Hydrophilic interaction liquid chromatography based method for simultaneous determination of purines and their derivatives in food spices.

This work presents method for separation and quantification of adenine, guanine, xanthine, hypoxanthine, uric acid, and creatinine in food spices using hydrophilic interaction liquid chromatography with UV detection. Optimized conditions allowed separation with mobile phases containing acetonitrile and additives ammonium acetate (90:10, v/v, pH 6.1) or formate (90:10, v/v, pH 3.2). In food spices no uric acid was detected, creatinine (16 ± 2 µg g-1) was found only in instant dried yeast. The highest content of purines was determined in dried yeast (xanthine 110 ± 8 µg g-1, hypoxanthine 441 ± 24 µg g-1, adenine 84 ± 16 µg g-1, guanine 163 ± 12 µg g-1), high in curry, herbal pepper, and chicken seasoning, the lowest concentration was in black pepper (hypoxanthine 12 ± 2 µg g-1, adenine 27 ± 3 µg g-1). To best of our knowledge, no such complementary method and obtained data have been reported so far.

Freeze Surface-Enhanced Raman Scattering Coupled with Thin-Layer Chromatography: Pesticide Detection and Quantification Case.

Thin-layer chromatography (TLC) is widely used in various branches of chemical science to separate components in complex mixtures because of its simplicity. In most cases, analyte spots are visually detected by fluorescence, and the retention factor (Rf) is determined from the distance traveled by the analyte. Further characterizations are often necessary to identify separated chemicals because molecular information other than Rf is not available. Surface-enhanced Raman scattering (SERS) has been coupled with TLC to complement molecular information. In previously reported TLC-SERS, metal nanoparticle suspension was dropped onto analyte spots to obtain SERS spectra. This approach is simple and efficient for SERS measurements on the TLC plate but has limited sensitivity for several reasons, such as the low solubility of analytes in the dropped solution, difficult control of nanoparticle aggregation, and interference from the stationary phase. We recently showed that freezing enhances SERS sensitivity by a factor of ∼103. Freezing simultaneously concentrates analytes and silver nanoparticles (AgNPs) in a freeze concentrated solution, where aggregation of AgNPs is facilitated, allowing sensitive freeze SERS (FSERS) measurements. Here, we discuss FSERS measurements on TLC plates to demonstrate the superiority of this combination, i.e. TLC-FSERS. Freezing enhances SERS sensitivity by freeze concentration and facilitated aggregation of AgNPs and, in addition, eliminates interference from the stationary phase. Under the optimized condition, TLC-FSERS enables the on-site detection of pesticides at the nM level. The use of the SERS signal from adenine added as the internal standard allows us to quantify pesticides. Applications to a commercial green tea beverage are also demonstrated.

Effects of Extraction Temperature on Efficacy of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), Aqueous Extract against Oxidative Stress.

This study was designed to understand the effect of extraction temperature, i.e., room temperature (GLRT), 50°C (GL50), 100°C (hot water; GL100), and 200°C (GL200) on antioxidant and biological activity of G. lucidum. The % yield obtained was 5.3%, 7.6%, 10.7%, and 13.2% at various extraction temperatures; room temperature, 50°C, 100°C and 200°C, respectively. Similarly, phenolic content (51.6, 57.9, 82.9, and 93.1 mg/g extract) and flavonoid content (18.8, 23.2, 34.3, and 36.3 mg/g extract) were observed to be increased with rise in extraction temperature. However, extraction temperature resulted in loss of antioxidant activities above 100°C as evident by chemical assays such as DPPH, FRAP, ABTS, and TRP conducted on extracts. In contrast, three bioactive compounds, i.e., adenine (3.26, 3.48, 2.16, and 1.45 mg/g extract), uracil (3.99, 3.21, 2.51, and 1.47 mg/g extract), and adenosine (5.92, 5.62, 2.22 and 0.7 mg/g extract), quantified by high performance thin layer chromatography showed decrease in their content with increasing extraction temperature. Extract prepared at room temperature and 50°C prevented loss of cell viability and generation of reactive oxygen species resulted after hydrogen peroxide exposure; however, cytoprotective efficacy was not significant at 100°C and 200°C The order of cytoprotective effects observed by these extract were in the following order: room temperature ≥ 50°C > 100°C > 200°C. Overall, the optimal temperature conditions for the efficient extraction of G. lucidum with water retaining bioactive compounds and biological activity was found to be below 100°C.

Artificial Sandwich Base for Monitoring Single-Nucleobase Changes and Charge-Transfer Rates in DNA.

Developing a convenient method to discriminate among different types of DNA nucleotides within a target sequence of the human genome is extremely challenging. We herein report an artificial ferrocene-base (Fe-base) that was synthesized and incorporated into different loci of a DNA strand. The Fe-base replacement on a nucleobase can interact with DNA bases and efficiently discriminate among A, T, G, and C DNA bases of the complementary locus on the basis of interacting electrochemical properties. Furthermore, cyclic-voltammetry (CV) studies demonstrated the electrochemical stability of DNA strands incorporated with Fe-bases and the reversibility of the incorporation. Square-wave voltammetry (SWV) was performed to measure current changes between Fe-bases and bases of interest in the DNA duplex. The changes in the charge-transfer rates appeared to be correlated with the position of the Fe-base in the DNA strand, allowing rapid and efficient sensing of single-nucleobase changes in DNA and showing promise for the design of Fe-oligomer chip technology as a tool for DNA sequencing.

Nitrogen compounds in Phacelia tanacetifolia Benth. honey: First time report on occurrence of (-)-5-epi-lithospermoside, uridine, adenine and xanthine in honey.

Lacy phacelia (Phacelia tanacetifolia Borkh.) honey composition was screened by UHPLC-DAD-QqTOF-MS. The targeted analysis revealed 6 major nitrogen compounds including aromatic amino acids (tyrosine, phenylalanine), purine derivatives (adenine, xanthine), nucleoside (uridine) and rare non-cyanogenic cyanoglucoside, (-)-5-epi-lithospermoside ((2Z)-2-[(4R,5R,6S)-4,5-dihydroxy-6-(ß-d-glucopyranosyl)oxycyclohex-2-en-1-ylidene]acetonitrile). Their identity was confirmed by different analytical tools: HRMS, co-chromatography with standard compound or comprehensive NMR experiments. All the compounds, except amino acids, were reported and determined in honey for the first time. The amount of the compounds was quantified in 16 unifloral phacelia samples: adenine (18.45 ±â€¯4.63 mg/kg), xanthine (10.53 ±â€¯2.98 mg/kg), uridine (42.84 ±â€¯9.26 mg/kg), tyrosine (14.66 ±â€¯10.22 mg/kg), (-)-5-epi-lithospermoside (70.61 ±â€¯31.37 mg/kg) and phenylalanine (20.41 ±â€¯11.99 mg/kg). The (-)-5-epi-lithospermoside content is significantly correlated with P. tanacetifolia pollen percentage (R2 = 0.5612, p < 0.001) and it is proposed as a potential marker of botanical origin for phacelia honey.

[Determination of Nucleosides and Nucleobases in Natural, Cultured and Tissue Culture Anoectochilus roxburghii Using LC-MS].

OBJECTIVE: To establish a method for simultaneous determination of nucleosides and nucleobases in natural, cultured and tissue culture Anoectochilus roxburghii by high performance liquid chromatography-electrospray ionization/ion trap mass spectrometry (HPLC-ESI/MS). METHODS: The separation was performed on a Welch Ultimate XB-C18 column (250 mm x 4.6 mm,5 µm). 20 mmol/L ammonium acetate solution and methanol were adopted as the mobile phase with gradient elution. The flow rate was 1.0 mL/min. The injection volume was 20 µL. The column temperature and UV wavelength were set at 30 degrees C and 260 nm, respectively. RESULTS: Cytosine, uracil, cytidine, uridine, hypoxanthine, adenine, inosine, guanosine,fl-thymidine and adenosine were identified in natural, cultured and tissue culture Anoectochilus roxburghii. The total content of nucleosides and nucleotides in Anoectochilus roxburghii were 1.6639, 1.8568 and 2.2013 mg/g,respectively. CONCLUSION: The contents of nucleosides and nucleobases in herb are affected by its growth pattern. The total content of nucleosides and nucleotides was tissue culture herb > cultured herb > natural herb. This investigation would provide the theoretic basis for quality standards and applications of Anoectochilus roxburghii in clinical research.

[Determination of four kinds of purines in deer fetus soft capsule by HPLC].

OBJECTIVE: To establish an HPLC method for simultaneous determination of four kinds of purines in deer fetus soft capsule. METHODS: Four kinds of purines were detected and determined by HPLC. The mobile phase was 0.02 mol/L KH2PO4 (containing 1 mmol/L heptane sulfonic acid sodium, pH = 3.8)-methanol = 97:3. Detection wavelength was 254 nm and flow rate was 1.5 ml min. The linear relationship of four kinds of purines was as follows: hypoxanthine: Y1 = 83695X1 + 355 (r1 = 0.9998), with the linear range 0.040-0.667 mg/mL; xanthine: Y2 = 50638X2 + 39 (r2 = 0.9989), with the linear range 0.008-0.119 mg/mL; guanine: Y3 = 30269X3-9562 (r3 = 0.9924), with the linear range 0.018 - 0.279 mg/mL; adenine: Y4 = 38975X4-8671 (r4 = 0.9989), with the linear range 0.027-0.399 mg/mL The average sample recovery rate of hypoxanthine, xanthine, guanine and adenine were 98.1%, 98.6%, 98.0% and 97.5%, with RSD 1.0%, 0.4%, 0.8% and 0.6%, respectively. RESULTS: The content of hypoxanthine, xanthine, guanine and adenine in 3 lots of deer fetus soft capsule were 116.5-132.0 microg/capsule, 21.2-23.0 microg/capsule, 48.6-54.3 microg/capsule and 68.9-75.2 microg/capsule, respectively. CONCLUSIONS: This method is simple,accurate and reproducible, which provides a basis for quality control of purines in deer fetus soft capsule.

Simultaneous determination of 10 nucleosides and nucleobases in Antrodia camphorata using QTRAP LC-MS/MS.

A liquid chromatography-triple-quadrupole linear ion trap mass spectrometry (LC-QTrap-MS) analysis has been developed for the identification and quantification of 10 nucleosides and nucleobases in extracts of Antrodia camphorata. The method was successfully used to qualitatively identify for six nucleosides namely, cytidine, uridine, inosine, guanosine, thymidine, adenosine and four nucleobases namely, uracil, guanine, xanthine, adenine in A. camphorata. Under optimized chromatographic conditions, good separation for 10 target compounds were obtained on an Agilent HC-C18(2) column (4.6 × 250 mm, 5 µm) eluted by a mobile phase of 5 mM ammonium acetate solution-methanol at a flow rate of 0.5 mL/min. Data acquisition was carried out in multiple reaction monitoring transition mode. Additional identification and confirmation of target compounds were performed using the enhanced product ion modus of the linear ion trap. It was the first report about simultaneous analysis of nucleosides and nucleobases in A. camphorata using this method. These results demonstrated that the QTRAP LC-MS/MS was a useful tool for quality evaluation of some medicinal plant products by using nucleosides and nucleobases as chemical markers. This method might also be utilized for the investigation of edible plant materials and agricultural products containing nucleosides and nucleobases.

Electrochemically fabricated self-aligned 2-D silver/alumina arrays as reliable SERS sensors.

A novel SERS sensor for adenine molecules is fabricated electrochemically using an ordered two-dimensional array of self-aligned silver nanoparticles encapsulated by alumina. Silver is electro-deposited on the interior surfaces at the bottom of nano-channels in a porous anodic aluminum oxide (AAO) film. After etching aluminum, the back-end alumina serves as a SERS substrate. SERS enhancement factor greater than 10(6) is measured by 532 nm illumination. It exhibits robust chemical stability and emits reproducible Raman signals from repetitive uses for eight weeks. The inexpensive mass production process makes this reliable, durable and sensitive plasmon based optical device promising for many applications.

[Determination of nucleosides in Rhizoma Pinelliae by high performance liquid chromatography].

A high performance liquid chromatographic (HPLC) method was established to determine nucleosides in Rhizoma Pinelliae, which is a dried stem tuber of Pinellia pedatisecta Schott in Pinellia plant belonging to Araceae family and has multiple efficiencies about down-bear counterflow and check vomiting, eliminating dampness and phlegm, etc. The separation of adenine, hypoxanthine, xanthine, uridine, thymine, adenosine and guanosine was achieved on a Lichrospher C18 column (150 mm x 4.6 mm, 5 microm) with the detection at 254 nm and gradient elution by acetonitrile-water containing 0.1% formic acid as the mobile phase. The linear ranges were from 1.6 mg/L to 50 mg/L for adenine, hypoxanthine, xanthine, uridine and guanosine, while from 1.2 mg/L to 40 mg/L for thymine and adenosine with correlation coefficients above 0.999 5. The average recoveries were between 98.9% and 101.2% with the relative standard deviations below 3%. The results of methodological study demonstrated that the method met the requirements of the determination. The nucleosides in Rhizoma Pinelliae from different districts were determined. The method is convenient and accurate with good reproducibility and can be used to evaluate the quality of Rhizoma Pinelliae.

Determination of the nucleosides and nucleobases in Tuber samples by dispersive solid-phase extraction combined with liquid chromatography-mass spectrometry.

A simple, fast and inexpensive method based on dispersive solid phase extraction (DSPE) combined with LC-MS was developed for simultaneous determination of 7 nucleosides and nucleobases (i.e., adenine, hypoxanthine, uridine, adenosine, guanine, guanosine, and inosine) in Tuber fruiting-bodies and fermentation mycelia. The DSPE procedure was firstly introduced to remove the protein interference from sample solutions, and D3520 macroporous resin was chosen as the DSPE sorbent because of its high removal capability on protein interferences, but low adsorption rate on analytes. Besides, key parameters on DSPE procedure (i.e., macroporous resin type, macroporous resin amount, methanol concentration, and vortex time) were optimized, and the protein removal efficacy could achieve about 95% after the process optimization. Though the method validation test, the DSPE-LC-MS method was confirmed to be precise, accurate and sensitive, and the column blinding problem was solved successfully. By using this established method, the total amount of nucleosides and nucleobases in the fermentation mycelia was determined to range from 4881.5 to 12,592.9µgg⁻¹, which was about 2-25 times higher than the fruiting-bodies (from 498.1 to 2274.1µgg⁻¹). The formulation of nucleosides and nucleobases in the fermentation mycelia maintained relatively constant, while the formulation in Tuber fruiting-bodies varied significantly with their species. Hierarchical cluster analysis (HCA) showed the formulation similarity of nucleosides and nucleobases between Tuber fermentation mycelia and the fruiting-bodies of Tuber indicum and Tuber himalayense. From the viewpoint of nucleosides and nucleobases, this work confirms the potentiality of Tuber fermentation mycelia as the alternative resource for its fruiting-bodies.

Simultaneous determination of five nucleosides and nucleobases of Rehmannia glutinosa Libosch. by high performance liquid chromatography.

This study is to establish a method for simultaneously determination of five nucleosides and nucleobases, including hypoxanthine, uridine, adenine, guanosine and adenosine in Rehmannia glutinosa Libosch. which was collected from different regions in China. A Diamonsil C18 column (250 mm x 4.6 mm, 5 microm) was used. Acetonitrile and 0.04 mol L(-1) potassium dihydrogen phosphate solution were adopted as mobile phase with gradient elution. The flow rate was 1 mL min(-1) and column temperature was 30 degrees C. The detection wavelength was at 254 nm. The method had good linearity over the range of 1.0 - 16.0 microg mL(-1) (r2 = 0.999 8), 5.0 - 80.0 microg mL(-1) (r2 = 0.999 8), 1.0 - 16.0 microg mL(-1) (r2 = 0.999 5), 1.25 - 20.0 microg mL(-1) (r2 = 0.999 8) and 1.0 - 16.0 microg mL(-1) (r2 = 0.999 8) for hypoxanthine, uridine, adenine, guanosine and adenosine, respectively. The average recoveries were between 98.8% and 100.7%. The content of hypoxanthine, uridine, adenine, guanosine and adenosine in Rehmannia glutinosa Libosch. from different regions was significantly different. This established method was sensitive and reliable for the quantification of five chemical constituents in Rehmannia glutinosa Libosch.

A classification model for G-to-A hypermutation in hepatitis B virus ultra-deep pyrosequencing reads.

MOTIVATION: G → A hypermutation is an innate antiviral defense mechanism, mediated by host enzymes, which leads to the mutational impairment of viruses. Sensitive and specific identification of host-mediated G → A hypermutation is a novel sequence analysis challenge, particularly for viral deep sequencing studies. For example, two of the most common hepatitis B virus (HBV) reverse transcriptase (RT) drug-resistance mutations, A181T and M204I, arise from G → A changes and are routinely detected as low-abundance variants in nearly all HBV deep sequencing samples. RESULTS: We developed a classification model using measures of G → A excess and predicted indicators of lethal mutation and applied this model to 325 920 unique deep sequencing reads from plasma virus samples from 45 drug treatment-naïve HBV-infected individuals. The 2.9% of sequence reads that were classified as hypermutated by our model included most of the reads with A181T and/or M204I, indicating the usefulness of this model for distinguishing viral adaptive changes from host-mediated viral editing. AVAILABILITY: Source code and sequence data are available at http://hivdb.stanford.edu/pages/resources.html. CONTACT: ereuman@stanfordalumni.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Testosterone affects hormone-sensitive lipase (HSL) activity and lipid metabolism in the left ventricle.

Fatty acids, which are the major cardiac fuel, are derived from lipid droplets stored in cardiomyocytes, among other sources. The heart expresses hormone-sensitive lipase (HSL), which regulates triglycerides (TG) breakdown, and the enzyme is under hormonal control. Evidence obtained from adipose tissue suggests that testosterone regulates HSL activity. To test whether this is also true in the heart, we measured HSL activity in the left ventricle of sedentary male rats that had been treated with testosterone supplementation or orchidectomy with or without testosterone substitution. Left ventricle HSL activity against TG was significantly elevated in intact rats supplemented with testosterone. HSL activity against both TG and diacylglyceride was reduced by orchidectomy, whereas testosterone replacement fully reversed this effect. Moreover, testosterone increased left ventricle free fatty acid levels, caused an inhibitory effect on carbohydrate metabolism in the heart, and elevated left ventricular phosphocreatine and ATP levels as compared to control rats. These data indicate that testosterone is involved in cardiac HSL activity regulation which, in turn, may affect cardiac lipid and carbohydrate metabolism.

On-line combination of single-drop liquid-liquid-liquid microextraction with capillary electrophoresis for sample cleanup and preconcentration: a simple and efficient approach to determining trace analyte in real matrices.

In order to improve the sensitivity of capillary electrophoresis (CE) and overcome the deficiency of commercial CE instruments in handling complex matrices directly, we proposed a novel technique which combined single-drop liquid-liquid-liquid microextraction (SD-LLLME) with CE on-line. In this technique, SD-LLLME was realized using a commercial CE instrument and, to further concentrate the target analyte, large-volume sample stacking combined sweeping without polarity switching was utilized. Even though without agitating the donor phase in the extraction process, the model compound, adenine was enriched 550-fold in only 10 min. The enrichment factors were 760 and 1030 when the extraction time was extended to 30 and 60 min, respectively. The relative standard deviations (RSDs) of adenine were 5.24% and 2.29% for peak area and migration time, respectively, which indicated that this method was much more reproducible compared to the existing methods that combined sample-preparation strategies with CE. In addition, this approach was selective while cleaning up target analyte. These mentioned advantages allowed the developed method to be an attractive approach to determining trace target compounds in complex real samples.

Sequence context outside the target region influences the effectiveness of miR-223 target sites in the RhoB 3'UTR.

MicroRNAs (miRNAs) are 21-22 nucleotide regulatory small RNAs that repress message translation via base-pairing with complementary sequences in the 3' untranslated region (3'UTR) of targeted transcripts. To date, it is still difficult to find a true miRNA target due to lack of a clear understanding of how miRNAs functionally interact with their targeted transcripts for efficient repression. Previous studies have shown that nucleotides 2 to 7 at the 5'-end of a mature miRNA, the 'seed sequence', can nucleate miRNA/target interactions. In the current study, we have validated that the RhoB mRNA is a bona fide miR-223 target. We have analyzed the functional activities of two miR223-binding sites within the RhoB 3'UTR. We find that the two miR-223 target sites in the RhoB 3'UTR contribute differentially to the total repression of RhoB translation. Moreover, we demonstrate that some AU-rich motifs located upstream of the distal miRNA-binding site enhance miRNA function, independent of the miRNA target sequences being tested. We also demonstrate that the AU-rich sequence elements are polar, and do not affect the activities of miRNAs whose sites lie upstream of these elements. These studies provide further support for the role of sequences outside of miRNA target region influencing miRNA function.

Sequence selective formation of 1,N(6)-ethenoadenine in DNA by furan-conjugated probe.

1,N(6)-Ethenoadenosine derivatives have been applied as fluorescence probes in various fields of biochemistry and molecular biology. We developed a 1,N(6)-ethenoadenosine-forming reaction at a target adenine in DNA duplex and applied it to a mutation diagnosis. Furan-derivatized oligodeoxyribonucleotides were synthesized and fluorescence properties were studied in the presence of complementary strand under oxidative conditions. Strong emissions at 430nm were observed in the presence of the complementary strand with an adenine in front of furan moiety.

The A-rich RNA sequences of HIV-1 pol are important for the synthesis of viral cDNA.

The bias of A-rich codons in HIV-1 pol is thought to be a record of hypermutations in viral genomes that lack biological functions. Bioinformatic analysis predicted that A-rich sequences are generally associated with minimal local RNA structures. Using codon modifications to reduce the amount of A-rich sequences within HIV-1 genomes, we have reduced the flexibility of RNA sequences in pol to analyze the functional significance of these A-rich 'structurally poor' RNA elements in HIV-1 pol. Our data showed that codon modification of HIV-1 sequences led to a suppression of virus infectivity by 5-100-fold, and this defect does not correlate with, viral entry, viral protein expression levels, viral protein profiles or virion packaging of genomic RNA. Codon modification of HIV-1 pol correlated with an enhanced dimer stability of the viral RNA genome, which was associated with a reduction of viral cDNA synthesis both during HIV-1 infection and in a cell free reverse transcription assay. Our data provided direct evidence that the HIV-1 A-rich pol sequence is not merely an evolutionary artifact of enzyme-induced hypermutations, and that HIV-1 has adapted to rely on A-rich RNA sequences to support the synthesis of viral cDNA during reverse transcription, highlighting the utility of using 'structurally poor' RNA domains in regulating biological process.

[Study on HPLC fingerprint characteristic analysis of Cordyceps sinensis and its similar products].

OBJECTIVE: To study on HPLC fingerprint characteristic analysis of Cordyceps sinensis and its similar products. METHODS: To determinate 13 samples of Cordyceps sinensis and its similar products by HPLC, and analyze the HPLC results with similar appraisal method and graphical methods of multivariate sample in two dimensional plane such as the methods of profile, radar chart and constellation graph. RESULTS: The similar appraisal method might synthesize the similar degree in quantification, while the graphical methods such as profile graph, radar chart and constellation graph could show more details about the classification and the characteristic of varieties directly. CONCLUSIONS: We recommend the combined application of similar appraisal method and the graphical methods due to its advantages on the judgment and characteristic analysis of fingerprint.

[Study on HPEC fingerprint of cultured Cordyceps militaris].

OBJECTIVE: To investigate the High Performance Capillary Electrophoresis (HPCE) fingerprint of cultured Cordyceps militaris (L.) Link. METHODS: Separation was performed on a 50 cm x 75 microm uncoated capillary with 0.5 mmol/L borate solution (pH 9. 18) as HPEC buffer. The run voltage was 20 Kv, temperature 25 degrees C and the DAD detection was set at 254nm. RESULTS: Fingerprint consisted of 11 common peaks. The validation of methods was satisfied with the requirements for SFDA's technical regulations. CONCLUSION: The method was accurate and simple and suitable to the quality control of cultured Cordyceps militaris (L.) Link.