tobacco use is among the most prevalent , 
 albeit preventable , human 
 carcinogen exposures .
cigarette smoking causes up to 90% of lung cancer , 
 the most common cause of cancer death in the united states , resulting 
 in a projected 159,480 deaths in 2013 .
tobacco - specific 
 nitrosamines ( tsna ) are among the most significant tobacco carcinogens ; 
 multiple studies clearly document their strong carcinogenicity in 
 laboratory animals as well as the occurrence of substantial amounts 
 of these carcinogens in both unburned tobacco and tobacco smoke .
one of the most prevalent of these compounds , 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone 
 ( nnk , 1 ) , is a remarkably effective lung carcinogen in laboratory 
 animals , inducing lung tumors in rodents independent of the route 
 of administration .
results of numerous investigations 
 indicate that nnk is a causative agent for lung cancer in smokers .
exposure to 
 nnk may also be causally related to nasal , oral , liver , pancreatic , 
 and cervical cancers .
nnk together with the related 
 nitrosamine nnn have been classified as group 1 carcinogens by the 
 international agency for research on cancer ( iarc ) .
therefore , the risk of lung cancer 
 in smokers 
 may be affected by the relative extent of nnk metabolic activation 
 and detoxification , with those who activate nnk more extensively being 
 at higher risk .
studies in laboratory animals show that nnk is metabolized 
 by three major routes : carbonyl reduction , pyridine oxidation , and 
 -hydroxylation .
carbonyl reduction produces 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol 
 ( nnal , 2 ) ( scheme 1 ) , which undergoes 
 metabolic transformations similar to those of nnk , except for the 
 formation of nnal - glucuronides ( nnal - glucs ) , an important pathway 
 of nnk and nnal detoxification .
the sum of nnal and its glucuronides can be measured in urine as 
  total nnal ,  which is an established biomarker of nnk 
 exposure in humans .
nnal can also be partially converted back to nnk ; 
 however , the nnk - nnal equilibrium favors nnal in rodents , primates , 
 and humans .
the second process , pyridine - n - oxidation , is a 
 minor metabolic pathway in humans and , 
 therefore , is not expected to play an important role in the relative 
 balance of metabolic activation and detoxification of nnk .
the third 
 major route , -hydroxylation , leads to the formation of dna 
 methylating and pyridyloxobutylating intermediates , and thus represents 
 metabolic activation of nnk and nnal ( scheme 1 ) .
formation of dna adducts from the reactive pyridyloxobutyl metabolite 
 has been well established ; some of these adducts have miscoding properties 
 and can activate oncogenes .
the major end products of the nnk and nnal -hydroxylation 
 pathway are 4-oxo-4-(3-pyridyl)butanoic acid ( keto acid , 3 ) and 4-hydroxy-4-(3-pyridyl)butanoic acid ( hydroxy acid , 4 ) ; urinary excretion of these metabolites has been shown in rodents , 
 primates , and humans . thus , the balance between urinary nnal - glucs , 
 as a biomarker of nnk exposure and detoxification , and the sum of 
 urinary 3 and 4 , as a biomarker of nnk metabolic 
 activation , could potentially provide a useful index for the relative 
 efficiency of these major pathways in individual smokers .
adapted with permission from 
 ref ( 38 ) . copyright 
 2008 american association for cancer research .
the main obstacle to using 3 and 4 as 
 biomarkers of nnk metabolic activation is nicotine in cigarette smoke , 
 which also generates these metabolites and is present at more than 
 1,000-fold higher levels than nnk .
therefore , in our 
 previous study we developed a novel approach to specifically identify 
 nnk - derived 3 and 4 by using deuterium - labeled 
 nnk . in that study , we added [ pyridine - d4]nnk ( [ d4]1 ) to a commercial cigarette , 
 which originally contained significantly lower levels of nnk than 
 regular brands , and then analyzed urine samples collected from individuals 
 who smoked these cigarettes . after treating 
 urine with nabh4 to convert 3 to 4
tandem 
 mass spectrometry ( lc - esi - ms / ms ) to distinguish the [ d4]1-derived urinary [ pyridine - d4]hydroxy acid 
 ( [ d4]4 ) from the corresponding unlabeled metabolite 
 and demonstrated for the first time that nnk is metabolically activated 
 in smokers .
future application 
 of our unique methodology can potentially provide 
 important insights into the role of interindividual differences in 
 nnk metabolic activation in cancer risk .
however , the analytical procedure 
 for the analysis of ( [ d4]4 ) in our original 
 study of 20 smokers was not optimized for robust application in larger 
 trials . therefore ,
our purpose in this study was to develop an improved 
 lc - esi - ms / ms method for the quantitation of total [ d4]4 in human urine .
[ pyridine - d4]nnk ( [ d4]1 ) is carcinogenic and mutagenic and should be handled with extreme 
 care , using personal protective equipment in a well - ventilated hood 
 at all times .
compounds 3 and 4 were purchased from toronto research chemicals 
 ( toronto , on ) , and 
 [ pyridine - d4]ethyl nicotinate and [ c6]ethyl nicotinate were purchased from cambridge isotope laboratories 
 ( tewksbury , ma ) and moravek biochemicals ( brea , ca ) , respectively . 

all aqueous solutions were prepared using h2o purified from a 0.22 m millipore system ( billerica , ma ) . 

briefly , 
 [ pyridine - d4]ethyl nicotinate or [ c6]ethyl nicotinate ( 0.75 mmol ) was added to a suspension of nah ( 3.7 
 mmol , 60% dispersion in mineral oil ) in 5 ml of refluxing benzene . 
 after the addition of ethanol ( 0.5 ml ) , diethyl succinate ( 3.75 mmol ) 
 was added into the stirred refluxing mixture dropwise , and the reaction 
 was allowed to take place for 45 min .
the mixture was then cooled 
 in an ice bath , and 0.5 ml of 5 m hcl was added slowly with constant 
 stirring , which was continued for another 10 min after the addition 
 was completed .
the aqueous phase was extracted with benzene and , after 
 discarding the organic layer , adjusted to ph 9 by the addition of 
 nh4oh and extracted again with chloroform .
the chloroform 
 layer was then dried over na2so4 and concentrated 
 under diminished pressure to obtain the crude diethyl -nicotinyl 
 succinate as a brown oil .
this crude product was heated under reflux 
 in 2 ml of 5 m h2so4 for 36 h. after the solution 
 was cooled to room temperature , naoh was added to adjust the ph to 
 45 , and the precipitated crude [ d4]3 or [ c6]3 was collected for 
 further purification by hplc using a linear gradient from 100% 15 
 mm nh4oac to 20% 15 mm nh4oac and 80% ch3cn over 50 min , at 0.7 ml / min . a luna c18 column ( 250  
 4.6 mm , 5 m particle size , phenomenex ) was used , and the uv 
 detector was set at 254 nm .
the products were collected at 17 min , 
 as determined by the retention time of unlabeled 3 .
the 
 products were treated with nabh4 to produce [ d4]4 and [ c6]4 , which 
 were purified using the same hplc system and collected at 15 min , 
 based on the retention time of unlabeled 4 .
the solvent 
 in the collected hplc fractions was removed by rotary evaporation , 
 and the products were dried overnight under vacuum yielding [ d4]4 and [ c6]4 [ 0.6 mg of each ( 11% ) ] .
co - injection on hplc of each product with 4 gave only one peak , and the purity of each was determined 
 to be approximately 98% .
ms analysis ( 20 ev ) of each product in the 
 positive esi mode further confirmed their identity : [ d4]4 , m / z 186 [ m + h ] ( relative intensity 52% ) and m / z 168 ( 100% ) ; and [ c6]4 , m / z 188 [ m + h ] ( 63% ) and m / z 170 ( 100% ) .
briefly , 1.13 mg ( 6.10 mol ) 
 of [ d4]4 , synthesized as described above , 
 was incubated in 1 ml of 3% ( v / v ) concentrated h2so4 in meoh at room temperature overnight .
samples were 
 then loaded onto 5 ml chemelut cartridges ( agilent tech . ) , and the 
 [ d4]4 methyl ester was eluted with 25 ml of 
 ch2cl2 .
after the removal of solvent , 100 l 
 of 50 mg / ml dmap solution in toluene and 500 l of hexanoic 
 anhydride were added , and the second esterification reaction was allowed 
 to take place at 70 c for 20 min . after cooling to room temperature , 
 1.5 ml of hexane / etoac ( 90:10 ) was added , and the product was extracted 
 thrice with 1 ml of 1 n hcl .
the aqueous phase containing the product 
 was neutralized ( nahco3 ) , and the ester was extracted with 
 3  5 ml hexane / etoac ( 90:10 ) , concentrated , and purified by 
 preparative hplc , yielding [ d4]6 ( 27% ) 95% 
 pure , ms ( in the positive esi mode ) m / z 298 [ m + h ] ( 15% ) , m / z 182 ( 100% ) , and m / z 122 ( 27% ) ; h nmr ( cdcl3 , no tms added , 500 mhz ) :  0.88 
 ( 3h , t , j = 7.0 hz , 6-ch3 ) , 1.231.34 
 ( 4h , m , 4-ch2 , 5-ch2 ) , 1.61 
 ( 2h , m , 3-ch2 ) , 2.122.29 ( 2h , m , 3-ch2 ) , 2.312.40 ( 4h , m , 2-ch2 , 2-ch2 ) , 3.67 ( 3h , s , coo 
ch3 ) , 5.82 ( 1h , dd , j = 8.2 , 5.5 hz , 4-ch ) .
the same method 
 as that described for [ d4]6 was used for the 
 preparation of [ c6]6 ( 36% ) , 95% 
 pure , m / z 300 [ m + h ] ( 5% ) , m / z 184 ( 100% ) , and m / z 124 ( 82% ) . [ d4]6 and
smokers 
 urine 
 samples analyzed here were collected from subjects recruited for a 
 chemoprevention trial that will be described elsewhere .
the study 
 and the urine sample collection were approved by the university of 
 minnesota institutional review board ( study # 0712m22651 ) . as part 
 of the trial design ,
subjects were asked to stop smoking their usual 
 cigarettes and switch to the study cigarettes to which [ d4]1 was added .
the cigarettes were prepared as previously 
 described , and their use was approved by the us food and drug administration 
 ( ind 74037 ) .
subjects were asked to collect 
 a 24-h urine sample after a 7-day period of smoking cigarettes containing 
 [ d4]1 and bring the sample to the tobacco 
 research programs clinic at the university of minnesota ; these urine 
 samples were analyzed here .
study subjects kept urine at room temperature 
 during the 24-h collection and delivered it to the clinic the next 
 day . an experiment in which urine of a study subject was incubated 
 overnight at 37 c showed no degradation of hydroxy acid . in 
 the laboratory ,
subjects further followed the trial protocol , which will 
 be described elsewhere along with data on additional biomarkers including 
 creatinine .
additional samples collected by the subjects in the study 
 were not analyzed for this report .
five microliters of 0.1 ng/l [ c6]4 internal standard solution was added to 3 ml of urine , followed 
 by 0.6 ml of 1 m naoh solution and 0.3 ml of basic nabh4 solution ( 7.2 mg nabh4 in 0.3 ml of 0.1 m naoh ) .
the 
 sample was mixed by vortexing and allowed to stand for at least 2 
 h at room temperature to convert all [ d4]3 to [ d4]4 .
excess nabh4 was destroyed 
 by adding 1 m hcl dropwise , and the ph of the sample was adjusted 
 to 6.57.5 .
the volume was reduced to 1 ml ( speedvac ) , and 
 the sample was applied to a 200 mg/3 ml strata - x cartridge ( phenomenex ) 
 activated with 2 ml of meoh .
the cartridge was washed with 1 ml of 
 h2o ( which was shown not to remove the analyte ) , and [ d4]4 was eluted by applying another 5 ml of h2o to the cartridge .
on 
 the next day , 1 ml of freshly prepared 3% ( v / v ) concentrated h2so4 in meoh was added to the dry residue , followed 
 by sonication and vortexing to completely dissolve the sample .
the 
 mixture was allowed to stand at room temperature for at least 2 h 
 to convert [ d4]4 to its methyl ester .
then , 
 2 ml of 50 mg / ml nahco3 was added dropwise to adjust the 
 ph to 7.08.0 .
the sample was loaded onto a 5 ml chemelut cartridge 
 ( agilent tech . ) , and the [ d4]4 methyl ester 
 was eluted with 2  8 ml of ch2cl2 .
the 
 eluant was concentrated on a speedvac for 1.5 h , and 500 l 
 of ch2cl2 was immediately added to the dry residue . 

this was applied to a ch2cl2-activated bondelut 
 cartridge ( agilent tech . ) , which was washed with 1 ml of ch2cl2 and 1 ml of ch2cl2/etoac ( 50:50 ) 
 sequentially .
the [ d4]4 methyl ester was finally 
 eluted with 2 ml of 100% etoac and dried by speedvac .
immediately after drying , 10 l 
 of dmap ( 50 mg / ml in toluene ) and 50 l of hexanoic anhydride 
 were added .
after sonication and vortex mixing , the mixture was incubated 
 at 70 c for 20 min , producing [ d4]6 . 
 to extract the product into the aqueous phase ,
0.5 ml of hexane / etoac 
 ( 90:10 ) and 0.5 ml of 1 n hcl were added sequentially with thorough 
 vortex mixing ( 30 s ) after each addition .
the mixture was centrifuged 
 at 500 g for 2 min , and the aqueous phase containing 
 the analyte was separated .
this extraction was repeated once , and 
 the aqueous phases from both extractions were combined and applied 
 to an activated 60 mg oasis mcx cartridge ( waters corp . ) , which was 
 washed with 3 ml of 1 n hcl , 3 ml of meoh/1 n hcl ( 40:60 ) , and 3 ml 
 of h2o / meoh / nh4oh ( 80:15:5 ) , sequentially .
the 
 hexanoate [ d4]6 was then eluted with 10 ml 
 of h2o / meoh / nh4oh ( 30:65:5 ) and dried overnight 
 ( speedvac ) .
the residue was dissolved in 2  100 l of 
 ch3cn , filtered on a spin - x centrifuge tube filter ( 0.45 
 m , corning life sciences ) , and transferred to a 300 l 
 fused lc autosampler vial with insert .
the sample was finally dried 
 on a speedvac and kept frozen at 20 c until lc - esi - ms / ms 
 analysis .
the dried 
 sample was redissolved 
 in 12 l of h2o and analyzed on a tsq vantage system 
 ( thermo scientific ) interfaced with an agilent 1100 capillary hplc 
 system and an agilent 1100 micro autosampler .
a zorbax eclipse pah 
 column ( 150  0.5 mm , 3.5 m particle size ) was maintained 
 at 40 c throughout the separation .
the chromatography was performed 
 with a ch3cn and 15 mm nh4oac solvent system 
 at a flow rate of 10 l / min .
a linear gradient started with 
 60% aqueous phase and 40% ch3cn and changed to 44.3% aqueous 
 phase and 55.7% ch3cn over 22 min .
the gradient was then 
 ramped to 35% aqueous phase and 65% ch3cn over 3 min , and 
 returned to the initial condition in 2 min .
the mass spectrometer was operated in the positive 
 ion electrospray mode with selective reaction monitoring ( srm ) .
the 
 transitions m / z 298 [ m + h ]  m / z 122 [ m(ch3(ch2)4coo + cooch3 + h ) ] and m / z 298 [ m + h ]  m / z 182 [ m  ch3(ch2)4coo ] were used for 
 [ d4]6 , and the corresponding transitions m / z 300  m / z 124 and m / z 300  m / z 184 were used for [ c6]6 .
the collision energy ( c.e . ) was 30 ev for m / z 298  m / z 122 and m / z 300  m / z 124 transitions , and 20 ev for m / z 298 m / z 182 and m / z 300  m / z 184 transitions .
the heated capillary 
 temperature was 285 c , and the spray voltage was 3000 mv .
the purpose of 
 this study was to 
 develop a sensitive and reproducible assay for the analysis of [ d4]4 in the urine of smokers who smoked cigarettes 
 containing [ d4]1 .
overall , our method includes 
 the reduction of urinary [ d4]3 to [ d4]4 , followed by conversion of the highly polar [ d4]4 to a stable methyl ester hexanoate ( [ d4]6 , figure 1 ) , which can 
 be further purified and analyzed by lc - esi - ms / ms with excellent separation 
 efficiency and ionization selectivity .
carbons 
 that covalently bond to hydrogens are labeled to assign nmr peaks 
 for 6 .
the lc - esi - ms / ms conditions were developed using 
 standard aqueous 
 solutions containing [ d4]6 and [ c6]6 .
the product ion spectra of [ d4]6 were obtained by collision - induced dissociation of 
 the ion at m / z 298 [ m + h ] as shown in figure 2 .
we examined a range 
 of collision energy values to investigate the fragmentation pattern . 
 the protonated molecular ion ( m / z 298 ) that was observed at 10 ev ( figure 2a ) decreased at 20 ev ( figure 2b ) and was 
 barely observed at 30 ev ( figure 2c ) .
a base 
 peak with m / z 182 [ m  ch3(ch2)4coo ] was observed 
 at 20 ev and a base peak with m / z 122 [ m  ch3(ch2)4coo  
 cooch3  h ] was observed at 30 ev .
the 
 transition m / z 298  m / z 182 ( 20 ev ) was chosen for quantitation 
 because of the overall better peak shape and sensitivity both in nonsmokers 
 urine samples to which [ d4]4 was added and 
 in smokers urine .
the transition m / z 298  m / z 122 
 ( 30 ev ) was monitored to confirm the identity of the analyte in urine 
 samples .
the corresponding transitions for [ c6]6 are m / z 300  m / z 184 and m / z 300  m / z 124 .
product 
 scan of [ d4]6 at different collision 
 energy values used in the method development : a , 10 ev ; b , 20 ev ; 
 and c , 30 ev .
the calibration curve 
 for the quantitation of [ d4]6 was built by 
 preparing a series of standard mixes containing 
 varying ratios of [ d4]6 to [ c6]6 ( internal standard ) .
as can be seen from figure 3 , which shows the calibration curve constructed 
 from five standard mixes ( six measurements performed for each on different 
 days ) , excellent linearity was observed for the analyte in the range 
 of 0.84 fmol 84 fmol [ d4]6 .
calibration 
 curve for the quantitation of [ d4]6 by lc - esi - ms / ms . 

the amount of [ c6]6 ( internal 
 standard ) was maintained at 51.1 pmol on 
 column , while the amount of [ d4]6 ranged from 
 0.84 fmol to 84 fmol on column .
the y axis plots the peak area ratio from the corresponding 
 transitions , m / z 298  m / z 182 and m / z 300  m / z 184 . 
 each data point represents
the average of six measurements performed 
 on different days with the error bars indicating standard deviation .
to determine precision , accuracy , 
 recovery , and limit of detection of the assay , we used a nonsmoker s 
 urine to which a known amount of [ d4]4 was 
 added .
a typical lc - esi - ms / ms trace obtained in this analysis is shown 
 in figure 4a .
the intraday precision of the 
 assay was assessed by analyzing six aliquots of a nonsmoker s 
 urine mixed with [ d4]4 at 360 fmol / ml urine . 
 the measured mean level of [ d4]4 was 342  
 8 ( sd ) fmol / ml ( coefficient of variation ( cv ) = 2.1% ) . the interday 
 precision was determined by analyzing positive controls , aliquots 
 of a nonsmoker s urine mixed with [ d4]4 at 225 fmol / ml urine , with six sets of assays performed during method 
 characterization .
the concentration of [ d4]4 in positive controls averaged 226  10 ( sd ) fmol / ml ( cv = 4.6% ) . 

assay accuracy was determined by analyzing a nonsmoker s urine 
 to which increasing amounts of [ d4]4 were 
 added , ranging from 11.3 fmol / ml to 1.12 pmol / ml .
the accuracy of 
 the measurements ranged from 99.0% to 115.8% . as demonstrated in figure 5 , the added and measured levels of [ d4]4 were highly correlated ( r = 0.9983 ) .
this experiment was performed at the method development 
 step and was further included with each set of smokers urine 
 samples analyzed later in the study .
for the 20 sets of accuracy analyses 
 performed in total , the average accuracy was 100.1%  14.7% ( n = 100 ) , and the average recovery of the [ c6]4 internal standard was 32.1%  27.6% 

additional accuracy experiments with each 
 sample set allowed us to determine the precision of the assay at [ d4]4 levels that are lower than those used in the 
 initial precision test and in the positive controls . at 11.3 fmol
/ ml 
 [ d4]4 addition , which is below the lowest 
 level measured in smokers urine samples , the coefficient of 
 variation was 14.2% . at the next [ d4]4 addition 
 level , 56.6 fmol / ml urine ,
the coefficient of variation was 5.8% , 
 similar to that determined for the positive controls .
at a 3:1 signal - to - noise 
 ratio , the limit of detection ( lod ) was 10 fmol / ml in nonsmokers 
 urine to which [ d4]4 was added .
the limit 
 of quantitation ( loq ) at a 5:1 signal - to - noise ratio was 25 fmol / ml 
 and was established based on smokers urine analyzed by this 
 method .
typical lc - esi - ms / ms chromatograms obtained upon analysis of a , 
 a nonsmoker s urine ( 3 ml ) to which 168 fmol of [ d4]4 was added , and b , urine of a study subject who smoked 
 cigarettes containing [ d4]1 . relationship between added and measured levels of [ d4]4 in a nonsmoker s urine : method accuracy 
 analysis .
the method was applied to the analysis of [ d4]4 in the urine of 87 smokers who smoked at least 10 
 [ d4]1-containing cigarettes per day for at 
 least 1 week . during sample preparation , a negative control , a positive 
 control , and the accuracy test samples , all nonsmoker s urine 
 with or without [ d4]4 ,
recovery of [ c6]4 was similar in nonsmokers and 
 smokers urine , indicating that the high levels of unlabeled 4 that are present in smokers urine do not interfere 
 with the analyses .
small peaks with retention time and m / z values matching [ d4]4 were occasionally observed in the nonsmoker s negative control 
 samples and also in a pooled urine sample from smokers who did not 
 smoke [ d4]1 ; however , the level of this background 
 interference in all cases was below lod ( 1/3 lod ) . in smokers 
 who smoked [ d4]1-containing cigarettes , urinary 
 [ d4]4 concentrations ranged from 25 fmol / ml 
 to 390 fmol / ml urine , averaging 130 fmol / ml .
results for individual 
 urine samples are summarized in supporting information , table s1 . a typical trace from an analysis of a smoker s 

the distribution 
 of the measured [ d4]4 concentrations in these 
 urine samples is illustrated in figure 6 .
histogram 
 demonstrating the distribution of [ d4]4 levels 
 in 87 urine samples from study subjects who smoked 
 cigarettes containing [ d4]1 for 1 week . for 
 the levels in individual samples
evaluation of the metabolic activation of 
 the tobacco - specific 
 lung carcinogen nnk in individual smokers may potentially provide 
 important insights for understanding interindividual variation in 
 the risk of developing lung cancer due to exposure to tobacco products . 

the addition of [ d4]1 to special study cigarettes 
 followed by the measurement of urinary total [ d4]4 in people who smoke these cigarettes is a promising novel 
 approach that can be used for this purpose .
this approach requires 
 a reliable and sensitive methodology for the measurement of the deuterium - labeled 
 urinary biomarkers . in this study
, we developed a sensitive and highly 
 reproducible lc - esi - ms / ms method for the analysis of [ d4]4 in human urine .
the method is characterized by excellent 
 sensitivity , precision , accuracy , and recovery .
the original 
 method for the analysis of [ d4]4 that was 
 used in our previous study included the conversion of the 
 acid to its methyl ester to enable a purification step that can not 
 be applied without the esterification ; this was followed by hydrolysis 
 of the ester back to [ d4]4 , which was analyzed 
 by lc - esi - ms / ms .
the resulting chromatographic 
 traces were characterized by relatively high background noise and 
 variable recovery of the analyte . in this study , we modified our protocol 
 by including the second derivatization step after the conversion of 
 [ d4]4 to its methyl ester , thus producing 
 [ d4]6 , a stable methyl ester hexanoate .
this additional esterification decreases the 
 polarity of the resulting product , allowing for more efficient removal 
 of the multitude of polar interfering compounds present in the urine 
 matrix , which ultimately results in better chromatography .
furthermore , 
 the ionization efficiency in the esi source is improved due to both 
 decreased compound polarity and reduced matrix suppression .
the second 
 esterification also increases the molecular weight of the analyte 
 by 98 amu , which increases the selectivity in the ms detection since 
 most interfering compounds from the urine matrix have lower molecular 
 weights .
another important modification is that in the new protocol , 
 we applied the isotope dilution approach by using [ c6]4 as the internal standard , instead of 5-methylhydroxy 
 acid ( 5 , figure 1 ) which was used 
 in our previous assay .
this approach 
 ensures more accurate quantitation of the analyte as compared to the 
 use of a surrogate internal standard . as shown in figure 4 , the analysis of both the nonsmoker s urine 
 to which [ d4]4 was added and the urine of 
 a smoker who switched to [ d4]1-containing 
 cigarettes , produced clean chromatograms with well - resolved peaks 
 for [ d4]6 and [ c6]6 .
the concentration range of urinary total [ d4]4 obtained in the current study for the 87 subjects 
 who smoked [ d4]1-containing cigarettes for 
 1 week is lower 
 than that reported previously .
thus , in our previous study , the levels 
 of urinary total [ d4]4 ranged from 0.43 to 
 8.7 pmol / ml urine , averaging 2.8 pmol / ml in 20 smokers who smoked 
 cigarettes to which [ d4]1 was added at the 
 same level as in the present study . this 
 could be due to differences in the analytical procedures and internal 
 standards used in the two studies , as well as the differences in the 
 brand of study cigarettes used ( marlboro virginia blend in this study 
 vs quest cigarettes in the previous study ) , which may have affected 
 the resulting smoking rates ( 21 vs 28 cigarettes per day in the current 
 and the previous study , respectively ) , and other potential differences 
 between the two studies and/or subject characteristics .
we applied 
 our new method to reanalyze a small set of urine samples that were 
 available from the previous study and stored at 20 c 
 since its completion .
we selected samples that , according to our previous 
 analyses , contained high levels of [ d4]4 : 
 3.5 , 4.2 , and 8.8 pmol / ml urine .
reanalysis of these samples by the 
 new method produced 1.4 , 1.7 , and 3.1 pmol / ml [ d4]4 , respectively . while there are discrepancies in urinary 
 [ d4]4 levels measured by the two methods , 
 which could be due to a combination of the method differences and 
 the prolonged storage of the samples from the previous study ( at least 
 6 years ) , the levels determined by the new method in the urine collected 
 in the previous study are still higher than those measured in the 
 current study .
importantly , the method presented here has been thoroughly 
 characterized and shows a broad dynamic range of accurate [ d4]4 quantification , making it applicable in future studies .
nnk intake in smokers , nonsmokers exposed to secondhand smoke , 
 and in smokeless tobacco users has been extensively analyzed by measuring 
 urinary total nnal , the end - product of the nnk carbonyl reduction 
 pathway .
it was also shown that pyridine - n - oxidation is only a minor metabolic pathway in humans .
in contrast , the information on the extent of 
 nnk -hydroxylation in humans is extremely limited .
it has been 
 established that some of the adducts produced by pyridyloxobutylation 
 of dna or globin release 4-hydroxy-1-(3-pyridyl)-1-butanone ( hpb ) .
while several studies detected and quantified hpb - releasing dna 
 and globin adducts in humans , it is not clear how much of the 
 total measured level of these adducts is derived from nnk versus the 
 related tobacco carcinogen n-nitrosonornicotine , 
 which shares common metabolic pathways with nnk and leads to the formation 
 of the same adducts .
moreover , it is 
 not clear how dna or globin adduct levels can be compared to the urinary 
 products of nnk detoxification , in order to estimate the balance between 
 the two pathways in the same individual .
therefore , availability of 
 a specific urinary biomarker of nnk metabolic activation offers practical 
 advantages for investigation of the relative extent of nnk metabolic 
 activation and detoxification in humans .
our previous study developed 
 a unique approach that provided such a biomarker , and our current 
 report describes a sensitive and reproducible assay for the measurement 
 of this biomarker in future studies .
a limitation of this study 
 is that we did not quantify [ d4]3 and [ d4]4 separately .
however , 
 the sum of the keto and hydroxy acids is an informative aggregate 
 biomarker for the quantitation of the total efficiency of the nnk 
 and nnal -hydroxylation pathways , and is expected to be used 
 in future large - scale studies .
the separate analysis of [ d4]3 and [ d4]4 can be easily achieved 
 by measuring [ d4]4 in the same urine sample 
 without the nabh4 reduction and then subtracting the measured 
 amount from the total [ d4]4 determined by 
 the method described here . it is also 
 important to note that , because the levels of natural nnk in regular 
 cigarettes are higher than the levels of [ d4]1 added to study cigarettes , the amount of the nnk - derived hydroxy 
 acid in the urine of regular smokers may be higher than the levels 
 of [ d4]4 measured in people who smoke cigarettes 
 containing [ d4]1 .
however , the ultimate goal 
 of the [ d4]1 approach is not to provide absolute 
 quantification of nnk metabolic activation but to compare the relative 
 ratio of biomarkers reflecting metabolic activation and detoxification 
 pathways within an individual . in summary ,
we have developed 
 a sensitive and robust assay for 
 the quantitation of total [ d4]4 in smokers 
 urine .
the assay incorporates a two - step derivatization procedure 
 and lc - esi - ms / ms analysis to achieve excellent precision , accuracy , 
 recovery , and limit of detection .
analysis of smokers urine 
 indicated that levels of [ d4]4 as low as 26 
 fmol / ml can be quantified .
the results of this work further confirm 
 that [ d4]4 could be used as the urinary biomarker 
 to study nnk metabolic activation .
the broad dynamic range of this 
 assay will be very useful in large studies , especially those that 
 may deal with potentially lower efficiency of nnk metabolic activation 
 in smokers due to polymorphisms in nnk metabolizing genes or in chemoprevention 
 trials .
 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone 
 ( nnk , 1 ) is a potent tobacco - specific lung carcinogen 
 believed to play a 
 key role in the development of lung cancer in smokers . 
 metabolic activation 
 of nnk to dna damaging reactive intermediates proceeds via -hydroxylation 
 pathways . 
 the end products of these pathways are excreted in the urine 
 of smokers as 4-oxo-4-(3-pyridyl)butanoic acid ( keto acid , 3 ) and 4-hydroxy-4-(3-pyridyl)butanoic acid ( hydroxy acid , 4 ) . 
 the sum of these biomarkers ( after nabh4 treatment ) , 
 referred to as total hydroxy acid , could potentially be used to measure 
 the extent of nnk metabolic activation in smokers . 
 however , the same 
 metabolites are formed from nicotine ; therefore , there is a need to 
 distinguish the nnk- and nicotine - derived keto and hydroxy acid in 
 smokers urine . 
 we previously developed a unique methodology 
 based on the use of [ pyridine - d4]nnk ( [ d4]1 ) , which metabolizes to the correspondingly labeled biomarkers . 
 in this study , we developed a sensitive and reproducible assay for 
 the detection and quantitation of total [ pyridine - d4]hydroxy 
 acid ( [ d4]4 ) in human urine . 
 a two - step derivatization 
 approach was used to convert [ d4]4 to [ pyridine - d4]methyl 4-hexanoyl-4-(3-pyridyl)butanoate ( [ d4]6 ) , and an lc - esi - ms / ms method was developed for the analysis 
 of this derivative with excellent sensitivity , accuracy , and precision . 
 
 the robustness and reproducibility of the assay was further confirmed 
 by its application for the analysis of urine samples from 87 smokers 
 who smoked [ d4]1-containing cigarettes for 
 1 week . 
 the measured level averaged 130 fmol / ml urine . 
 the developed 
 assay can be used in future studies that may require evaluation of 
 the relative efficiency of nnk metabolic activation in humans . 
Introduction
Experimental Procedures
Results
Discussion