dna oxidation plays a significant role 
 in the pathophysiology of 
 cancers , aging , and inherited diseases , with epidemiological 
 studies demonstrating a strong association between the reactive oxygen 
 ( ros ) and nitrogen species ( rns ) of chronic inflammation and increased 
 cancer risk . this potential for dna damage to play a causative role in mutation 
 and carcinogenesis has prompted the chemical characterization of oxidatively 
 induced dna damage and the quest to develop biomarkers that reflect 
 this damage and serve as surrogates for short - lived ros and rns . as the most readily oxidized site in dna , guanine 
 is a major target for dna oxidants , and its oxidation produces a wide 
 spectrum of end products , including 8-oxo-7,8-dihydro-2-deoxyguanosine 
 ( 8-oxodg ) spiroiminodihydantoin ( sp ) guanidinohydantoin ( gh ) , 2-amino-5-[2-deoxy--d - erythro - pentofuranosyl)amino]-4h - imidazol-4-one 
 ( imidazolone ) and its hydrolysis product , 2,2-diamino-4-[(2-deoxy--d - erythro - pentofuranosyl)amino]-5(2h)-oxazolone 
 ( ox ) ( scheme 1(9 ) ) . although 8-oxodg is produced abundantly in dna exposed to rns and 
 ros , it is several orders - of - magnitude more susceptible to further oxidation 
 than dg itself , yielding more stable 
 secondary oxidation products , including sp and gh ( scheme 1 ) . ox and its precursor imidazolone have been observed as oxidation 
 products of dg or 8-oxodg in oligodeoxynucleotides and calf thymus 
 dna treated with mn - tmpyp / khso5 , peroxynitrite ( onoo ) , and photochemical oxidants ( scheme 1 ) . all of these dna 
 oxidation products are promutagenic , with both diastereoisomers of sp strongly inhibiting dna polymerase and causing mutation frequencies that are at least an order of magnitude 
 higher than those for 8-oxodg . therefore , as part of our current focus on inflammation - related 
 dna damage , including assessment of potential biomarkers , we developed 
 a liquid chromatography - coupled isotope - dilution triple - quadrupole 
 mass spectrometry method ( lc - ms / ms ) to simultaneously measure 8-oxodg , 
 sp , gh , and ox in dna and applied the method to dna treated in vitro with onoo , singlet oxygen derived 
 from photoactivated rose bengal , and -radiation . singlet 
 oxygen , with a high quantum yield in aqueous solution , 
 is an important reactive oxygen species in vivo and , 
 although accompanied by low levels of other reactive species , is the 
 primary oxidant arising via rose bengal photosensitization . -irradiation of aqueous solutions , however , yields hydroxyl 
 radicals and hydrated electrons , with the former causing oxidation 
 of both 2-deoxyribose and nucleobases in dna . onoo is the product of a reaction of macrophage - derived 
 nitric oxide and superoxide , and its protonated form undergoes homolytic 
 bond scission to yield a hydroxyl radical and a nitrogen dioxide radical , 
 which are strong and weak oxidants , respectively . the new lc - ms / ms method , which is both more efficient and 
 more sensitive than previous methods , also revealed novel features 
 of the oxidant - specific distribution of these g oxidation products . uniformly labeled dg ( c and n > 98% ) and uniformly 
 labeled n dg ( 98% ) were obtained from cambridge 
 isotope laboratories ( andover , ma ) . benzonase nuclease , deoxyribonuclease 
 ( dnase ) , calf intestine alkaline phosphatase , and calf thymus dna 
 were from sigma aldrich ( st . louis , mo ) , onoo solution 
 from cayman chemical company ( ann arbor , mi ) , phosphodiesterase 1 
 from usb products ( cleveland , oh ) , nanosep omega 10 kd exclusion filter 
 columns from pall corporation ( ann arbor , mi ) , and sep - pak c18 cartridges from waters ( milford , massachusetts ) . all other chemicals , 
 reagents , and solvents were purchased from sigma - aldrich . uv / vis spectroscopy 
 measurements were made on an hp8452 diode - array spectrophotometer 
 ( agilent technologies , palo alto , ca ) or a beckman du640 diode - array 
 spectrophotometer ( beckman coulter , inc . high - performance 
 liquid chromatography ( hplc ) analyses were carried out on an agilent 
 1100 series hplc system with binary pumps , a degasser , and an autoinjector . quantitative liquid chromatography  tandem mass spectrometry 
 ( lc - ms / ms ) analyses were conducted on an agilent 6430 triple quadrupole 
 mass spectrometer interfaced with an agilent capillary 1200 binary 
 pumping system or an ab sciex api 3000 triple quadrupole mass spectrometer 
 ( ab sciex , framingham , ma ) interfaced with an agilent 1100 binary 
 hplc . electrospray ionization mass spectrometry analyses for isotopic 
 purities and structure confirmations by exact mass were done on an 
 agilent msd - tof . experiments were designed to yield similar levels 
 of sp across the different oxidation conditions in order to facilitate 
 comparisons . this approach provides a means for comparing relative 
 amounts of the other lesions rather than total amounts of damage . n - labeled 8-oxodg 
 was synthesized as described earlier , and its identity 
 was confirmed by comparison of its reversed phase hplc retention time 
 with that of commercially available 8-oxodg , its characteristic uv 
 spectrum ( max= 248 and 292 nm ) , and by esi - ms / ms , 
 which gave a protonated molecular ion with m / z 289 and two fragment ions at m / z 117 and m / z 173 , which 
 correspond to the protonated 2-deoxyribose and n5 - 8-oxo - guanine . the standard was quantified by uv spectroscopy 
 using an extinction coefficient of 10,300 m cm at 293 nm . the c- , n - labeled , and unlabeled sp , gh , and ox 
 were synthesized from labeled dg as described previously , and structures were confirmed by uv spectra , 
 hplc retention times , and esi - ms / ms . for sp , esi - ms / ms analysis yielded 
 a protonated molecular ion at m / z 300.1 , along with characteristic fragment ions including m / z 184 and m / z 117.1 ( figure s1b ) . the molar extinction coefficient for 
 the sp was determined to be 10,500 m cm at max = 230 nm , which is consistent with the 
 reported value . for gh , the esi - ms / ms 
 spectrum ( figure s1c ) showed m / z 158.0 as the major fragment released from the protonated gh ( m / z 274.1 ) . for ox , esi - ms / ms analysis 
 revealed a protonated molecular ion at m / z 246.7 , with fragmentation occurring by both loss of 2-deoxyribose 
 ( m / z 131 ) and loss of co2 ( [ m + h this distinct fragmentation 
 pattern reflects the loss of aromaticity upon oxidation of dg to oxazolone , 
 accompanied by the incorporation of a relatively labile lactone group 
 in the molecule . working 
 solutions of onoo were prepared in 0.1 m naoh by 
 dilution of the commercial stock solution of 100 mm in 0.3 m naoh , 
 the concentration of which was verified by measuring the absorbance 
 at 302 nm (  = 1670 m cm ) . aliquots of the working stock solutions 
 of onoo were placed on the side walls of tubes 
 containing a solution of calf thymus dna ( 300 g in 0.4 ml of 
 25 mm sodium phosphate buffer , ph 7.2 ) to produce final concentrations 
 of 01000 m . the oxidation reaction was initiated by 
 rapidly vortexing the tube for 1 min and then allowing the reaction 
 to proceed at ambient temperature for 1 h. calf thymus 
 dna ( 300 g , 1 g/l in 50 mm sodium phosphate buffer , 
 ph 7.4 ) was irradiated in the presence or absence of 10 m rose 
 bengal ( added from a dimethyl sulfoxide ( dmso ) stock solution ) with 
 uv light in a rayonet rmr-600 reactor with rpr-3500 bulbs (  max = 350 nm ) for 0 , 15 , 30 , 60 , or 90 min . the control samples 
 were treated with an equal volume of dmso and irradiated with uv light 
 for 90 min . calf thymus dna ( 250 g , 
 1 g/l ) in chelex - treated 50 mm sodium phosphate buffer 
 at ph 7.4 was exposed to co -radiation delivered 
 at 133 gy / min in a gammacel-220 ( mds nordion , ottawa , ontario , canada ) , 
 at ambient temperature , for total doses of 0 , 7.5 , 20 , 33 , 70 , or 
 133 gy . following each of the above treatments , dna was precipitated by the addition of ethanol to 70% , washed with 
 70% ethanol , resuspended in 100 l of water , and quantitated 
 by uv / vis spectroscopy . the dna was then digested to nucleosides with 
 4 units of benzonase nuclease , 0.1 units phosphodiesterase 1 , 4 units 
 dnase , and 20 units calf intestine alkaline phosphatase in 10 mm tris 
 buffer ( ph 7.9 ) and 1 mm magnesium chloride . deferoxamine mesylate 
 ( 3 mm ) and butylated hydroxytoluene ( 1 mm ) were added as antioxidants 
 to inhibit artifactual oxidation . n5 - 8-oxodg 
 ( 1 pmol ) , c8n4-oxazolone 
 ( 4.25 pmol ) , c9n5-guanidinohydantoin 
 ( 5 pmol ) , and c10n5-spiroiminodihydantoin ( 2.9 pmol ) were added as internal standards 
 and the samples digested for 4 h at 37 c . samples were then 
 filtered through 10 kd exclusion filters to remove enzymes and , finally , 
 dried under reduced pressure . digested samples were prepurified 
 using a varian microsorb - mv c18 reversed - phase column ( 5 m 
 particle , 100  pore size , 250  4.6 mm ) with an acetonitrile/8 
 mm ammonium acetate gradient . individual 
 fractions were then dried and subjected to lc - ms / ms analysis . the 
 column was maintained at 10 c with 0.6 ml / min flow rate , and 
 the gradient conditions are detailed in supporting 
 information , table s1 . sp , gh , and ox were collected in a single 
 fraction eluting between 3 and 6 min , and 8-oxodg was collected at 
 14.615.6 min ( supporting information , table s2 ) . the fractions were dried under reduced pressure . to control for dna losses sustained during dna workup , canonical 
 2-deoxyribonucleosides were quantified by integrating the 
 uv absorbance peaks obtained during hplc fractionation and summing 
 areas to compare to the 2-deoxyribonucleoside calibration 
 curves run with each experiment . the dried samples containing 
 8-oxodg and the corresponding isotopomer were dissolved in 100 l 
 of deionized water , and 10 l were analyzed on an agilent 6410 
 tandem quadrupole , or an ab sciex api 3000 , interfaced with an agilent 
 1100 series hplc system . residual dg was separated from 8-oxodg using 
 isocratic reversed phase chromatography with 200 l / min , 25 
 c , 3% acetonitrile in water with 0.1% acetic acid on a c18 thermo 
 hypersil gold aq column ( 150  2 , 1 mm , 3 m particles ) . it is important to note that 8-oxodg can be formed from dg in - source 
 during ionization ; therefore , separation of dg from 8-oxodg is essential 
 for accurate quantitation . the mass spectrometers 
 were operated in the positive ion mode , with all instrument parameters 
 optimized for maximum sensitivity . samples were analyzed in the multiple 
 reaction monitoring mode ( mrm ) , which monitors collision - induced dissociation 
 of a precursor ion to an abundant characteristic product ion . the 
 optimized parameters for the api 3000 were ion source , turboionspray ; 
 200 ms dwell time ; declustering potential , 15 v ; focusing potential , 
 90 v ; entrance potential , 5 v ; collision cell exit potential , 15 v ; 
 nebulizer gas , 13 au ( arbitrary unit ) ; curtain gas , 7 au ; collision 
 gas , : 9 au ; temperature , 400 c ; and ion spray voltage , 4,000 
 v. samples were analyzed with the following transitions : m / z 284  168 for 8-oxodg , m / z 289  m / z 173 for n5-labeled 8-oxodg , and m / z 268 the fraction containing 
 sp , gh , ox , and their corresponding isotopomers was dried and then 
 redissolved in deionized water ( 30 l ) . esi - ms / ms analyses were 
 done on an agilent 6430 triple quadrupole mass spectrometer interfaced 
 with an agilent capillary 1200 series binary pumping system with a 
 hypercarb column ( 100 mm  1.0 mm , 5 m particles ; thermo 
 scientific , torrance , ca ) . the gradient was 0.3% formic acid in water 
 ( a ) and 0.3% formic acid in acetonitrile ( b ) from 2% to 70% b over 
 15 min at 20 l / min ( microionization spray capillary ) . the mass spectrometer was 
 operated in positive ion mode , with instrument parameters optimized 
 for maximal sensitivity ( supporting information , table s3 ) . samples were analyzed in multiple reaction monitoring 
 mode ( mrm ) , using the following transitions : m / z 300  m / z 184 
 and m / z 315  m / z 194 for sp and uniformly c , n - labeled sp ; m / z 274  m / z 158 and m / z 288  m / z 167 
 for gh and uniformly c , n - labeled gh ; m / z 247  m / z 87 and m / z 259  m / z 93 for ox and uniformly c , n - labeled ox , respectively ( figure s1 
 and supporting information , figure s4 ) . calibration 
 curves for the labeled and unlabeled forms of each of the four dna 
 oxidation products were constructed by plotting the mrm signal ratios 
 between the labeled and unlabeled forms against their corresponding 
 concentration ratios . measurements of 8-oxodg , sp , gh , and ox were 
 based on the mrm signal ratios between analytes of interest and their 
 isotope - labeled internal standards and their respective response curves 
 ( table 1 and supporting 
 information , figure s5 ) . lod , limit of detection ; loq , limit 
 of quantitation . the limits of detection ( lod ) 
 and quantification ( loq ) of 8-oxodg , ox , sp , and gh were determined 
 by adding known amounts of the corresponding labeled nucleosides to 
 deionized water , followed by lc - ms / ms analysis as described above . the nominal signal - to - noise ratios were 3 ( lod ) and 10 
 ( loq ) . assuming approximately equal responses for the unlabeled and 
 the labeled nucleosides in the mrm mode , the limit of detection of 
 each oxidation product ( the unlabeled nucleoside ) was in the range 
 of 1 to 3 fmol , with limits of quantification around 3 to 10 fmol , 
 corresponding to approximately 13 nucleoside/10 nucleosides in 100 g of dna ( table 1 ) the first critical step in the 
 method involves artifact - free processing of the oxidized dna . in this case , treated dna samples were purified by ethanol precipitation , 
 followed by the addition of the antioxidants , deferoxamine mesylate 
 and butylated hydroxytoluene , along with isotopically labeled internal 
 standards . dna was then enzymatically hydrolyzed with benzonase , phosphodiesterase 
 1 , and deoxyribonuclease ( dnase ) , with dephosphorylation to nucleosides 
 by calf intestine alkaline phosphatase . following the removal of enzymes 
 by filtration a second critical step in the method involves 
 hplc prepurification 
 of the various damaged 2-deoxyribonucleosides . the hydrophilic 
 sp , gh , and ox eluted early on the reversed - phase hplc system and 
 were collected as a single fraction , while 8-oxodg was well retained 
 on the column . the two groups of 2-deoxyribonucleosides were 
 collected at the predetermined retention times noted in experimental procedures . to quantify the total dna injected 
 on the hplc column , canonical 2-deoxyribonucleosides were 
 quantified by integrating the area under the peaks of the uv absorbance 
 profiles obtained during the prepurification steps , with analysis 
 by calibration curves . the final step of the method involves 
 lc - ms / ms analysis of the 
 individual purified 2-deoxyribonucleosides . the hydrophilic 
 sp , gh , and ox were optimally resolved on a hypercarb graphite column 
 using an acetonitrile / water gradient , while 8-oxodg was best resolved 
 using a c18 reversed phase hplc column , as in the prepurification . both hplc systems were coupled to a triple quadrupole mass spectrometer 
 operated in positive ion mode . the sp and ox diastereomers were observed 
 with this approach ( supporting information , figure s4 ) . typical detection limits for modified dna nucleosides 
 were 310 fmol ( table 1 ) . for 8-oxodg 
 analysis , the use of a reversed phase column eluted with acetic acid / acetonitrile 
 provided similar ms / ms quantification properties as those reported 
 previously . several control studies 
 were performed to assess oxidative artifacts 
 during dna processing and analysis . the detection and quantitation 
 limits of the guanine oxidation products are listed in table 1 . the dg transition m / z 268  m / z 152 was monitored 
 to confirm the separation between 8-oxodg and dg and thus to ensure 
 that artifactual production of 8-oxodg in the ion source is not included 
 in the analysis . [ c10,n5]-8-oxodg was not detected in the pilot experiments 
 ( 1 nmol , in triplicate ) spiked with [ c10,n5]-dg , which suggests that artifactual formation 
 of 8-oxodg during the sample processing was minimal . to assess the 
 oxidation of 8-oxodg during workup , [ n5]-8-oxodg 
 was added to a dna sample before enzymatic digestion . subsequent analysis 
 for [ n5]-sp , [ n5]-gh , 
 and [ n4]-ox was below the detection limits , 
 which demonstrates minimal artifactual formation of sp , gh , and ox 
 during the digestion and workup process . the analytical method was now applied to quantify sp , gh , ox , and 
 8-oxog in dna samples oxidized by four well studied oxidants : onoo , sin-1 , singlet oxygen arising from photoactivated 
 rose bengal , and -radiation . the results are shown in figures 1 and 2 , table 2 , and in supporting information tables s2 and s3 . the background levels of damage products in the 
 commercial calf thymus dna were found to be 2.8  1.4 8-oxodg 
 per 10 nt , 4.7  1.9 sp per 10 nt , 0.52 
  0.11 gh per 10 nt , and 3.6  6.2 ox per 10 nt . the background of 8-oxodg in the calf thymus dna is higher 
 than the levels observed in cells and tissues but is consistent with literature values . as shown in figure 1 , sin-1 produced nonlinear 
 dose  response curves for all damage products , with onoo , -radiation , and rose bengal photosensitization 
 producing more linear dose  response curves . dna was treated with ( a ) -radiation , ( b ) onoo , ( c ) o2 from photooxidation 
 of rose bengal , and ( d ) sin-1 , and the g oxidation products 8-oxodg , 
 sp , gh , and ox were quantified as described in experimental 
 procedures . data are plotted as the lesion level without exponential 
 scaling on the y - axis , with the exponential scaling 
 noted in the inset . average amounts of each oxidation product across all measured 
 doses 
 for each oxidant were calculated and presented as a stack plot . the 
 per nucleotide levels chosen were the same as those plotted in figure 1 . during chronic inflammation , locally produced 
 ros and rns can attack 
 dna to cause mutation and cell death as part of the pathophysiology 
 of cancer and other diseases . as the most readily oxidized of the nucleobases , guanine ( g ) is 
 preferentially targeted by oxidants and produces a wide spectrum of 
 end products , including 8-oxodg , sp , gh , and ox . 8-oxodg is used widely 
 as a biomarker of g oxidation , whereas other more stable oxidation 
 products , such as sp , gh , and ox , have received relatively little 
 attention in spite of their toxicity and mutagenicity . a previously published method for lc - ms / ms analyses of 
 8-oxodg , sp , gh , and ox was 10-times 
 less sensitive for sp and gh than the present method , with an lod 
 of about 1 lesion per 10 nt . further , the lc - ms / ms 
 method of sugden and co - workers , which used a reversed - phase hplc 
 resolution and no prepurification , yielded high background levels 
 of sp in wild - type e. coli cells on the order of 
 1 sp per 10 to 10 nt . in the present studies , we have decreased the detection limits by 
 at least 10-fold to the level of 1 lesion per 10 nt by employing a prepurification step and appropriate chromatographic 
 systems for resolving the various damage products . in addition , our 
 method gives similar results to those of earlier studies , e.g. , for 
 -radiation , confirming that our method is accurate . the more noteworthy result in the analysis of g oxidation products 
 was the strong oxidant dependence of the product spectra . this is 
 illustrated by the dose  response curves in figure 1 and stacked distribution plots of the average g oxidation 
 products under the different oxidizing conditions shown in figure 2 . both figures were plotted to permit easy comparisons 
 between oxidizing conditions and relative changes in g oxidation products . 
 clearly , 8-oxodg is the most abundant product formed with the four 
 oxidants ( figure 1 ) , with ox consistently observed 
 at higher levels than the other two secondary g oxidation products . these results are consistent with published reports , in which ox was found to be the 
 major secondary g oxidation product generated from one- electron oxidants , 
 onoo , -radiation , and singlet oxygen . among individual oxidants , the lowest relative levels of 8-oxodg 
 are present with photosensitized o2 , which 
 correlates with the high relative generation of sp ( figure 1 , figure 2 ) . reported 
 that sp was the major secondary oxidation product when guanosine was 
 exposed to rose bengal - photosensitized o2 at 
 neutral ph . our results corroborate the 
 prominence of sp in o2-mediated chemistry , 
 though more ox was formed in our experiments than in the previous 
 work . this difference is possibly due to the fact that ye et al . performed 
 their oxidation studies with 2-deoxynucleosides , whereas we 
 used calf thymus dna . previous studies have shown differences in relative 
 ratios of different g oxidation products when using 2-deoxyribonucleosides 
 and single - stranded and double - stranded oligonucleotides . for -radiation , figure 1a demonstrates linear increases in each g oxidation product 
 examined , with ox as the predominant product with relatively small 
 proportions of sp . onoo appears to produce 
 the most even distribution 
 of g oxidation products under the conditions used , as shown in figure 2 . however , ox remains the most abundant secondary 
 g oxidation product ( table 2 ) and exists in 
 higher relative proportion as compared to untreated dna ( figure 2 ) . the high rate of ox production with onoo is substantiated by the reaction of dna with sin-1 
 that generates no and o2 that combine to form onoo . the rate of onoo formation from sin-1 decomposition varies with the 
 medium . in phosphate buffer , the half - life 
 of sin-1 is estimated to be 1426 min , with the release of 
 onoo at a rate of 1012 m / min . ox is formed in high relative rates ( figure 2 ) and rapidly increases with higher onoo concentrations . the concentrations of sin-1 used in this experiment were relatively 
 high compared to those in other studies examining dna oxidation , which is likely the reason that onoo resulted 
 in the highest overall levels of oxidation among the various oxidants 
 ( table 2 ) . differences in the relative amounts 
 of g oxidation products for onoo and sin-1 are 
 not surprising given the varying responses observed for the two oxidizing 
 agents by kim et al . , though that study 
 was looking at more generalized damage . these differing responses 
 are possibly due to other reactive and toxic products formed during 
 the decomposition of sin-1 in addition to the 
 effects of sustained lower doses of onoo over time 
 rather than bolus addition . response curve for 8-oxodg formation 
 by onoo oxidation of dna , with levels of 8-oxodg 
 increasing up to 2 equivalents of onoo , then decreasing 
 until 10 equivalents of onoo were added , and finally 
 increasing again with higher concentrations of onoo . further , they observed that sp and gh were formed in dna in a dose - dependent 
 manner at low onoo concentrations but that the 
 levels of these lesions dropped significantly at high onoo concentrations . in contrast , the lower concentrations of onoo used in the present studies produced a nonlinear but 
 smooth increase in the amounts of 8-oxo - dg , sp , and gh in dna treated 
 with increasing onoo concentrations . the differences 
 could be partially due to the different dosage points monitored , increased 
 number of experimental replicates performed , and higher ratio of dg 
 to onoo in our studies . yu et al . also observed 
 more artifactual oxidation during digestion and workup as evidenced 
 by the oxidation of labeled dg included in the analysis . when we performed 
 a similar experiment , no oxidation of labeled dg was observed . the 
 onoo used by yu et al . synthesized via ozonolysis 
 could have different impurities than that purchased for these studies . in conclusion , a sensitive and specific lc - ms / ms method was developed 
 for the quantification of 8-oxodg , sp , gh , and ox lesions in dna . 
 application of the method to dna oxidized in vitro with four different oxidants revealed a clear oxidant dependence 
 of the relative quantities of the g oxidation products .
oxidative damage to dna has many origins , including irradiation , 
 inflammation , and oxidative stress , but the chemistries are not the 
 same . 
 the most oxidizable base in dna is 2-deoxyguanosine ( dg ) , and 
 the primary oxidation products are 8-oxodg and 2-amino - imidazolone . 
 the latter rapidly converts to 2,2-diamino - oxazolone ( ox ) , and 8-oxodg 
 is further oxidized to spiroiminodihydantoin ( sp ) and guanidinohydantoin 
 ( gh ) . in this study , we have examined the dose  response relationship 
 for the formation of the above four products arising in calf thymus 
 dna exposed to gamma irradiation , photoactivated rose bengal , and 
 two sources of peroxynitrite . in order to carry out these experiments 
 , 
 we developed a chromatographic system and synthesized isotopomeric 
 internal standards to enable accurate and precise analysis based upon 
 selected reaction monitoring mass spectrometry . 
 8-oxodg was the most 
 abundant products in all cases , but its accumulation was highly dependent 
 on the nature of the oxidizing agent and the subsequent conversion 
 to sp and gh . among the other oxidation products , ox was the most 
 abundant , and 
 sp was formed in significantly greater yield than gh .