the united nation 's office on drugs and crimes ( unodc ) and the world health organisation ( who ) recently estimated that 149272 million people used psychoactive substances at least once in the past 12 months [ 1 , 2 ] .
the most commonly used substance was cannabis ( 129190 million people ) , followed by amphetamine type stimulants , opioids , and cocaine .
cannabis and cocaine analyses by gc - ms are two of the most frequently used drug assays [ 3 , 4 ] .
research in analytical sciences has shown a sustained effort to develop methods with improved sensitivity and to facilitate fast , reliable , and cost - effective methods to identify users .
the specific reasons behind the need for detection range from current risk to self and others , to future noncompliance .
for example , authorities often require evidence of abstinence from drugs before regranting driving licence [ 6 , 7 ] , allowing child custody , returning to workplace [ 9 , 10 ] , or licensing to practice . in these cases
the drug detection window is one of the main analytical challenges , since most drugs can only stay in our body system for shorts periods for example , the plasma elimination half - life of tetrahydrocannabinol ( thc ) and thccooh is ca .
other considerations include the accuracy , reproducibility , sample quantity required , and limits of detection / quantification for a developed method .
drug analysis in hair has become a very common part of forensic and clinical toxicology ( doping , work - place drug testing , rehabilitation programs , and treatment centres ) [ 3 , 13 , 14 ] .
hair testing for drugs of abuse is a developing technique that offers the possibility of a longer detection window than is commonly obtained from urine or blood analysis [ 15 , 16 ] and thus distinguishes between long - term use and short - term single exposure .
hair testing improves drug analysis by being noninvasive ; samples are easy to store at room temperature and there is a negligible risk of infection .
drugs incorporate into hair through several mechanisms , either endogenously , by ingestion ( via the blood during hair formation or through sweat and sebum ) , or exogenously , through external contamination ( deposition of drugs on the surface of the hair and passive inhalation ) . due to the extremely low incorporation rate of the psychoactive ingredient of cannabis , ( -thc ) , cocaine , and other drugs in hair ( especially in blonde , brown , and thin hair ) , the development of sensitive techniques is essential for quantitative analysis . the concentration of the drug in hair may reflect the amount of drug used ; that is , if the rate of consumption is high or low .
in addition , when a sample shows the absence of drug , it does not always mean that the sample is free from the drug , only that the concentration may be too low to be detected .
the interpretation of hair analysis involves finding the correct concentrations to distinguish between the common or occasional consumer .
most methods include decontamination , digestion , drug extraction , reconstitution , and derivatisation for sample preparation preceding hair analysis by gc - ms .   in this paper , we report lower limits of detection for -thc and cocaine for the first time without derivatisation .
this is an improvement on previously reported methods that achieved a lower limit of detection of 2.5  ng / mg for -thc but with derivatisation .
further method developments in hair digestion and extraction have also been made , thus permitting direct measurement of the drug without issues such as contamination from reagents , formation of byproducts , and reduction in recovery , chromatographic resolution , and ionisation efficiency that may arise from derivatisation .
delta-9-tetrahydrocannabinol ( -thc ) and cocaine ( 1  mg / ml ) and their deuterated analogues -thc - d3 and cocaine - d3 ( 100  g / ml ) were obtained from lgc standards ( teddington , uk ) .
proteinase k enzyme and hplc grade pentane were obtained from sigma aldrich ( dorset , uk ) , dtt cleland 's reagent , and tris hcl buffer were purchased from vwr , ( leicestershire , uk ) .
hexane , dichloromethane , and all the other organic solvents were hplc grade from fisher ltd .
hair samples ( 50  mg ) were collected from the posterior vertex [ 21 , 22 ] ( posterior cortex hair rarely varies during growth and the hair number in this area is more constant and less affected by age and sex differences ) and decontaminated by washing with 3  ml dichloromethane .
they were vortex mixed for 2  min ( this step repeated 3 times ) .
this cleans the hair from contaminants such as colouring , sebum , shampoo , and so forth , which may interfere with analysis . after decontamination
, the hair samples were pulverised to ca . 0.5  mm long segments by hand scissors .
the samples were screened for the presence of -thc or cocaine [ 21 , 22 ] . for enzymatic digestion , the pulverized hair
was added to proteinase k enzyme in a ratio of 1  mg hair : 1  mg enzyme ; followed by 100  mg of dtt cleland 's reagent and 1  ml of tris hcl buffer .
hair was digested with enzyme in the presence of -thc - d3 and cocaine - d3 ; a spiked concentration of 1  ng / mg was used as internal standard .
a comparative extraction was conducted using naoh digestion of spiked hair samples using 1  ml of 1  m naoh at 95c for 10  mins followed by neutralisation with 1  ml of 1  m hcl and 3  ml of phosphate buffer ( ph = 7 , 0.2  m ) .
the analytes were extracted by liquid - liquid extraction ( lle ) using 6  ml of hplc grade pentane .
after vortex mixing and centrifugation ( 10  min at 2383  g ) , the supernatant organic layer was transferred into a fresh glass tube using a pasteur pipette and the hair residue pellet was discarded .
the organic layer was mixed with a 25  l aliquot of 2  m hcl which had been diluted to 1% in phosphate buffer ( ph = 7 , 0.2  m ) to prevent drug loss during evaporation .
the organic layer was evaporated under a gentle stream of nitrogen gas at 50c using a hotplate concentrator techno db-3 ( cambridge , uk ) . spiked samples of 0.021.50  ng / mg
the extracted residue was reconstituted with 60  l hexane , transferred to autosampler vials , and 3  l was injected into the gc - ms system .
the extracts were analysed using an agilent technology 7890a gas chromatograph , in combination with an agilent 5975 xl ei / ci msd triple axis detector mass spectrometer connected to a 7683b autosampler ( agilent ltd . ,
ca , usa ) operating in electron impact ionisation ( ei ) mode using helium carrier gas with a flow rate of 1.3  ml / min .
the analytical column for gc was a bp - x5 sge forte capillary column ( victoria , australia ) ( 30  m length  0.25  m film thickness  0.25  mm internal diameter ) ( 5% phenyl polysilphenylene - siloxane ) .
pulsed , splitless injection was performed for a purge time of 1  min , a purge flow rate of 53  ml / min , and an initial pulse pressure of 20  psi reducing to 15  psi .
the gc oven temperature for -thc was programmed to start at 50c , held for 1  min , then increased to 200c at 40c / min , held for 2  min , and increased to 280c at 80c / min , held for 3  min , to a final step of 310c at 80c / min , held for 4  min .   for cocaine analysis ,
the initial temperature was 50c , held for 1  min , then increased to 200c at 100c / min , held for 2  min , then to 280c at 80c / min held for 3  min , to a final step of 310c at 80c / min , held for 4  min .
the quadrupole temperature was 150c ; electron multiplier voltage ( emv ) 2,200  v. the analysis was performed in selected ion monitoring mode ( sim ) .
the solvent delay time was 7  min ; elution window 713  min . the precursor and product ions of -thc , -thc - d3 internal standard ( is ) , cocaine , and cocaine - d3 ( is ) were , -thc , m / z 314 , 299 ; -thc - d3 , m / z 317 , 302 cocaine , m / z 303 , 182 ; cocaine - d3 , m / z 306 , 185 ( precursor ions , product ions ) .
the retention times were 7.9  min and 12.9  min , respectively , for cocaine and -thc . between samples
blank hair ( 50  mg ) was spiked with solutions of the analytes in methanol resulting in calibrator concentrations of 0.02 , 0.05 , 0.1 , 0.5 , 1 , and 1.5  ng / mg -thc and cocaine and standard curves were linear with r values of 0.995 and 0.997 , respectively .
the lower limit of detection ( llod ) , lower limit of quantification ( lloq ) , intraday precision , interday precision , accuracy , and recovery for each analyte were also calculated .
extraction recovery was determined by comparing the area ratio of -thc and cocaine extracted from 0.5  ng / mg spiked blank hair samples with the area ratio of standard neat solutions prepared in hexane of the same concentration .
linear calibration graphs ( -thc r = 0.995 ; cocaine 0.997 ) were established at concentrations ranging from 0.021.00  ng / mg for -thc and 0.051.00  ng / mg for cocaine .
the calibration curves were prepared by spiking known concentrations of -thc or cocaine to blank hair samples at 0.02 , 0.05 , 0.10 , 0.50 , 1.00 , and 1.50  ng / mg , with a constant amount of -thc - d3 and cocaine - d3 ( 1  ng / mg ) .
the calculated peak area from gc chromatograms for spiked concentrations was divided by the peak of the internal standard to determine the abundance ratio .
the gc chromatograms ( figures 1 and 2 ) show peaks representing -thc and cocaine eluting at rt = 12.9  0.2 and 7.9  0.2  mins , respectively .
results shown in figure 3 were obtained with the enzymatic and alkaline ( naoh ) digestions . for these tests
-thc ( 5  ng / ml ) was spiked into two blank hair samples and digested with proteinase k at 37c or by naoh at a temperature not greater than 95c . this process was repeated 6 times for each digestion method .
use of proteinase k resulted in an average concentration of 4.85  ng / ml  0.23 with 95% recovery , whilst the samples digested by naoh resulted in a mean of 3.15  ng / ml  0.1 and 62% recovery .
one of the explanations for the reduced extraction recovery when using naoh is the drug degradation caused by the strong basic naoh conditions and high temperature .
this enzymatic hydrolysis method is an improvement over other approaches , which could easily cause drug degradation in the presence of sodium hydroxide ( naoh ) , hydrochloric acid ( hcl ) and high temperatures , and thus improve the stability of the method and therefore accuracy .
detection was feasible for -thc concentrations as low as 0.015  ng / mg and for cocaine at 0.02  ng / mg and quantification was possible at the concentrations of 0.02  ng / mg and 0.05  ng / mg for -thc and cocaine respectively using a signal to noise ratio > 3 .
all qualitatively identified positives ( samples prescreened by elisa ; 9 positive for only -thc and 1 positive for both -thc and cocaine ) were confirmed and quantified by gc - ms .
ten hair samples were positive for -thc and one also for cocaine ( table 2 ) . using the abundance ratio , the actual drug concentrations ( ng / mg ) in the unknown hair samples ranged between 0.050.35  ng / mg hair for -thc . the only positive sample for cocaine was measured to be 0.1  ng / mg .
the number of publications describing analytical procedures relating to drug incorporation into hair , decontamination and analysis has increased in recent years [ 2 , 2326 ] , but in this paper an improved digestion and gc - ms method has been proposed which enhances drug analysis capabilities .
the sensitivity achieved for -thc ( lod 0.01  ng / mg  0.01 and loq 0.02  ng / mg  0.01 ) and for cocaine ( lod 0.02  ng / mg  0.015 & loq 0.05  ng / mg
 0.01 ) is better than previous reports which have been obtained from derivatised samples ( lod 0.0252.5  ng / mg , loq 0.057.5  ng / mg for -thc and lod 0.030.5  ng / mg , loq 0.051  ng / mg for cocaine ) which is higher than what we achieved [ 20 , 2730 ] .
negative results could also mean that the concentration of drug in hair is below the detection limit of the method .
an additional advantage of the increased sensitivity is that the analysis required a reduced amount of hair , thus making the method more feasible for drug testing .
although derivatisation is an important factor to improve sensitivity , it can be problematic in complex matrices .
derivatisation is sometimes time consuming , can add possible contamination to the sample mixture , and could result in a decrease in the sensitivity of the method .
the performance in terms of reliability , feasibility , and length of analysis can be improved by using enzymes . in this study ,
gc - ms has been performed on underivatised , informed positive hair samples for cocaine and -thc .
all the 10 samples in table 2 were positively confirmed ( by elisa ) for -thc ( 10 samples ) and 1 sample was found to be also positive for cocaine .
hair analysis has proved to be a useful method of public health research in the case of -thc and cocaine screening .
the method that has been developed is capable of detecting exceptionally low levels of -thc and cocaine in human hair when only ca .
enzymatic digestion and the given chromatographic and mass spectrometric conditions were essential for reproducible and accurate analysis of these psychotropic drugs in hair without any interference .
an additional advantage of this method is that unlike previously published work , it does not require derivatisation [ 17 , 20 , 23 , 24 ] .
thus , it is a convenient and potentially less problematic method which can be employed for routine drugs testing .
this method can complement conventional blood and urine analysis with the advantages of noninvasiveness of sample collection , negligible risk of infection ( blood analysis ) , facile sample storage ( small sample size , limited biohazard and adulteration / contamination risks ) , and negligible sample degradation . also , hair analysis could help prevent false negative elisa results that can be encountered from the higher limit of detection or even previously developed gc - ms methods by detecting ultra - low concentrations .
the limit of detection for elisa screening was found to be 0.5  ng / mg for the cocaine kit and 0.3  ng / mg for -thc .
 gas chromatography - mass spectrometric ( gc - ms ) methods for drug analysis routinely employ derivatising reagents . 
 the aim of this paper was to develop a method for the analysis of two recreational drugs , delta-9-tetrahydrocannabinol ( 9-thc ) and cocaine in hair samples using gc - ms , without prior derivatisation , thus allowing the sample to be reanalysed in its original form . 
 an enzymatic digestion technique was also developed . 
 ten hair samples , that were known positive for either 9-thc and/or cocaine , were enzymatically digested , extracted , and then analysed by gc - ms . 
 all samples measured contained 9-thc and one sample contained cocaine . 
 the limits of detection ( lod ) and quantification ( loq ) were 0.02  ng / mg and 0.05  ng / mg , respectively , for cocaine and 0.015  ng / mg and 0.02  ng / mg , respectively , for 9-thc . 
 the wide detection window , ease of direct analysis by gc - ms , lower detection limits of underivatised samples , and the stability of drugs using this technique may offer an improved method of analysis . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions