries.

Evaluation of Ficus nitida Allelopathic Potential and the Most Efficient Application Method for Controlling Weeds Associated with Sunflower Plant

Allelopathy is a phenomenon by which plants positively or negatively affect neighboring plants by releasing allelopathic compounds. These allelochemicals are secondary metabolites found in different concentrations in shoots, roots, leaves, flowers, and even pollen grains. Allelochemicals have potential as natural bioherbicides for controlling weeds when applied in the form of extracts or through intercropping, cover cropping, and mulching. The present study was conducted to investigate the allelopathic potential of Ficus nitida leaves against Echinochloa crus-galli L. and Corchorus olitorius L weeds associated with sunflower plant via two application methods (mixing of F. nitida leaf powder with soil and foliar spray of F. nitida leaf powder alcoholic extract). Two pot experiments were carried out for the two summer seasons of 2020 and 2021 in the greenhouse of the National Research Centre (NRC), Dokki, Giza, Egypt. Nine treatments were applied in a completely randomized block design. Three treatments were applied before sowing, namely F. nitida leaf powder was mixed with the soil at rates of 15, 30, and 45 g/pot. The other three treatments of alcoholic leaf powder extract of F. nitida were sprayed twice on both plants and weeds at 10, 20, and 30% (w/v) concentrations. Additionally, three check treatments—healthy (sunflower only), unweeded, and both weeds only—were applied for comparison. The recorded results showed that F. nitida had an allelopathic bioherbicidal effect on both weeds. By increasing the concentration of F. nitida extract, the bioherbicidal potential increased. Moreover, the recorded results showed that foliar spray of alcoholic extract at 30% concentration was the superior application method for controlling weeds. Mixing of F. nitida leaf powder at 45 g/pot ranked second after this superior treatment. A noticeable result is that these two superior treatments improved sunflower growth parameters and yield traits. Quantitative estimation of phenolic compounds and flavonoids demonstrated that the concentration of these allelochemicals is higher in alcoholic extract than in water extract. Moreover, phenolic acid concentrations as detected by high-performance liquid chromatography fractionation are higher in alcoholic extract than in water extract.

High-throughput capillary electrophoresis analysis of biopharmaceuticals utilizing sequential injections.

The complexity of biotherapeutic products implies an ever-increasing list of product quality attributes that need to be monitored and characterized. In addition, the growing interest in implementing process analytical technology in biopharmaceutical production has further increased the testing burden, together with the need for rapid testing that can facilitate real-time or near-real-time decision-making. Capillary electrophoresis (CE) has made a place in biopharmaceutical analysis but is regarded as a low-throughput method, with the instrument dead time constituting more than 80% of the total time of analysis. In this study, the dead time of CE was utilized to analyse 3mAb samples in a single-CE run. This approach resulted in an up to 77% reduction in the total analysis time and increased the productivity by up to 300%, compared to traditional single CE-ultraviolet runs, without compromising resolution or relative peak areas. Additionally, good method reproducibility was observed. The compatibility of the method has been demonstrated with protein A eluate and cation exchange chromatography fractions. We, thus, propose that sequential injections can be applied for fast and robust CE analysis of biopharmaceuticals.

Diversity of sesquiterpenoids from Stellera chamaejasme with neuroprotective effects

Chromatographic fractionation of the 95% EtOH extract of the roots of Stellera chamaejasme yielded 20 sesquiterpenoids of four different types, guaiane-, carotane-, sesquicarane-, and alpiniane-types. Among them, sesquistrachanoids A−F were previously undescribed ones, whose structures including absolute configurations were elucidated by spectroscopic methods, the Mo2(OAc)4-induced ECD experiment, and analysis of experimental and calculated 1D NMR and ECD data. Sesquistrachanoid A is a 2,3-seco-guaiane-type sesquiterpenoid with a α-pyrone core and sesquistrachanoid B is the first example of 8,9-seco-guaiane-type sesquiterpenoid featured with an 1,8-δ-lactone core. Sesquistrachanoid C is a guaiane sesquiterpenoid characterized by a peroxide bridge between C-8 and C-10. All sesquiterpenoids were evaluated for their neuroprotective effects on cell damage induced by sodium nitroprusside in PC-12 cells. The bioassay results showed that six compounds at 10 μM could restore the cell viability, being comparable to that of the positive control edaravone. The mechanistic study on the most pronounced activity compound, stelleraguaianone B, demonstrated that it played a neuroprotective role by promoting the mRNA expression of antioxidant enzymes to reduce oxidative stress.

Commentary on 'Identification and characterization of select oxysterols as ligands for GPR17'.

Commentary on 'Identification and characterization of select oxysterols as ligands for GPR17'.

Bioassay-guided fractionation of mushroom (Schizophyllum commune) extract using vacuum column chromatography

This study was carried out to evaluate the tyrosinase inhibition activity, which represents anti-pigmentation/browning property and bioactive compounds of sub-fractions of Schizophyllum commune extracts. In this study, two types of crude extracts, namely K4C and K30C, were subjected to chromatographic fractionation in vacuum column chromatography packed with silica gel. The columns were stepwise eluted with different ratios of solvent acetone: methanol (A:M) to obtain several fractions. Anti-pigmentation activity was measured by tyrosinase inhibition assay. The fraction with the highest activity for K4C extract was 7A:3M (30.46±3.22%) while fraction 100M (34.32±2.14%) was the highest for K30C extract. Biological components such as organic acids, amino acids, as well as phenolic acids were also assessed by ultra and high-performance liquid chromatography (UPLC and HPLC). The results revealed that the amounts of bioactive compounds were varied; the major organic acids detected were citric acid (313.142±1.069 mg/L), tartaric acid (37.656±8.807 mg/L) and L-malic (23.748±2.565 mg/L) in the fraction K4C-7A:3M compared to fraction K30C-100M. The same fraction showed the highest amount of amino acids while phenolic acids were detected in low amounts in K4C -7A:3M and K30C-100M fractions. The fraction with the highest biological activity has the potential to be used as a bio-ingredient in food applications and nutricosmetic product formulations. The potential product derived from this project may diversify mushroombased downstream products available in Malaysia, and subsequently further expand our mushroom industry.

Proteomic analysis of Taenia solium cysticercus and adult stages.

Taenia solium (T. solium) cysticercosis is a neglected parasitic zoonosis that occurs in developing countries. Since T. solium has a complex life cycle that includes eggs, oncospheres, cysticerci, and adults, presumably many proteins are produced that enable them to survive and establish an infection within the host. The objectives of this study were to perform a comparative proteomic analysis of two ontogenetic stages of T. solium (cysticerci and adult) and to analyze their differential expression of proteins. Methods proteins were separated by High Performance Liquid Chromatography (HPLC) fractionation, and protein samples were also digested in liquid and identified by liquid chromatography tandem mass spectrometry (LC-MS/MS); the differentially expressed proteins were then processed by a bioinformatics analysis and verified by parallel reaction monitoring (PRM). Results we identified 2,481 proteins by label-free quantitative proteomics. Then differentially expressed proteins were screened under P values < 0.05 and 2 fold change, we found that 293 proteins up-regulated and 265 proteins down-regulated. Discussion through the bioinformatics analysis, we analyzed the differences types and functions of proteins in the Taenia solium and cysticercus, the data will provide reference value for studying the pathogenic mechanism of the two stages and the interaction with the host, and also support for further experimental verification.

Baccharis dracunculifolia DC (Asteraceae) Root Extract and Its Triterpene Baccharis Oxide Display Topical Anti-Inflammatory Effects on Different Mice Ear Edema Models.

B. dracunculifolia is popularly used to treat skin diseases. This work aimed to evaluate the topical anti-inflammatory properties of B. dracunculifolia root extract (BdR) and its major compound baccharis oxide (BOx) on mice ear edema models. BdR was analyzed by GC-MS, and BOx was isolated by chromatographic fractionation. Topical anti-inflammatory activities were determined by using the croton oil, capsaicin, histamine, and phenol-induced mouse ear edema models. N-acetyl-β-D- glucosaminidase (NAG) and myeloperoxidase (MPO) activities, as well as NO dosage and histopathological analyses, were also evaluated. Phytochemical analysis of BdR showed BOx as one of the major constituents. BdR and BOx (both at 0.1, 0.5, and 1.0 mg/ear) significantly reduced croton oil, histamine, and phenol-induced ear edema, while only BOx was effective in reducing capsaicin-induced edema. MPO and NAG activities, as well as NO production, were significantly inhibited by BdR and BOx. Histopathological analysis confirmed the topical anti-inflammatory properties of BdR and BOx. Our findings showed that BdR and BOx demonstrated significant topical anti-inflammatory effects in mouse ear edema induced by different agents, suggesting their possible application on skin inflammatory diseases.

On the co-elution of benzo[a]pyrene and dibenzo[a,l]pyrene in chromatographic fractions and their unambiguous determination in tobacco extracts via laser-excited time resolved Shpol'skii spectroscopy.

High performance liquid chromatography is widely used for the analysis of polycyclic aromatic hydrocarbons in a wide variety of samples. Of particular concern are benzo[a]pyrene and dibenzo[a,l]pyrene, two of the most toxic polycyclic aromatic hydrocarbons ever tested. Under EPA method 610, these two compounds co-elute with almost identical retention times. Our studies demonstrate the feasibility of directly determining them in a chromatographic fraction without further separation. Their unambiguous determination is based on spectral and lifetime information with a two-step experimental procedure consisting of the evaporation of the chromatographic fraction followed by the dissolution of the residue with microliters of n-octane. With the aid of a 77 K fiber optic probe, limits of detection at the parts-per-billion concentration level (ng mL-1) are obtained from the microliter sample via laser excited time resolved Shpol'skii spectroscopy. This approach is then applied to the analysis of benzo[a]pyrene and dibenzo[a,l]pyrene in tobacco extracts.

In-Depth Blood Proteome Profiling by Extensive Fractionation and Multiplexed Quantitative Mass Spectrometry.

Blood in the circulatory system carries information of physiological and pathological status of the human body, so blood proteins are often used as biomarkers for diagnosis, prognosis, and therapy. Human blood proteome can be explored by the latest technologies in mass spectrometry (MS), creating an opportunity of discovering new disease biomarkers. The extreme dynamic range of protein concentrations in blood, however, poses a challenge to detect proteins of low abundance, namely, tissue leakage proteins. Here, we describe a strategy to directly analyze undepleted blood samples by extensive liquid chromatography (LC) fractionation and 18-plex tandem-mass-tag (TMT) mass spectrometry. The proteins in blood specimens (e.g., plasma or serum) are isolated by acetone precipitation and digested into peptides. The resulting peptides are TMT-labeled, separated by basic pH reverse-phase (RP) LC into at least 40 fractions, and analyzed by acidic pH RPLC and high-resolution MS/MS, leading to the quantification of ~3000 unique proteins. Further increase of basic pH RPLC fractions and adjustment of the fraction concatenation strategy can enhance the proteomic coverage (up to ~5000 proteins). Finally, the combination of multiple batches of TMT experiments allows the profiling of hundreds of blood samples. This TMT-MS-based method provides a powerful platform for deep proteome profiling of human blood samples.