morphine and codeine are naturally occurring alkaloids in opioid plants , have long been used as a drug , and are also abused . while the presence of illicit drugs or their metabolites in urine is an evidence of intake , their concentrations in blood are expected to correlate with their effects on the central nervous system .
codeine is a potent -opioid receptor agonist which is used for the treatment of adult cough .
simultaneously , there have been athletes in sports competitions who use a larger dose in order to improve performance .
this practice is contrary to the principle of fair competition and also harmful to the health of the athletes ' body .
heroin as one of the most widely abused drug , rapidly metabolized to 6-monoacetylmorphine ( 6-mam ) once inside the human body .
this specific heroin metabolite 6-mam is detected at a higher concentration usually within 2 to 4 hours , and after six hours , has not been detected in the urine .
the absence of 6-mam in urine , however , morphine is both a well - known pharmaceutical agent and an important metabolite of codeine and heroin which have relatively long a detection time .
morphine and codeine analysis of urine is used in forensic toxicology to study drug addiction .
there are numerous papers published about the simultaneous determination of morphine and codeine in human fluids , including the micellar electrokinetic chromatography ( mekc ) method , disposable pipette extraction ( dpx ) method , high performance liquid chromatography method , liquid chromatography - mass spectrometry , and liquid chromatography / triple quadrupole tandem mass spectrometry ( lc / ms / ms ) method [ 68 ] .
several gas chromatography - mass spectrometry ( gc - ms ) methods have been developed for the analysis of codeine , morphine , or other opiates .
much attention has been directed to the confirmation of morphine and codeine in urine by gc - ms .
a few methods have been developed specifically for the analysis of 6-acetylmorphine ( 6-am ) with morphine and codeine because all three drugs are often present after heroin use .
assays of morphine and codeine by gc - ms are capable of high sensitivity , specificity , and selectivity .
gc - ms is superior to other analytical methods which provide important diagnostic value to study the drug abuse .
the aim of this study was to establish methods and seek out more reliable identification and quantitation of morphine and codeine for detection addicts sample .
although through in saliva is another approach ; the reliability of saliva analysis is limited by the fact that analyte levels , and even the availability of required sample volume , are again dependent on several physiological factors , nutrition and fluid intake , while the biological effects of the consumed illicit substance may also be a significant factor .
the identification of chronic consumers or the late verification of a single intake is feasible using hair as a matrix , but it is not suitable for the early verification of consumption .
urine is a preferable matrix for analytical purposes in comparison with saliva because of the minimal discomfort caused to sampled individuals , so it is widely available .
the simple and effective ethyl acetate extraction was employed in our work , and ethyl acetate was adopted because of its high extraction efficiency .
propionic anhydride was chosen as the derivatization reagent because it exhibited better effect than acetic anhydride or trifluoroacetic acid anhydride , which could provide preferable stability , and the disadvantage of acetyl derivatives indistinguishable from morphine and the 6-am can be avoided .
.   evaluated propionic anhydride , mbtfa , hfaa , and bstfa for gc - ms analysis of 6-am .
they concluded that propionic anhydride gave accurate , precise , and sensitive results while providing compatibility with other methods on the same gc - ms instrument .
residual derivatization reagent in the injector will react with drugs in other methods not intended for derivatization .
the derivatization procedure accommodates the analysis of opioids commonly requiring gc - ms confirmation in urine .
concentrations of the analytes in the samples were expected to be smaller than the low end of the therapeutic range ( 25  ng / ml ) , which highlighted the importance of efforts aimed at increasing the sensitivity of detection .
the relative standard deviation of the retention parameters of the target compound was required not to exceed 5% relative standard deviation .
morphine [ 10  g / ml in methanol ] and codeine [ 10  g / ml in methanol ] solutions were obtained from the institute of forensic science under the ministry of justice ( shanghai , china ) .
sodium hydroxide ( purity > 98.0% ) was purchased from sigma - aldrich trading co ( shanghai , p.r . ,
china ) , and ethyl acetate ( purity > 98.0% ) was purchased from siyou chemical reagent co. , ltd ( tianjin , china ) , and propionic acid anhydride ( purity > 98.0% ) was purchased from sinopharm chemical reagent co. , ltd ( beijing , china ) .
pyridine was from shenbo chemical co. , ltd ( shanghai , china ) . while methanol was obtained from siyou chemical reagent co. , ltd ( tianjin , china ) .
ultrapure water was prepared by a milli - q purification system from millipore ( bedford , usa ) .
analysis was performed on an agilent 6890n gas chromatograph ( gc ) coupled with an agilent 5975b mass spectrometer ( ms , agilent technologies , wilmington , de , usa ) .
the capillary column used was a hp-1ms [ 30  m  0.25  mm , 0.25  m ] .
helium was the carrier gas at a flow rate of 1.0  ml / min .
the temperature program was : initial temperature , 100c for 1.5  min ; ramp at 25c / min to 280c and held for 15  min ; injection temperature , 250c ; and transfer line , 280c .
electron impact ionization was performed at 70  ev energy and at a 230c ion source temperature .
sim mode was applied to quantify analyzes using target ions at m / z 341 , 397 , and 268 for morphine propionyl compound and m / z 229 , 355 , and 282 for codeine propionyl compound ( figure 1 ) .
the primary standard stock solutions of morphine ( 100  g / ml ) and codeine ( 100  g / ml ) were separately prepared in 10  ml volumetric flasks with urine ; 10% naoh was added dropwise until ph 9.09.2 was reached , and 1.0  ml of borax buffer solution was added . to this , 3  ml of extraction solvent ( ethyl acetate ) was added and vortex - mixed on a vortexer for 2.0  min , followed by centrifugation at 3000  r / min for 5 min .
the supernatant organic layer was transferred into a 5  ml glass test tube and dried under air stream at 60c .
the dried residue was reconstituted in 50  l of propionic anhydride and 20  l of pyridine .
all reagents were vortex - mixed , then heating for 3  min at 80c and dried under air stream at 60c . the dried residue was reconstituted in 50  l of methanol , and 1  l of this solution was injected into gc - ms .
specificity was determined by analysis of blank urine , without addition of morphine and codeine to determine possible interference with these compounds . to evaluate the linearity ,
the calibration curves were generated using the analyte peak area by linear regression on three consecutive days .
the lloq was estimated in the process of calibration curve construction and was defined as the lowest concentration for which precision ( rsd ) was better than 20% .
qc samples at three concentration levels ( 50 , 200 , and 1600  ng / ml for morphine and codeine ) were analyzed to assess the accuracy and precision of the method .
again , the assays were performed on three separate days , and on each day six replicates of the qc samples at each concentration level were analyzed .
the assay precision for each qc level was determined as the relative standard deviation ( rsd ) of the measured concentrations .
the intra- and interday precisions were required to be below 15% , and the accuracy was required to be within 15% .
stability in urine was assessed in the autosampler at room temperature for 12  h. the effect of three freeze - thaw cycles was also investigated .

 figure 2 shows the typical chromatograms of a blank urine sample spiked with morphine and codeine .
no interfering endogenous substances were observed at the retention times of the morphine and codeine .
calibration curves for morphine and codeine were generated by linear regression of peak area ratios against concentrations , respectively .
the regression equation for the calibration plot were y = 2270.9c + 202.3 with r = 0.9974 for morphine , and y = 3099.0c + 31625.7 with r = 0.9958 for codeine ( y is the peak area of analyte , and c is the concentration of analyte in human urine ) , and concentrations are in the range 252000  ng / ml for morphine and codeine , respectively .
the lloq for morphine in human urine was 25  ng / ml and the precision and accuracy at lloq were 10.5% and 87.6% , respectively .
the lloq for codeine in human urine was 25  ng / ml and the precision and accuracy at lloq were 13.8% and 88.9% , respectively .  
the precision of the method was determined by calculating rsd for qcs at three concentration levels over three validation days .
intraday precision was 12% or less and the interday precision was 13% or less at each qc level .
the aforementioned results demonstrate that the values are within the acceptable range and the method is accurate and precise .
the recovery of morphine and codeine was evaluated by comparing peak area ratios of extracted qc samples with those of reference qc solutions reconstituted in blank urine extracts .
all the stability studies of morphine and codeine in human urine were conducted at three concentration levels ( 50 , 200 , and 1600  ng / ml for morphine and codeine ) with three replicates for each concentration .
the stability results showed that morphine and codeine in human urine were stable during three freeze - thaw cycles .
stability of morphine and codeine extracts in the sample solvent on autosampler was also observed over a 12  h period .
a stable , selective , and sensitive gc - ms method has been developed for the simultaneous determination of codeine and its metabolite morphine in human urine .
this developed method with derivatization for sample preparation was successfully applied for the determination of morphine and codeine in human urine for methodological study .
 a sensitive and selective gas chromatography - mass spectrometry ( gc - ms ) method was developed and validated for the determination of morphine and codeine in human urine . 
 the gc - ms conditions were developed . 
 the analysis was carried out on a hp-1ms column ( 30  m  0.25  mm , 0.25  m ) with temperature programming , and helium was used as the carrier gas with a flow rate of 1.0  ml / min . 
 selected ion monitoring ( sim ) mode was used to quantify morphine and codeine . 
 the derivation solvent , temperature , and time were optimized . 
 a mixed solvent of propionic anhydride and pyridine ( 5  :  2 ) was finally used for the derivation at 80c for 3  min . 
 linear calibration curves were obtained in the concentration range of 252000.0  ng / ml , with a lower limit of quantification of 25  ng / ml . the intra- and interday precision ( rsd ) values were below 13% , and the accuracy was in the range 87.2108.5% . 
 this developed method was successfully used for the determination of morphine and codeine in human urine for forensic identification study . 