nicotine is a chemical that is present in the mount of about 1% of the weight of cigarette tobacco .
nicotine is the main active ingredient of tobacco and is also the main factor that leads to smoking addiction or dependence producing .
cotinine , a major degradation product of nicotine metabolism , has been an important recognized specific biomarker for evaluating cigarette smoke exposure .
urine specimens are commonly employed in biological monitoring because urine collection is noninvasive and poses minimal infectious disease risk to participants and researchers .
continuous and complete 24  h urine collection yields more accurate results , because spot urine sampling may not provide a valid overview of the entire toxicant exposure profile .
urine sample integrity and completeness is essential to exposure assessment research , and absence of compliance with the collection protocol is a fundamental concern to the researcher .
because creatinine is excreted in urine at a relatively constant rate through glomerular filtration , its measurement is an evaluation of sample integrity and completeness [ 6 , 7 ] . in addition
, urinary creatinine is commonly used in a ratio format to normalize analyte quantification for specimen concentration [ 8 , 9 ] .
the normalization process involves dividing the concentration of the analyte of interest by the creatinine concentration obtained in the same urine sample , with the result reported as the concentration of target analyte per millimol of creatinine .
recently , as a normalization basis , urinary creatinine was used to consider the excretion of a variety of xenobiotics related to smoking , ranging from cotinine to mercapturic acids .  
there are numerous papers published about the determination of creatinine in human fluids , including the jaffe method [ 11 , 12 ] , enzymatic method , flow injection analysis , high - performance liquid chromatography [ 1522 ] , capillary electrophoretic [ 2325 ] , zone electrophoresis , gas chromatography - mass spectrometry , or liquid chromatography combined with mass spectrometry ( lc - ms - ms ) [ 2830 ] .
recent determination of creatinine , delta - aminolevulinate , and tyrosine in biological fluids with a direct injection by lc - ms - ms was performed , and isotope dilution tandem mass spectrometry was used to assess the accuracy of creatinine determination in serum , plasma , or mouse plasma [ 28 , 29 , 3133 ] .
developed a rapid method for the analysis of creatinine in urine by solid - phase extraction ms - ms .
though a time - consuming solid - phase extraction was used for sample preparations , the selectivity of tandem mass spectrometry can not eliminate all interferences in urine .
another modified lc - ms - ms method was introduced by park et al . which allowed direct analysing creatinine in 24  h urine after diluted with methanol .
however , the analyte was eluted from the column at 0.59  min which can not be separated from the water dissolved urinary proteins and macromolecules ( retention time < 1  min on c18 column ) .
the methods based on the color reactions and enzymatic assay are confined by the lack of selectivity .
the round robin results revealed considerable and unsatisfying variations between laboratories and methods .   in order to eliminate the interferences from different instruments for urinary creatinine and use urinary creatinine to normalize smoking related biomarkers in human biological fluids ,
a sensitive and selective lc - ms - ms method for determining creatinine in urine was developed and verified with enzymatic colorimetric assay .
the data was applied to adjust cotinine values and was undertaken to explore whether any improvement occurred in the concordance with tar to relate it to tobacco exposure .
creatinine was obtained from the united states pharmacopeial convention ( rockville , md , usa ) .
creatinine - d3 ( n - methyl - d3 ; purity : 98% ; isotopic purity : 99% , toronto research chemicals inc . ,
primary stock solutions of creatinine and creatinine - d3 for the preparation of standard and quality control ( qc ) samples , were prepared by weighing separately . the primary stock solutions ( 0.21 and 0.1  mg / ml ) of the creatinine and creatinine - d3 ,
respectively , prepared in water and stored at 80c were found to be stable for three months ( data not shown ) .
appropriate dilutions were made in water to produce the working stock solutions of 100 , 1,000 , and 10,000  ng / ml for creatinine for the preparation of calibration curve .
calibrators ( 1 , 2 , 5 , 10 , 20 , 50 , 200 , 500 , and 2,000  ng / ml ) were freshly prepared by the addition of different aliquots of the working stock solution of the analytes and 25  ng / ml of creatinine - d3 to water .
quality control samples for creatinine at three different concentrations ( 50 , 200 , and 400  ng / ml ) were also prepared with human urine .
frozen urine samples were thawed to room temperature and mixed to suspend any settled precipitate .
a 10  l formic acid was added to 1  ml aliquot of human urine sample , stirred , and centrifuged at 10000  rpm for 10  min .
the mixture was filtered through a 0.22  m polyethersulfone membrane and a 5  l urine aliquot was transferred to an amber volumetric flask and brought to a total volume of 10  ml with water after being spiked with 100  l of creatinine - d3 internal standard solution ( 1  g / ml ) .
a 5  l aliquot was injected on - column for lc - ms - ms .
the enzymatic colorimetric method was performed in a hitachi modular automatic analyzer ( roche ) .
enzymatic method is based on the enzymatic degradation of creatinine and its reaction products by creatininase , creatinase , and sarcosine oxidase .
all samples were analyzed using an agilent 1200 liquid chromatograph ( agilent technologies , wilmington , de , usa ) coupled with an api 4000 triple quadruple mass spectrometer equipped with a turboionspray source ( applied biosystems , foster city , ca , usa ) .
esi was performed in the positive ion mode ( ionspray voltage 4500  v ) with nitrogen as nebulizing ( gas 1 ) , heater ( gas 2 ) , curtain , and collision gas .
gas flow parameters were optimized ( nebulizer 40  psi , heater 40  psi and curtain gas 30  psi ) by making successive flow injections while introducing mobile phase into the ionization source at 200  l / min . the declustering potential ( 73  v ) ,
entrance potential ( 10  ev ) , collision energy ( 29  v ) , and cell exit potential ( 8  v ) were optimized for creatinine by integrated springe pump at a constant flow rate of 10  l / min . the turbo ion spray temperature was set at 480c .
quantitative analysis was performed in the multiple reaction monitoring ( mrm ) mode with a dwell time of 100  ms .
an agilent zorbax eclipse xdb - c18 column ( 2.1  150  mm , 3.5  m particle size , agilent technologies , wilmington , de , usa ) was used with a flow rate of 200  l / min at ambient temperature .
isocratic separation was performed with 50% solvent a ( 0.1% formic acid in water ) and 50% solvent b ( 0.1% formic acid in acetonitrile ) .
solvents were filtered through a 0.45  m membrane and degassed by a vacuum before use .
aliquots ( 5  l ) of the standard or diluted urine samples containing internal standard were injected onto the lc - ms - ms system .
total urinary cotinine among smokers and nonsmokers was analyzed according to a previously published lc - ms - ms method .
several performance parameters were tested to validate the proposed method according to food and drug administration ( fda ) guidelines for bioanalytical methods . these were linearity of calibration plots , goodness of fit of calibration plots to the linear regression model , specificity , selectivity , thawing stability , recovery , matrix effects , and precision .  
246 24  h - urine samples from 82 smokers in three separate days and 57 blank 24  h - urine samples of nonsmokers were obtained at baseline from ongoing studies ( urinary biomarkers related to smoke exposure ) in the institute of clinical pharmacology of zhengzhou university .
during the initial course of method development and validation , several different lc columns and relevant solvent systems were evaluated for the best chromatographic separations of analytes from background interference .
poor peak shapes were observed on most columns , except those with xdb c18 column .
the initial mobile phase was chosen as water and methonol ( v / v ) , while poor peak shapes were also observed . in order to get good peak shapes and separation , solvent a ( 0.1% ammonium acetate in water ) and solvent
the detection parameters of ms were optimized using a syringe pump at a flow rate of 10  l / min .
an esi mass spectrum of creatinine was shown in figure 1 . under the conditions of esi , the protonated molecules ( [ m + h ] ) of creatinine and creatinine - d3
collision - induced dissociation of both compounds yielded one major fragment ion at m / z 86 for creatinine and m / z 89 for creatinine - d3 , respectively , corresponding to the neutral loss of co [ m + h  co ] . at the same time , the fragment ion [ m + h  co]could yield another main product ion at m / z 44 for creatinine and m / z 47 for creatinine - d3 ( figure 1 ) . for each analyte
these ion pairs are 114/86 and 114/44 for creatinine for the confirmation and quantification and 117/47 for creatinine - d3 .
no significant interfering peaks from endogenous compounds were observed at the retention times of creatinine and creatinine - d3 .
the retention time of creatinine and creatinine - d3 was 1.4 and 1.3  min , respectively .
a typical mrm chromatogram of creatinine - d3 and creatinine dissolved in water was presented ( a1 ) , ( a2 ) , and ( a3 ) in figure 2 .
both compounds were detected in a diluted urine sample ( ( b1 ) , ( b2 ) , and ( b3 ) in figure 2 ) .  
urine matrix underwent a thaw / refreeze cycle during each validation experiment , accumulating six such cycles by the end of the validation .
the average values over the six validation experiments for the concentrations of creatinine were 10.09  0.31  mmol / l ( cv = 2.1% ) .
the cvs were of similar magnitude to interday cvs , indicating that fast thawing of urine in chilled water did not influence the concentration of analytes .
the effect of urine constituents over the ionization of analytes and is was determined by comparing the responses of the postextracted urine standard qc samples ( n = 6 ) with the response of analytes from neat samples at equivalent concentrations .
matrix effect was determined at same concentration of analyte and is as in recovery experiment .
reported that pretreatment of urine with solid - phase extraction was not a necessary step for urinary creatinine measurement and that simple dilution of urine without pretreatment provided high selectivity for creatinine . on account of this , a simple dilution with water and methanol was applied in this study . as shown in figure 3 , urine samples dilution with water could get clear chromatograms of creatinine .
the signal - to - noise ratio in urine diluted with water is higher than with methanol for the narrow creatinine chromatogram peek and low noise .
creatinine was easily detected in all urinary specimens and it also could be better analyzed when the urine samples were diluted to 2000-fold in our assay .
in addition , applying a diluted urine sample to the lc - ms - ms could reduce the impact of the matrix effects and thus allow more samples to be processed before cleaning .
analytical recovery rates were obtained by spiking a nonsmoker pool urine sample with three concentrations of creatinine ( 50 , 200 , and 400  ng / ml ) .
recoveries ranged from 98.6 to 106.0% ( n = 6 ) ( table 1 ) . the peak - area ratios of creatinine to is were plotted versus creatinine concentration to construct calibration curves .
the calibration curve created using creatinine dissolved in water was linear ( y = 0.0106x + 0.00615 , r = 0.9995 ) in the analytical range from 1 to 2000  ng / ml .  
lod and loq were determined based on the instrument response with the integrated function of the analyst 1.5 software ( applied biosystems ) .
these calculations were based on signal / noise ratios of 3 and 10 for lod and loq , respectively .
the corresponding concentrations were calculated from the ratio to the internal standard area on the calibration curve .
the lod and loq for creatinine dissolved in water were 0.30 and 0.99  ng / ml . the intraday precision ( rsd ) of the methods was established by replicate analyses ( n = 10 ) of samples containing low , medium , and high concentration of creatinine .
the interday precision ( rsd ) was established by replicate analyses of the same samples on 10 separate days .
intra - day and interday precisions determined were 1.01.8% and 1.52.9% , respectively ( table 2 ) .
published hplc method , the presence of protein in the injected samples can cause modification of the column end in biased analytical results . thus , extensive sample cleanups including liquid - liquid ( l - l ) extraction and spe were needed to get low sample matrix effects and good hplc separation for the target compound . however ,
solid - phase extraction was not a necessary step for urinary creatinine measurement and that simple dilution of the urine sample without pretreatment provided high selectivity for creatinine . in this experiment ,
a simple dilution with water was used after acid precipitation , centrifugation , and filtration .
the creatinine concentration could be well determined and the matrix effect could be extraordinaire light after diluting 2000-fold with water .
compared with published lcms / ms methods , the new methods required fewer samples ( 5  l compared to 50  l urine ) thanks to the lower limit of detection ( 0.3  ng / ml compared to 22.8  ng / ml , 1  ng / ml , 6  ng / ml , and 3.7  ng / ml ) .
in addition , with the lower limit of detection , this method can be used to measure creatinine concentrations as low as 1  ng / ml .   to check the effectiveness of lc - ms - ms , 28 24  h - urine samples ( 16 smokers and 12 nonsmokers ) were measured by using the lc - ms - ms with a simple one - step dilution and the enzymatic colorimetric method ( for details for enzymatic colorimetric method see table 1 in supplementary material available at doi:10.1155/2012/245415 ) for the same set of urine samples run .
the creatinine values measured by the colorimetric and lc - ms - ms methods were positively associated ( pearson r = 0.984 , r = 0.968 , p < 0.0001 , figure 4 ) ( for original data see table  1 in supplementary material ) .
however , lc - ms - ms has advantage of low detection limits and high selectivity compared with enzymatic colorimetric method .
data from the same instrument could provide more accurate and stable results for creatinine normalization technique .
cotinine , a major metabolite of nicotine , is the most appropriate parameter to evaluate tobacco exposure and smoking status due to its higher stability and half life when compared to nicotine [ 9 , 39 ] .
urinary creatinine and cotinine concentrations were determined in 24  h - urine samples ( n = 246 ) from 82 smokers and 24  h - urine samples ( n = 57 ) from 57 nonsmokers ( lc - ms - ms method for the determination of cotinine see supplementary material ) .
the normalization process involves dividing the concentration of cotinine by the creatinine concentration obtained in the same urine sample , and the result was expressed as the concentration of cotinine per millimol of creatinine . the difference between creatinine normalized and nonnormalized cotinine values and the correlation of total cotinine in 24  h - urine and cotinine creatinine ratio was evaluated .
the normalized cotinine values are statistically significantly correlated with nonnormalized concentration ( pearson r = 0.858 , p < 0.0001 ) .
total cotinine in 24  h - urine and cotinine creatinine ratio were also positively associated (
pearson r = 0.942 , p < 0.0001 ) ( original data see tables  25 in supplementary materia ) . 24  h urine cotinine as a means of assessing exposure to xenobiotics is considered the  gold standard ,  which presumably represents the best information on urinary cotinine excretion .
24  h urinary cotinine was positively correlated with tar as in figure 5(a ) ( table 3 ) .
adjusting cotinine values was undertaken to explore whether any improvement occurred in the concordance with tar , and the result was showed in figure 5(b ) ( table 3 ) .
it is obvious that the corresponding bubbles in figure 5(a ) were concentrated more than that in figure 5(b ) , except 8  mg  :  10  mg . there was no use in improving the concordance , except 8  mg  :  10  mg ( 0.716 versus 0.722 ) .
a simple and specific method was developed for the determination of urinary creatinine by the lc - ms - ms .
the sample preparation only involves centrifugation and filtration of diluted urine , which not only allows a high sample throughput but also reduces creatinine background noise and urine salts concentration .
in addition , urinary creatinine was used in a ratio format to normalized cotinine concentration .
the normalized cotinine values are statistically significantly correlated with total cotinine in 24  h - urine and cotinine creatinine ratio were also positively associated . because cotinine  :  creatinine ratio varied significantly across smoking groups for the difference of individual , 24  h - urinary cotinine was more appropriate for expressing correlation with tar than cotinine  :  creatinine ratio .
 a simple and sensitive high performance liquid chromatography - tandem mass spectrometry ( hplc - esi - ms - ms ) method was developed and validated for the quantification of creatinine in human urine . 
 the analysis was carried out on an agilent zorbax eclipse xdb - c18 column ( 2.1  150  mm , 3.5  m ) . 
 the mobile phase was 0.1% formic acid in water and 0.1% formic acid in acetonitrile ( 50/50 , v / v ) . 
 linear calibration curves were obtained in the concentration range of 12000.0  ng / ml , with a lower limit of quantification of 0.99  ng / ml . the intra- and interday precision ( rsd ) values were below 3% . 
 the method was successfully applied to a bioequivalence study of creatinine in chinese smokers and nonsmokers . 
 the total cotinine in 24  h urine and cotinine  :  creatinine ratio were also positively associated ( pearson r = 0.942 , p < 0.0001 ) 
 . however , cotinine  :  creatinine ratio varied significantly across smoking groups for the difference of individual . 
 24  h urinary cotinine was more appropriate for expressing correlation with tar than cotinine  :  creatinine ratio . 
1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusions