This research was funded by the Health Canada Genomics Research and Development Initiative (GRDI) and the Canadian Regulatory Strategy for Biotechnology (CRSB). The authors have no conflict of interests.

Ensure that all animal husbandry, housing, handling and experimentation adhere to local rules and regulations and that experimentation protocols are approved prior to commencing any study. For the described experiments, animal care, handling, and treatment were approved by the Health Canada Animal Care Committee. See the &#8220;Reagents table&#8221; for the suppliers&#8217; information.
1. Collecting Biological Samples
<ol>
	<li>Cells or Animal Tissues
	<ol>
		<li>Grow human alveolar adenocarcinoma A549 cells in F12-K media containing 10% fetal bovine serum, 100 units/ml of penicillin and 100 &#181;g/ml of streptomycin.</li>
		<li>Seed cells at approximately 1 million cells per a 10-cm plate. For each experiment, use a set of 12 plates in total (three plates per one biological replicate x four doses) to provide enough DNA (80 &#956;g) for enzymatic digestion and HPLC analysis.</li>
		<li>When cell density becomes greater than approximately 70% of the plate surface area, remove media and wash cells twice with 4 ml of phosphate-buffered saline (PBS), pH 7.4.</li>
		<li>For cultured animal cells, use KBrO3 as a positive control. 
		NOTE: A positive linear relationship between KBrO3 concentration and 8-oxo-dGuo frequency has been reported in the literature9.</li>
		<li>Dissolve KBrO3 in PBS. Ensure that the final concentration in the cell culture medium is greater than 1 mM to obtain a statistically significant increase in 8-oxo-dGuo relative to un-exposed cells. Add the same concentration of KBrO3 to each plate in the series (e.g., 12 10-cm plates).</li>
		<li>Incubate the plates for 3 hr at 37 &#176;C. Remove the media and wash once with PBS.</li>
		<li>Add 1 ml of trypsin solution (stock concentration 2.5 g/ml) and incubate for 3 min at 37 &#176;C.</li>
		<li>Wash with 4 ml PBS, collect the cells from each set into a single 50 ml conical polystyrene centrifuge tube, and centrifuge at 1,000 x g for 5 min at 4 &#176;C.</li>
		<li>Remove PBS and store the cell pellet at -80 &#176;C until further analysis. Artificial DNA oxidation can be further minimized by nuclear isolation, as described elsewhere10.</li>
	</ol>
	</li>
	<li>Animal Tissues
	<ol>
		<li>Dose animals as required. For this protocol, adult (9 weeks of age) treat male Muta Mouse by oral gavage daily for three consecutive days with 20 mg DBC/kg bw per day dissolved in olive oil. 
		NOTE: This transgenic animal harbors engineered &#955;-bacteriophage and mutation reporter gene lacZ from E. coli11 is used for transgenic rodent mutation assays12.</li>
		<li>Perform animal necropsy e.g., anesthetized the mice can using isoflurane and then euthanized via cervical dislocation followed by chest cavity opening. Immediately flash freeze tissues in liquid nitrogen. Store tissues at -80 &#176;C until analysis. &#160; 
		NOTE: The timing of euthanasia after the treatment may be another important variable (for instance, DNA oxidation was maximal at 72 hr after noise exposure in the rat brain and liver13).</li>
	</ol>
	</li>
</ol>
2. DNA Extraction, Precipitation and Wash (For Tissues Proceed Directly to 2.2)
<ol>
	<li>Homogenize the collected cells in a 50 ml conical polystyrene centrifuge tube with 1 ml of DNA isolating agent such as DNAzol. Use a 1,000 &#956;l pipette tip to disperse the cell pellet until the solution is homogeneous. Transfer homogenate to a new 1.5 ml microcentrifuge tube. Incubate on ice for 10-20 min. Proceed to 2.3.</li>
	<li>Homogenize 15-20 mg of tissue in a 1 ml handheld nonstick glass homogenizer containing 500 &#956;l DNA isolating agent. Gently raise and lower homogenizer for about 1 min; softer tissues will require less time. Gentle treatment ensures less shearing of DNA. Store the homogenate on ice for about 10-20 min.</li>
	<li>Pellet the homogenate by centrifugation for 10 min at 10,000 x g and 4 &#176;C in a 1.5 ml conical microcentrifuge tube.</li>
	<li>Carefully transfer the resulting supernatant into a new 1.5 ml microcentrifuge tube paying careful attention to avoid contacting the pellet.</li>
	<li>Precipitate DNA from the homogenate by adding 0.5 ml 100% EtOH per 1 ml of homogenate. Invert tubes 10 times to ensure isolating reagent and EtOH are sufficiently mixed.</li>
	<li>At this point, DNA precipitate is viscous, spool it onto a plastic pipette tip (e.g., with a maximum holding capacity of 200 &#956;l) and then transferred to a new 1.5 ml conical microcentrifuge tube. &#160; 
	NOTE: Quick centrifugation can be employed to remove any remaining lysate from the isolated DNA. 
	(DNA Wash and Solubilization)</li>
	<li>Add 1 ml of 75% EtOH to the isolated DNA. Suspend the DNA pellet thoroughly by inverting the tubes 10 times.</li>
	<li>Carefully decant the ethanol from the tube. Ensure that the DNA pellet sticks to the side of the tube. Store the tubes vertically for 1-2 min and use a pipette to remove any excess EtOH from the bottom of the tube.</li>
	<li>Repeat the DNA wash once more, and either store the sample in EtOH at -20 &#176;C (stable for several months) or immediately proceed to digestion.</li>
	<li>Dissolve DNA in digestion buffer (described below) and then quantify using standard spectroscopic methods (i.e., absorbance at 260 nm using NanoDrop spectrophotometer).</li>
</ol>
3. Enzymatic Digestion
<ol>
	<li>Prepare &#8220;digestion buffer&#8221; by combining appropriate volumes (depending on the number of samples to be digested) of 50 mM monobasic Na phosphate (containing 2.0 mM KCl, 1.0 mM desferal (DFO) and 200 mM MgCl2) with 50 mM dibasic Na phosphate (also containing 2.0 mM KCl, 1.0 mM DFO and 200 mM MgCl2). Each sample requires 4.0 &#956;l monobasic and 17.0 &#956;l dibasic phosphate solution.</li>
	<li>Remove any remaining EtOH from the bottom of the tube and air-dry samples for about 5 min. Ensure that the DNA does not dry out completely.</li>
	<li>Dissolve 80 &#956;g extracted DNA in 21.0 &#956;l of the digestion buffer, add 1 unit of DNase I dissolved in 2.0 &#956;l of digestion buffer.</li>
	<li>Vortex lightly to achieve thorough mixing and incubate for 1.5 hr at 37 &#176;C.</li>
	<li>At the end of the incubation period, add 216.4 &#956;l of dibasic 50 mM Na phosphate solution containing 2.0 mM KCl, 1.0 mM DFO and 200 mM MgCl2.</li>
	<li>Prepare &#8220;digestion buffer-2&#8221; by combining one volume of digestion buffer with nine volumes of dibasic, 50 mM Na phosphate (with 2.0 mM KCl, 1.0 mM DFO and 200 mM MgCl2).</li>
	<li>Add 0.025 units of phosphodiesterase (PDE) I enzyme in 16.3 &#956;l of digestion buffer-2. Pipette up and down for several seconds, then vortex lightly to achieve thorough mixing and incubate for 1.5 hr at 37 &#176;C.</li>
	<li>Add 0.4 units of alkaline phosphatase (AP) in 8.0 &#956;l of digestion buffer-2. Pipette up and down for several seconds, then vortex lightly to achieve thorough mixing and incubate for 1.5 hr at 37 &#176;C. Add 33.0 &#956;l HPLC grade MeOH.</li>
	<li>Run digested DNA samples on HPLC, as described below, right away or store at -80 &#176;C until use to minimize oxidation. &#160;NOTE: The 1.5-hr digestion steps suggested here are based on such intervals suggested elsewhere9 and on the findings that similar protocols achieve complete DNA digestion9. Therefore, it was assumed that the only limiting factor to achieve complete digestion was time and that enzyme inhibition and sample inhomogeneity were negligible.</li>
</ol>
4. HPLC Run: Preparation of Mobile Phase, Instrumentation Setup and Maintenance
<ol>
	<li>Use ultrapure water to prepare all buffer solutions and treated with high bond strength resin (e.g., Chelex 100: styrene divinylbenzene copolymer with iminodiacetate ions that chelate transition metals with high affinity) to minimize metal contamination and DNA oxidation during sample preparation.</li>
	<li>Prepare 250 mM Na phosphate buffer, pH 6.2. Use a ratio of 1 : 3.6957 of dibasic sodium phosphate (FW = 177.99 g/mol) to monobasic sodium phosphate (FW = 119.98 g/mol). 
	Example: Therefore, for a 3 L stock solution combine 70.81 g of monobasic and 28.43 g of dibasic powder, add water up to e.g., 2.8 L and verify that the pH of the solution is 6.2. Adjust pH with 250 mM mono or dibasic sodium phosphate solutions, prepared separately and not by concentrated acids or bases. Add 2.24 g of KCl to obtain a concentration of 10 mM KCl.</li>
	<li>Prepare mobile phase in three bottles, at least 1 L each, containing: solvent B &#8211; HPLC-grade methanol, solvent &#8211; B ultrapure water, and solvent C &#8211; phosphate buffer from step 4.2. Insert HPLC mobile phase supply tubing into the bottles; remaining tubes must be also dipped in one of the bottles (e.g., solution C) and primed even if they are not required for mixing.</li>
	<li>During the run, mix the mobile phase solutions as 6% solvent A, 74% solvent B and 20% solvent C to achieve final solution of 50 mM sodium phosphate buffer (pH 6.2) with 2.0 mM KCl, and 6% MeOH.</li>
	<li>Detach the electrode from the electrochemical detector, disassemble it and clean reference and working electrodes with the electrode cleaning solutions and polishing discs recommended by the manufacturer. Rinse the cleaned surfaces with ultrapure water, wipe the water off gently and ensure the detector is dry before turning the system on.</li>
	<li>Reassemble the detector and connect the mobile phase inlet to the electrode. Turn on the flow and allow the mobile phase to fill up the electrode before attaching the outlet mobile phase tube. Install a new column prior to starting optimization. Ensure that installation is in the proper direction and follows all manufacturer&#8217;s recommendations.</li>
	<li>Turn on the HLPC instrumentation well ahead (e.g., 1 hr) of sample analyses to allow equilibration and to reduce baseline noise. See Table 1 for suggested settings; the back pressure limit should be set to 3,000 psi to avoid column damage. Turn on the degasser. Proceed with priming the pumps and setting up solvent gradient.</li>
	<li>Dry prime the pumps according to manufacturer&#8217;s instructions. Do this to wet the lines when a mobile phase has been replaced or the tubing inlet is exposed to air.</li>
	<li>Locate the priming disc and insert a 10-15 ml plastic syringe there. Make sure the syringe is fully inserted before opening the line. To open the lines simply turn the disc counter clockwise for one turn.</li>
	<li>Start the prime with the appropriate HPLC interface selection. Gently pull the syringe to draw fluid into the lines. Once fluid is flowing, the prime is complete; allow the program to finish the run. Repeat for all lines (usually four, depending on the instrument).</li>
	<li>Alternatively, wet prime when the lines are already wetted from an earlier dry prime (see below). &#160; 
	NOTE: When a large amount of time has elapsed between uses (e.g., 2 weeks or more), a dry prime is recommended.</li>
	<li>Change the composition of the mobile phase solvent components to 25% each using the HPLC user interface. From the interface, select &#8220;Direct Function&#8221; option, then select &#8220;Wet Prime&#8221; option. This will prime all the lines at the same time.</li>
	<li>Make sure all the air is out of the system by observing the wet prime line as it enters the waste container. After a good prime, ensure that no bubbles are left in the line. If bubbles persist, repeat priming.</li>
	<li>Once the pump is primed, begin moving the HPLC mobile phase. Use the HPLC interface to manually change the composition of the mobile phase to 6% methanol (solvent A) and 94% water (solvent B), and select a flow rate of 0.9 ml/min. Allow 5 min to clear any methanol from the column cleaning step.</li>
	<li>After 5 min, change the composition to 6% methanol (solvent A), 74% water (solvent B), 20% buffer (solvent C), increase the flow rate to 1.0 ml/min. Set other parameters as summarized in Table 1, and set the back pressure limit to 3,000 psi to avoid column damage. &#160; 
	NOTE: Potential problems with HPLC setup include drifting baseline, split peak and diminished signal intensity. These can usually be overcome by cleaning the column after each use, frequent electrode cleaning, and ensuring that the buffer solutions are free of contamination (e.g., particulate matter or microbial growth).</li>
	<li>Run the mobile phase for about 1-1.5 hr to achieve a stable baseline signal.</li>
	<li>Using the instrument software interface, select parameters as indicated in Table 1 and set run time to 15 min. Inject the sample. Use increased run times for complex samples (determine this empirically by observing the presence or absence of peaks beyond 15-min interval). &#160; 
	NOTE: Instrument software allows programming of sample run conditions and setup for autosampling.</li>
	<li>After the runs have been completed, turn off the detector cell using the interface of the detector. It is important to follow manufacturer&#8217;s recommendations regarding powering down the detector as improper handling and shut-down can damage the instrument. DO NOT turn off the cell by switching the back of the detector as this could damage the instrument.</li>
	<li>Manually change the mobile phase composition to 6% methanol and 94% water. Run for 5 min.</li>
	<li>Manually reduce the flow rate to 0.7 ml/min, immediately change the composition to 50% MeOH and 50% water. Run for at least 20 min. Failure to run this step for the recommended time could result in damage to the column and the detector. Turn off the flow, and then turn off the degasser.</li>
</ol>
5. Preparation of Standards
<ol>
	<li>Prepare mobile phase buffer as outlined in steps 4.1&#8211;4.3 above. It is important to use the HPLC mobile phase for preparation of standards to avoid peaks resulting from mixing of dissimilar solutions after sample injection.</li>
	<li>Dilute this solution one in five with ultrapure water prior to use, and the final solution must contain 6% MeOH.</li>
	<li>dGuo Standard 
	NOTE: High oxidation potential required for dGuo detection may increase the risk of measuring interfering electroactive compounds. This can be verified by, for example, a standard curve of UV detection of dGuo at 260 nm and comparing it to the standard curve created with EC detection. If both align well (e.g., Supplementary Figure 1) and the sample matrix effects have been taken into account, one may proceed with dGuo detection by UV at 260 nm, rather than EC detection.
	<ol>
		<li>Measure 1.0 mg of dGuo in a 1.5 ml microcentrifuge tube. Add 1.0 ml HPLC grade MeOH.</li>
		<li>Vortex at high speed until dGuo dissolves completely. This is the primary stock; store at -20 &#176;C for several weeks.</li>
		<li>To create the secondary dGuo stock (0.5 mM), dilute 143 &#181;l of primary dGuo stock in 857 &#181;l of HPLC mobile phase. Store on ice until use and prepare fresh daily.</li>
		<li>Make further dilutions to create a standard curve (e.g., Table 2). Store solutions on ice and prepare fresh daily. Run standards in series with experimental samples.</li>
		<li>Prior to run, vary the detector voltage using the highest concentration of the stock to find optimal voltage.</li>
	</ol>
	</li>
	<li>8-oxo-dGuo Standard
	<ol>
		<li>Measure 1.0 mg of 8-oxo-dGuo in a 1.5 ml microcentrifuge tube. Add 0.1 ml HPLC grade MeOH. Vortex on high speed until 8-oxo-dGuo dissolves completely. This is the primary stock of 8-oxo-dGuo. Store at -20 &#176;C for several weeks.</li>
		<li>In a separate tube, combine 2.9 &#956;l of primary stock and 997.1 &#956;l of HPLC mobile phase buffer, vortex. This is the secondary stock (10,000 nM).</li>
		<li>To create a working solution for the standard curve (250 nM), combine 24.4 &#956;l of secondary stock and 975.6 &#956;l of mobile phase buffer.</li>
		<li>Perform further dilutions to provide the solutions required to create a standard curve (e.g., Table 3). Store solutions on ice and prepare fresh daily. Run standards with the experimental samples.</li>
	</ol>
	</li>
	<li>Quantification
	<ol>
		<li>Integrate the area under the curve for both 8-oxo-dGuo and dGuo using standard software (as a part of HPLC-computer interface; see Supplementary Figure 2A).</li>
		<li>Construct standard curve (known concentration of each analyte vs. area under the curve (Supplementary Figure 2B). Using the equation of the standard curve, calculate the ratio for 8-oxo-dGuo/dGuo for each sample.</li>
	</ol>
	</li>
</ol>
6. Agarose Gel Electrophoresis
NOTE: Agarose gel electrophoresis may be performed to verify the completeness of DNA digestion.
<ol>
	<li>Prepare 50x Tris-acetate-EDTA (TAE) buffer, by dissolving 242 g Tris base (FW = 121.14) in 750 ml ultrapure water. Add 57.1 ml glacial acetic acid and 100 ml of 0.5 M EDTA (pH 8.0; EDTA will completely dissolve when the solution pH is adjusted to 8.0 with e.g., NaOH) and add ultrapure water to a final volume of 1 L. Store at RT, dilute this solution 50x prior to use.</li>
	<li>Weigh out 1 g of agarose and dissolve it in 100 ml 1x TAE buffer, heating in the microwave for 1-2 min. Wear goggles and protective equipment to avoid contact with boiling agarose.</li>
	<li>Cool the solution down until approximately 50 &#176;C, add 5 &#956;l ethidium bromide (caution: mutagen) and pour it into the agarose gel running apparatus. Insert the comb.</li>
	<li>Wait until the solution solidifies, pour TAE buffer over the gel and load the samples (volume depends on the comb size; typically, 10-20 &#956;l of DNA sample is loaded, mixed with 6x running dye to visualize and facilitate loading).</li>
	<li>Run the gel until the dye migrates approximately 2/3 the length of the gel (e.g., 45 min at 150 V). Visualize DNA bands under the UV light.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+8-oxoguanine+in+DNA+by+gas+chromatography-mass+spectrometry+and+HPLC-electrochemical+detection:+overestimation+of+the+background+level+of+the+oxidized+base+by+the+gas+chromatography-mass+spectrometry+assay.">Determination of 8-oxoguanine in DNA by gas chromatography-mass spectrometry and HPLC-electrochemical detection: overestimation of the background level of the oxidized base by the gas chromatography-mass spectrometry assay.</a> Chem. Res. Toxicol. 8 (8), 1039-1045 (1995).</li><li>Kawanishi, S., Murata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+DNA+damage+induced+by+bromate+differs+from+general+types+of+oxidative+stress.">Mechanism of DNA damage induced by bromate differs from general types of oxidative stress.</a> Toxicology. 221 (2-3), 172-178 (2006).</li><li>Tahara, S., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+mouse+splenic+cells+to+oxidative+DNA+damage+by+x-ray+irradiation.">Susceptibility of mouse splenic cells to oxidative DNA damage by x-ray irradiation.</a> Biol. Pharm. Bull. 27 (1), 105-108 (2004).</li><li>Garratt, L. W., Mistry, V., Singh, R., Sandhu, J. K., Sheil, B., Cooke, M. S., Sly, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpretation+of+urinary+8-oxo-7,8-dihydro-2'-deoxyguanosine+is+adversely+affected+by+methodological+inaccuracies+when+using+a+commercial+ELISA.">Interpretation of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine is adversely affected by methodological inaccuracies when using a commercial ELISA.</a> Free Radic. Biol. Med. 48 (11), 1460-1464 (2012).</li><li>Cooke, M. S., Collins, A., Olinski, R., Rozalski, R., Loft, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harmonising+measurements+of+8-oxo-7,8-dihydro-2&#8242;-deoxyguanosine+in+cellular+DNA+and+urine.">Harmonising measurements of 8-oxo-7,8-dihydro-2&#8242;-deoxyguanosine in cellular DNA and urine.</a> Free Radic. Res. 46 (4), 541-553 (2012).</li><li>Cadet, J., Douki, T., Ravanat, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+oxidatively+generated+base+damage+in+cellular+DNA.">Measurement of oxidatively generated base damage in cellular DNA.</a> Mutat Res. 711 (1-2), 3-12 (2011).</li><li>Chomczynski, P., Mackey, K., Drews, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNAzol:+a+reagent+for+the+rapid+isolation+of+genomic+DNA.">DNAzol: a reagent for the rapid isolation of genomic DNA.</a> Biotechniques. 22 (3), 550-553 (1997).</li><li>Collins, A. R., Cadet, J., M&#246;ller, L., Poulsen, H. E., Vi&#241;a, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+we+sure+we+know+how+to+measure+8-oxo-7,8-dihydroguanine+in+DNA+from+human+cells.">Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells.</a> Arch Biochem Biophys. 423 (1), 57-65 (2004).</li><li>Badouard, C., M&#233;n&#233;zo, Y., Panteix, G., Ravanat, J. L., Douki, T., Cadet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+new+types+of+DNA+lesions+in+human+sperm.">Determination of new types of DNA lesions in human sperm.</a> Zygote. 16 (1), 9-13 (2008).</li><li>Cadet, J., Douki, T., Gasparutto, D., Ravanat, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+damage+to+DNA:+formation,+measurement+and+biochemical+features.">Oxidative damage to DNA: formation, measurement and biochemical features.</a> Mutat Res. 531 (1-2), 1-2 (2003).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Validation of analytical procedures: text and methodology Q2(R1). , (2015).</li></ol>

No conflict of interest or competing financial interests declared.

Although 8-oxo-dGuo has been reported as a useful biomarker of DNA oxidation, its reliable quantification can pose a challenge. Although several published methods exist, there is a need for a comprehensive, descriptive overview of protocol to permit researchers to deploy the method in their laboratories. Here we present a detailed overview of an HPLC-based protocol that will permit new users to establish an effective method for 8-oxo-dGuo detection and quantification.
Three major methods that ...

Reactive oxygen species (ROS), whose steady-state levels can increase during many pathological conditions and xenotoxic metabolism, contribute to an increased frequency of oxidative DNA damage. Among several possible nucleobases oxidation products, oxidative DNA damage can readily be measured using the stable marker 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo), which is one of the oxidized forms of 2&#8217;-deoxyguanosine (dGuo)1. 8-oxo-dGuo is the most abundant DNA lesion2 and, therefore, has been studied to greater detail as a DNA oxidation biomarker despite the existence of multiple DNA oxidation products 3. In humans, this damage can be repaired via base excision repair by 8-oxoguanine glycosylase 1 (hOGG1)4. If left unrepaired, 8-oxo-dGuo can contribute to the formation of base pair-substitution mutations (i.e., G to T transversions)4. Importantly, 8-oxo-dGuo is an established marker for DNA damage in relation to the initiation and promotion of carcinogenesis2. Therefore, accurate quantification of 8-oxo-dGuo is a useful and desirable biomarker of oxidative DNA damage5.
There is widespread confusion in the literature regarding the correct names for oxidatively-damaged forms of 2-deoxyguanosine and, moreover, the correct name of the compound(s) routinely measured as a biomarker of oxidative DNA damage6. The 6,8-diketo and 6-enol,8-keto tautomeric forms of 8-oxo-dGuo (shown in Figure 1) are the two most prominent tautomers discussed in the literature5,7. The 6,8-diketo form is the most prominent form at physiological pH of 7.4, and is the most prominent DNA oxidation product7. Therefore, 8-oxo-dGuo, rather than 8-hydroxy-dGuo is the most appropriate name for this oxidation product6. It is also important to note that 2-deoxyguanosine (dGuo), rather than nucleobase guanine (Gua) or ribonucleoside guanosine (Guo), respectively, is detected by most methods6.
Accurate detection and quantification of 8-oxo-dGuo is challenging due to: i) variability in the digestion of the DNA sample, ii) adventitious oxidation of dGuo to 8-oxo-dGuo that can occur during sample preparation, and iii) the need for effective validation of the analytical HPLC-ED method8. In this protocol, we aimed at achieving i) by providing conditions, favorable for complete DNA digestion and ii) by the inclusion metal chelator and chelator-treated solutions and a special DNA-isolating reagent, while iii) was only partially addressed by inclusion of positive controls and thus providing that the method is capable of detecting 8-oxo-dGuo in biological samples. Further validation is beyond the scope of this paper. However, we are confident that this protocol will help the prospective users determine the extent to which they need to formally validate the protocol, depending on their purposes. A list of steps required for the formal validation of the method is provided further. During the development and deployment of a method for 8-oxo-dGuo detection, published methods were reviewed and consolidated. Thus, this method eliminates the need to gather information from several published sources that often lack important experimental details while also providing rapid and straightforward means of testing if the method for the detection and quantification of 8-oxo-dGuo has been adopted successfully. This adapted method was employed to successfully analyze DNA samples from cultured cells and murine tissue. This video article will assist other groups in establishing an effective method for reliable detection and quantification of 8-oxo-dGuo by HPLC-ED.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>8-oxo-dGuo standard</td>
			<td>Cayman Chemical Company</td>
			<td>89320</td>
			<td>Inappropriately referred to as &#34;8-hydroxy-2&#39;-deoxy Guanosine&#34; - see Fig. 1 and text for details</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P5931</td>
			<td>From E.coli</td>
		</tr>
		<tr>
			<td>Chelex 100</td>
			<td>Sigma-Aldrich</td>
			<td>C7901</td>
			<td>Chelates heavy metals</td>
		</tr>
		<tr>
			<td>Desferoxamine mesylate</td>
			<td>Sigma-Aldrich</td>
			<td>D9533</td>
			<td></td>
		</tr>
		<tr>
			<td>dGuo standard</td>
			<td>Sigma-Aldrich</td>
			<td>D7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibasic sodium phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>S9390</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA from salmon sperm</td>
			<td>Sigma-Aldrich</td>
			<td>D1626</td>
			<td>Sodium salt</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich</td>
			<td>D4527</td>
			<td>TypeII, from bovine pancreas</td>
		</tr>
		<tr>
			<td>DNAzol</td>
			<td>Invitrogen</td>
			<td>10503-27</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E4884</td>
			<td>The compound would not completely dissolve until solution pH is adjusted to 8.0 with e.g. NaOH</td>
		</tr>
		<tr>
			<td>F12-K media</td>
			<td>ATCC</td>
			<td>30-2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum</td>
			<td>ATCC</td>
			<td>30-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Guard column</td>
			<td>Chromatographic Specialties</td>
			<td>YBA 99S03 0204GC</td>
			<td>Protects colum from contamination; may also lead to pressure build-up</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Monobasic sodium phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Invitrogen</td>
			<td>15190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphodiesterase I enzyme&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P3243</td>
			<td>Type II from Crotalus adamaneus venom</td>
		</tr>
		<tr>
			<td>Teflon homogenizer</td>
			<td>Thomas Scientific</td>
			<td>7724T-1 or 7724T-5 for 1 or 5 mL, respectively</td>
			<td>Volume (holding capacity) depends on the amount of sample to be processed.</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Invitrogen</td>
			<td>15050-065</td>
			<td></td>
		</tr>
		<tr>
			<td>YMC-BASIC column with bonded spherical silica</td>
			<td>Chromatographic Specialties</td>
			<td>YBA 99S03 1546WT</td>
			<td></td>
		</tr>
	</tbody>
</table>

hplc measurement of the dna oxidation biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in cultured cells and animal tissues

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of biomacromolecules, including DNA. Therefore, efficient detection of DNA oxidation is important for a variety of research disciplines, including medicine and toxicology. A common biomarker of oxidatively damaged DNA is 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo; often erroneously referred to as 8-hydroxy-2&#39;-deoxyguanosine (8-OH-dGuo or 8-oxo-dG)). Several protocols for 8-oxo-dGuo measurement by high pressure liquid chromatography with electrochemical detection (HPLC-ED) have been described. However, these were mainly applied to purified DNA treated with pro-oxidants. In addition, due to methodological differences between laboratories, mainly due to differences in analytical equipment, the adoption of published methods for detection of 8-oxo-dGuo by HPLC-ED requires careful optimization by each laboratory. A comprehensive protocol, describing such an optimization process, is lacking. Here, a detailed protocol is described for the detection of 8-oxo-dGuo by HPLC-ED, in DNA from cultured cells or animal tissues. It illustrates how DNA sample preparation can be easily and rapidly optimized to minimize undesirable DNA oxidation that can occur during sample preparation. This protocol shows how to detect 8-oxo-dGuo in cultured human alveolar adenocarcinoma cells (i.e., A549 cells) treated with the oxidizing agent KBrO3, and from the spleen of mice exposed to the polycyclic aromatic hydrocarbon dibenzo(def,p)chrysene (DBC, formerly known as dibenzo(a,l)pyrene, DalP). Overall, this work illustrates how an HPLC-ED methodology can be readily optimized for the detection of 8-oxo-dGuo in biological samples.

dGuo was observed to have a retention time of 4.7 min whereas 8-oxo-dGuo had a retention time of approximately 6.4 min (Figure 2A and B). There is about 1,000-fold difference in the peak heights between the two analytes, as seen in Figure 2C. Voltammograms for 8-oxo-dGuo and dGuo were obtained by running standards at a working potential in the range of +0.2 to +1.1 V. The optimum working potential for 8-oxo-dGuo was determined to be +0.5 V, and +0.9 V for dGuo (F...

Watch this Scientific Journal Video about HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2â€™-deoxyguanosine, in Cultured Cells and Animal Tissues at JoVE.com

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&amp;#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

The goal of this protocol is the detection of the DNA oxidation marker, 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo) by HPLC-ED, in DNA from cultured cells or animal tissues.

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of ...

hplc-measurement-dna-oxidation-biomarker-8-oxo-78-dihydro-2

Research

JoVE Journal

Chemistry

26.2K vistas.  Health Canada.  El objetivo de este protocolo es la detección del marcador de la oxidación del ADN, 8-oxo-7,8-dihidro-2 'desoxiguanosina (8-oxo-dGuo) por HPLC-ED, en el ADN de células cultivadas o tejidos animales. 

Video: HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

Determining the Chemical Composition of Corrosion Inhibitor/Metal Interfaces with XPS: Minimizing Post Immersion Oxidation

An approach for acquiring more reliable X-ray photoelectron spectroscopy data from corrosion inhibitor/metal interfaces is described. More specifically, the focus is on metallic substrates immersed in acidic solutions containing organic corrosion inhibitors, as these systems can be particularly sensitive to oxidation following removal from solution. To minimize the likelihood of such degradation, samples are removed from solution within a glove box purged with inert gas, either N2 or Ar. The glove box is directly attached to the load-lock of the ultra-high vacuum X-ray photoelectron spectroscopy instrument, avoiding any exposure to the ambient laboratory atmosphere, and thus reducing the possibility of post immersion substrate oxidation. On this basis, one can be more certain that the X-ray photoelectron spectroscopy features observed are likely to be representative of the in situ submerged scenario, e.g. the oxidation state of the metal is not modified.

A protocol to avoid the oxidation of metallic substrates during sample transfer from an inhibited acidic solution to an X-ray photoelectron spectrometer is presented.

An approach for acquiring more reliable X-ray photoelectron spectroscopy data from corrosion inhibitor/metal interfaces is described. More specifically, ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

Quantitative Atomic-Site Analysis of Functional Dopants/Point Defects in Crystalline Materials by Electron-Channeling-Enhanced Microanalysis

A novel elemental and chemical analysis scheme based on electron-channeling phenomena in crystalline materials is introduced, where the incident high-energy electron beam is rocked with the submicrometric pivot point fixed on a specimen. This method enables us to quantitatively derive the site occupancies and site-dependent chemical information of impurities or intentionally doped functional elements in a specimen, using energy-dispersive X-ray spectroscopy and electron energy-loss spectroscopy attached to a scanning transmission electron microscope, which is of significant interest to current materials science, particularly related to nanotechnologies. This scheme is applicable to any combination of elements even when the conventional Rietveld analysis by X-ray or neutron diffraction occasionally fails to provide the desired results because of limited sample sizes and close scattering factors of neighboring elements in the periodic table. In this methodological article, we demonstrate the basic experimental procedure and analysis method of the present beam-rocking microanalysis.

We provide a general outline of quantitative microanalysis methods for estimating the site occupancies of impurities and their chemical states by taking advantage of electron-channeling phenomena under incident electron beam-rocking conditions, which reliably extract information from minority species, light elements, oxygen vacancies, and other point/line/planar defects.

A novel elemental and chemical analysis scheme based on electron-channeling phenomena in crystalline materials is introduced, where the incident ...

HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis

A method for identifying Chinese medicinal materials and their related adulterants was constructed by taking Clematidis Armandii Caulis (Chuanmutong, a universally used traditional Chinese medicine) as an example. Ten batches of genuine Chuanmutong varieties and five batches of related adulterants were analyzed and compared based on the high-performance liquid chromatography (HPLC) fingerprints combined with chemometrics, including cluster analysis (CA), principal component analysis (PCA), and orthogonal partial least-squares discrimination analysis (OPLS-DA). In addition, the content of &#946;-sitosterol was determined. The control chemical fingerprint of Chuanmutong was established, and 12 common peaks were identified. The similarity between the fingerprint of 10 batches of genuine Chuanmutong varieties and the control fingerprint was 0.910-0.989, while the similarity of five batches of adulterants was only 0.133-0.720. Based on the common peaks in the chromatogram, 15 batches of samples were classified into three content levels by PCA, and were aggregated into four categories by CA, achieving a clear distinction between authentic Chuanmutong and adulterants of Chuanmutong. Further, seven differential components that can effectively identify authentic Chuanmutong and adulterants of Chuanmutong were found through OPLS-DA. The &#946;-sitosterol content of 10 batches of genuine Chuanmutong varieties was 97.53-161.56 &#181;g/g, while the &#946;-sitosterol content of the five batches of adulterants varied greatly, among which the &#946;-sitosterol content of Clematis peterae Hand.-Mazz. and Clematis gouriana Roxb. Var. finetii Rehd. et Wils. was significantly lower than that of authentic varieties of Chuanmutong. The HPLC index component content and chemical fingerprint multi-pattern recognition method established in this study provide a new strategy for effectively identifying authentic Chinese medicinal materials and related adulterants.

HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis

Here, we present a protocol to establish high-performance liquid chromatography (HPLC), coupled with chemical fingerprint multi-pattern recognition, which provides a new strategy for effectively identifying the genuine varieties of Clematidis Armandii Caulis and its adulterants.

A method for identifying Chinese medicinal materials and their related adulterants was constructed by taking Clematidis Armandii Caulis (Chuanmutong, a ...