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Clinical Chemistry 53: 300-309, 2007. First published December 7, 2006; 10.1373/clinchem.2006.074237
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(Clinical Chemistry. 2007;53:300-309.)
© 2007 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Drug Screening of Preserved Oral Fluid by Liquid Chromatography–Tandem Mass Spectrometry

Elisabeth Leere Øiestada, Unni Johansen and Asbjorg Solberg Christophersen

Norwegian Institute of Public Health, Division of Forensic Toxicology and Drug Abuse, Oslo, Norway.

aAddress correspondence to this author at: Norwegian Institute of Public Health, Division of Forensic Toxicology and Drug Abuse, P.O. Box 4404 Nydalen, NO-0403 Oslo, Norway. Fax 47-23383233; e-mail elisabeth.oiestad{at}fhi.no.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Background: Oral fluid is an alternative matrix with potential applications in road-side drug screening, work-place testing, drug treatment programs, and epidemiological surveys. Development of methods for extensive drug screening in oral fluid is warranted.

Methods: We developed a liquid chromatography– tandem mass spectrometry (LC-MS/MS) method for drug screening of preserved oral fluid collected with the Intercept® collection device. Samples were prepared by liquid–liquid extraction with ethylacetate/heptane (4:1). LC-separation was achieved with an Atlantis dC18-column (2.1 x 50 mm, 3 µm particle). Mass detection was performed by positive ion mode electrospray LC-MS/MS and included the following drugs/metabolites: morphine, 6-monoacetylmorphine, codeine, buprenorphine, methadone, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, cocaine, benzoylecgonine, {Delta}-9-tetrahydrocannabinol, lysergic acid diethylamide, alprazolam, bromazepam, clonazepam, 7-aminoclonazepam, diazepam, N-desmethyldiazepam, 3-OH-diazepam, fenazepam, flunitrazepam, 7-aminoflunitrazepam, lorazepam, nitrazepam, 7-aminonitrazepam, oxazepam, zopiclone, zolpidem, carisoprodol, and meprobamat.

Results: Screening of 32 drugs was performed with a run time of 14 min. Within- and between-day relative CVs varied from 2.0% to 31.8% and from 3.6% to 39.1%, respectively. Extraction recoveries were >50% except for morphine (30%) and benzoylecgonine (0.2%). The concentrations of the lowest calibrator were 1 nmol/L (0.28 µg/L) to 500 nmol/L (68 µg/L), depending on the drug.

Conclusion: The method allowed rapid and sensitive oral fluid screening for the most commonly abused drugs in Norway and will be used for a road-side survey of drug use in normal traffic.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Oral fluid has become an important alternative to blood and urine as a matrix for drug analysis (1)(2)(3)(4). In contrast to blood samples, oral fluid samples can be collected in a simple, noninvasive manner by nonmedical personnel. Oral fluid samples can be collected under close supervision to prevent substitution or adulteration, which can be a problem with urine sampling. The collection of oral fluid for specific analysis has been an important tool for the evaluation of on-site drug testing devices used for screening for suspected drug use while driving [e.g., the European Union Roadside Testing Assessment (ROSITA)1 project, http://www.rosita.org].

Opiates, amphetamines, cannabis, and cocaine are readily detectable in oral fluid, with pharmacokinetics similar to plasma (5). Other papers have described the detection of opiates (6)(7)(8)(9), amphetamines (10)(11)(12), cocaine(13)(14)(15), and cannabis (16)(17)(18) in oral fluid. Although illicit drugs have been the main focus of oral fluid analysis, prescription drugs such as benzodiazepines should be included because of their frequent misuse (19)(20). In Norway, benzodiazepines are among the most frequently detected drugs in blood samples from suspected drug-impaired drivers (20), often in combination with illegal drugs, psychoactive compounds, or alcohol. Sensitive detection methods are required for benzodiazepines because of their low concentrations in oral fluid (5)(21). Methods for determination of a number of common benzodiazepines in oral fluid have been described (19)(22)(23).

Passage of a substance from the blood to saliva is dependent on the pH of the blood and oral fluid, protein binding, rate of oral fluid flow, pKa, and the molecular weight of the substance (24)(25). Flow rate and pH depend on the degree of oral fluid stimulation during collection. Recovery and drug stability also depend on the manner in which the sample has been collected (23)(26), e.g., with different collection devices or expectoration. Standardization of sample collection is therefore important. Often the amount of material available after oral fluid collection with a standard sampling device is limited. Multicomponent methods are therefore advantageous, and an increasing number of such methods have been described (27)(28)(29)(30)(31)(32).

Liquid chromatography–tandem mass spectrometry (LC-MS/MS) is increasingly being used in forensic toxicology for the identification and quantification of a wide range of compounds in biological samples (33). Easier sample preparation, no required derivatization, and short analysis time are the major advantages, and an LC-MS/MS method for opiates, cocaine, and some benzodiazepines has recently been shown to be a viable replacement for immunoassay drug screening of oral fluid in an addiction clinic setting, although this method did not test for tetrahydrocannabinol (THC) or amphetamines (34). Another comprehensive LC-MS/MS method for the investigation of drugs in drivers has been described, but this method did not include THC or low-dose benzodiazepines (35).

We describe a rapid and sensitive LC-MS/MS method for oral fluid screening of illegal and medicinal drugs important to traffic safety. The Intercept® collection device used in this method was selected as the reference collection device by the European Commission Roadside Testing Assessment project (ROSITA II; http://www.rosita.org).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
chemicals and reagents
We obtained reference compounds from multiple pharmaceutical companies: 7-aminonitrazepam, 7-aminoflunitrazepam, methamphetamine, benzoylecgonine, 3,4-methylenedioxyamphetamine (MDA), lysergic acid diethylamide (LSD), and {Delta}-9-THC from Cerilliant Corp; 7-aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, alprazolam, lorazepam, 6-monoacetylmorphine (6-MAM), N-desmethyldiazepam, and 3-hydroxydiazepam from Lipomed; alprazolam, bromazepam, 3-hydroxydiazepam, oxazepam, amphetamine, cocaine, benzoylecgonine, codeine, meprobamat, and carisoprodol from Sigma-Aldrich; oxazepam, 7-aminoclonazepam, N-desmethyldiazepam, clonazepam, nitrazepam, flunitrazepam, MDA, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDEA), 6-MAM, and THC from Alltech; buprenorphine and diazepam from RBI; morphine and methadone from NMD; zolpidem from Synthélabo Groupe; zopiclone from Council of Europe; and fenazepam from Chiron. We purchased the internal standards morphine-d3, amphetamine-d11, metamphetamine-d11, benzoylecgonine-d8, MDMA-d5, 7-aminoflunitrazepam-d7, N-desmethyldiazepam-d5, and methadone-d9 from Cerilliant Corp. and THC-d6 from High Standards Products Corp. Standard compounds were stored according to supplier recommendations (solid substances mainly at room temperature, ampules at 4 °C). Intercept collection devices and Negative Calibrator Oral Fluid were purchased from Orasure Technologies Inc., and Flag Blue Liquid Food Color was obtained from Chef’s Classic. HPLC-grade acetonitrile and methanol were purchased from Lab-scan Ltd.; analytical grade n-heptane, ammonium acetate, ethanol absolute and acetic acid, extra pure ammonia 32%, and HPLC-grade ethyl acetate from Merck; and ammonium carbonate from BDH Laboratory Supplies.

solutions
We prepared stock solutions in methanol, with the exception of zopiclon and zolpidem, which were dissolved in acetonitrile, and THC, which was dissolved in ethanol. Calibrator and QC solutions were prepared by appropriate dilution of stock solutions with water. The stock and aqueous solutions were stored at –20 °C and 4 °C, respectively. During initial method validation, we observed that peak heights for THC decreased substantially over time compared with freshly made standards, a finding that was interpreted as a problem with stability, and THC was therefore prepared in ethanol:water 1:2 and not combined with the rest of the compounds. The Intercept collection devices contain salts and preservatives. A solution with the same contents as the sample sets, Negative Calibrator Oral Fluid, was therefore purchased from Orasure Technologies Inc. Because the sample sets contained a blue dye that was not present in the Negative Calibrator Oral Fluid, the same blue dye was added (100 µL per 100 mL) to mimic the content of the samples. The resulting blue solution, hereafter referred to as zero calibrant solution, was used to prepare calibrators and QC samples. The sampling sets contained 0.8 mL of preservative buffer, and the expected volume of oral fluid was ~0.4 mL, giving a 2:1 dilution that was compensated for in the preparation of calibrators and QC samples. The internal standard solution was diluted with water to yield final concentrations in oral fluid, adjusted for dilution, of 0.015–0.89 µmol/L.

We prepared a 50 mmol/L stock solution of ammonium acetate, adjusted to pH 5 with acetic acid. From this stock a 5 mmol/L buffer solution was prepared by a 1:10 dilution with water. A 0.2 mol/L ammonium carbonate buffer, adjusted to pH 9.3 with ammonia, was also used in the assay.

sample collection and pretreatment
We collected oral fluid samples according to the instructions from Intercept (http://www.4intercept.com/procedure). The device consists of a collector pad on a plastic handle and a vial that contains 0.8 mL of stabilizing buffer solution. The collector pad is treated with sodium chloride, citric acid, sodium benzoate, potassium sorbate, gelatin, sodium hydroxide, and deionized water. The vial contains chlorhexidine digluconate, Flag Blue dye, Tween 20 (nonionic surfactant), and deionized water. The collector pad is wiped between gum and cheek to stimulate saliva production and placed in the supplied vial after a 2-min sampling time. The volume of oral fluid obtained with the Intercept device can vary from 0.05 to 0.7 mL, with an expected mean value of 0.4 mL. According to the manufacturer, results are valid as long as analysis is performed within 21 days.

We stored the collected samples at 4 °C before processing. The collection devices were weighed, and the contents of the Intercept sampling sets were transferred to 15-mL polypropylene tubes (Greiner Bio-One GMbH) after centrifugation at 1400g for 15 min. We then transferred 0.5-mL aliquots of the preserved oral fluid to separate 5-mL polypropylene tubes (Sarstedt AG & Co.) and stored them at 4 °C until the time of analysis. Surplus preserved oral fluid remaining in the 15-mL polypropylene tubes was stored at –20 °C.

extraction procedure
We mixed 0.5 mL of the calibrator, QC sample, or oral fluid sample with 50 µL of internal standard solution and 250 µL of 0.2 mol/L ammonium carbonate buffer. The concentrations in the internal standard solution were 0.018 µmol/L 7-aminoflunitrazepam-d7, 3.0 µmol/L amphetamine-d11, 0.31 µmol/L benzoylecgonine-d8, 0.77 µmol/L MDMA-d5, 0.85 µmol/L metamphetamine-d11, 0.27 µmol/L methadone-d9, 0.51 µmol/L morphine-d3, 0.045 µmol/L N-desmethyldiazepam-d5, and 0.05 µmol/ L THC-d6. The samples were extracted with 1.3 mL of ethylacetate:heptane (4:1) by mixing for 10 min. After centrifugation at 1400g for 5 min, the organic phase was transferred to total recovery vials (Waters) and evaporated to dryness under N2 at 40 °C (Zymark Turbovap). The residue was then dissolved in 60 µL of acetonitrile/water (10:90 v:v).

hplc conditions
We used a Waters Alliance 2695 system for LC. Separation was performed with a Waters Atlantis dC18 (2.1 x 50 mm, 3.5 µm) column, with gradient elution at a flow rate of 0.3 mL/min with 100% acetonitrile (mobile phase A) and 5 mmol/L aqueous ammonium acetate, pH 5 (mobile phase B; Table 1 ). The precolumn volume was set to 0.45 mL, and the column temperature held at 35 °C. The injection volume was 10 µL.


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  		Table 1. Gradient table.1

ms/ms
A Waters Quattro Ultima Pt tandem mass spectrometer, equipped with a Z-spray electrospray interface, was used for all analyses. Positive ionization was performed in the multiple reaction monitoring (MRM) mode, with one transition for each compound. The capillary voltage was set to 1.0 kV, the source block temperature was 120 °C, and the desolvation gas (nitrogen) was heated to 400 °C and delivered at a flow rate of 500 L/h. The cone gas (nitrogen) was set to 50 L/h, and the collision gas (argon) pressure was maintained at 0.5 psi. The appropriate MRM transitions, cone voltages, and collision energies for the individual analytes were determined by direct infusion into the mass spectrometer. The MRM transitions with the corresponding scan segment, cone voltage, and collision energy for the measurement of the analytes and the internal standards are shown in Supplemental Data Table 1Up . System operation and data acquisition were controlled using Mass Lynx 4.0 software. Analytes were identified by comparing the retention times of the respective MRM transitions with the retention times of the corresponding calibrators and QC samples. Data were processed with the QuanLynx program, using peak height for quantification.

method validation
The 5-point calibration curves (3 replicates of each standard) were based on peak-height ratios of the analyte relative to the corresponding internal standard. The concentration ranges for the calibrator solutions shown in Table 2 correspond to concentrations in oral fluid. The prepared solutions were one third of this concentration, to correct for dilution by the preservative liquid in the collection devices. The extraction recovery (Table 2 ) was determined with 10 replicates at 3 concentrations (low, medium, and high). We estimated extraction recovery by comparing peak heights obtained when the analytes were added before extraction and internal standards were added after with peak heights obtained when both the analytes and internal standards were added after the extraction step. Within-day precision was estimated by analysis of separate preparations of QC samples at 3 concentrations in a single assay (n = 10). Between-day precision was determined by analysis of preparations of 3 replicates of each QC concentration on 6 different days. Recovery was calculated in terms of bias as the percent deviation of the measured mean from the corresponding theoretical concentration. Drugs were added to the zero calibrant buffer solution at concentrations down to one tenth of the lowest calibrator and analyzed in 6 replicates to determine the limit of quantification (LOQ), which was defined as a mean signal-to-noise ratio of 10. For compounds for which the LOQ was less than one tenth of the lowest calibrator, the LOQ was referred to as less than the tested concentration in Table 2 , and no further experiments were performed to determine the exact LOQ.


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  		Table 2. Calibration range, correlation coefficient, LOQ, extraction recovery, within-day precision, between-day precision, and bias.1


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  		Table 2A. Continued

specificity
To investigate the specificity of the method, we fortified zero calibrant solution with high concentrations of selected prescription drug and extracted the samples as described earlier for the calibrators. The drugs tested were antidepressants, analgesics, antipsychotics, and other compounds commonly evaluated in forensic samples at our laboratory. A listing of these drugs and the concentrations tested is provided in Supplemental Data Table 2Up Up .

matrix effects
Matrix effects (MEs) were evaluated by the method proposed by Matuszewski et al. (36). The analyte signal in the fortified mobile phase was compared with the analyte signal in the matrix fortified after extraction, and the ME was defined as ME% = (extracted matrix height/mobile phase height) x 100. Five replicates of mobile phase and 10 replicates of oral fluid extracts were analyzed. Because our method used zero calibrant solution for the preparation of calibrators, 5 replicates of extracted zero calibrant solution were also fortified and used for comparison.

collection device recovery
We evaluated the possibility of loss of sample due to adsorption to the collection device. Aliquots of pooled oral fluid, obtained by expectoration, were put in polypropylene tubes and divided into 2 sets. In set I, the oral fluid was fortified with the analytes before placing the collection pad in the test tube. Set II was prepared with oral fluid without added analytes, and the analytes were added to the recovered preserved oral fluid after centrifugation. Analysis was performed with 4 replicates of each set.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
method validation
Calibration curves were made for each compound in the concentration range listed in Table 2Up Up . A weighted (1/x) 2nd-order regression line, which included the origin, was applied for each compound, and the resulting correlation coefficients are listed in Table 2Up Up . The ion chromatograms of the analytes and internal standards from the lowest calibrator are shown in Fig. 1 . The within-day precision, between-day precision, recovery, extraction recoveries, and LOQs for the analytes are presented in Table 2Up Up . The between-day CVs were 3.6%–39.1%, and the within-day CVs were 2.0%–31.8%.


Figure 1
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  		Figure 1. Ion chromatograms from the lowest concentration calibrator.

The extraction recoveries were sufficient for screening purposes. The compounds with recoveries <50%, i.e., morphine and benzoylecgonine, were analyzed with deuterated analogs as internal standards to partly compensate for the low recovery. Relative SDs were <10% for morphine (Table 2Up Up ). For benzoylecgonine, results must be viewed with caution because of the extremely low recovery (0.2%), and a high CV of ≤39%, primarily in connection with a positive cocaine finding. For both compounds, however, the required sensitivity was achieved.

specificity
Of the 47 substances tested for interfering peaks (Supplemental Data Table 2Up Up ), only 1 gave a possible false positive. High promethazine, at a tested concentration equaling 4.3 mg/L in oral fluid, was found to give a response for diazepam. Because the identity of diazepam also includes an added confirmation, due to simultaneous analysis of the metabolite N-desmethyldiazepam, this interference can be recognized. For cases with positive diazepam and no visible trace of N-desmethyldiazepam, the result should be viewed as a possible false positive and be reanalyzed with another method.

MES
A large ME was seen for several compounds (Table 3 ). Because the calibrators were prepared in zero calibrant solution, the ME relative to this solution was also investigated. A large improvement was seen for diazepam, whereas for buprenorphine, methadone, and morphine, the matrix effect worsened significantly compared with zero calibrant solution instead of the mobile phase. A substantial relative matrix effect was also observed for several compounds. The use of deuterated internal standards can partly overcome the problem of matrix effects. Morphine, methadone, and THC all have their own deuterated analogs as internal standard. Inclusion of a deuterated internal standard for buprenorphine and one of the nitrobenzodiazepines should be considered.


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  		Table 3. Evaluation of matrix effects.

The biologic variation in drug concentrations in saliva can be substantial. For example, the individual ratio between saliva and plasma (varied between 1.1 and 17.4 in individuals given a low dose of codeine in a controlled study (37). Oral fluid pH can also lead to large variations: saliva/plasma ratio variations from 273 at pH 5 to 0.44 at pH 7.8 have been reported for cocaine (25). Considering this inherent variation, the measured variations due to the matrix effect are acceptable for screening purposes.

collection device recovery
We observed a reduced concentration for several substances when we compared the results from analytes recovered from the collection devices (set I) with those fortified after centrifugation (set II), indicating adsorption by the device (Supplemental Data Table 3Up ). The problem of adsorption of THC to collection devices has been reported for the Salivette® collection device (18). In our study a >50% loss of THC added to the Intercept device was observed. In addition, 7-aminoclonazepam, buprenorphine, LSD, methadone, and zolpidem had recoveries <70%. Previously described results (32) found less adsorption (recovery >80%) for buprenorphine, methadone, and zolpidem, and similar results for THC (recovery 40%–50%); LSD and 7-aminoclonazepam were not evaluated.

According to the manufacturer, the volume of oral fluid obtained with the Intercept device can vary from 0.05 to 0.7 mL. Because the collected oral fluid is diluted in the preservative liquid, a sample will be available for analysis even when the original oral fluid volume is very small, which is advantageous. Addition of 0.4 mL of oral fluid from 8 different individuals to collection devices, performed in our laboratory, gave a mean value of 1.0 g of preserved oral fluid recovered, with a relative SD of 1.7%. A test of the weight of 41 unused collection devices gave a relative SD of 0.9%. Other authors, however, have reported that the volume of preservative liquid varied from device to device (19), making it difficult to evaluate the volume of collected oral fluid. Our procedure does include weighing, because at least some correction for very low or high volumes of oral fluid is necessary.

applicability of the method for road-side testing
Large interindividual differences in the measured saliva/plasma ratio have been reported (5)(24)(25). In addition, possible uncertainties related to the sampling process (26), including the amount of sample collected, analyte stability, dilution by and possible adsorption to the collection device, and differences in analyte concentrations for some compounds depending on pH, call for caution in quantitative evaluation of measured oral fluid concentrations. Nevertheless, good qualitative predictions of positive serum results from positive oral fluid concentrations, and to some extent correlation with symptoms of impairment, have previously been demonstrated for cannabis, cocaine and metabolites, opiates, and amphetamine and derivatives (28)(29)(38)(39).

A test using a preliminary version of our method was performed on 33 real samples from the ROSITA II project. Volunteer participants provided oral fluid samples. The ethics board was informed and had no objection to the study. In this test 95 positive whole blood results for specific compounds had corresponding positive oral fluid results, and only 4 positive whole blood results showed negative oral fluid results (1 clonazepam sample, 1 flunitrazepam sample, and 2 THC samples). The flunitrazepam sample had positive results for the metabolite 7-aminoflunitrazepam in both whole blood and oral fluid. In addition, 24 oral fluid positive results had corresponding negative whole blood results. Closer inspection of the data revealed that this discrepancy was largely attributable to a higher concentration and lower cutoff for oral fluid than whole blood. Chromatograms from a real sample are shown in Fig. 2 . The measured concentration ranges in oral fluid (in µmol/L) were as follows: amphetamine, 0.26–243 (n = 12); metamphetamine, 0.24–84 (n = 8); MDMA, 0.15–13 (n = 5); MDA, 0.27–3.2 (n = 4); morphine, 0.040–11 (n = 12); codeine, 0.024–20 (n = 12); cocaine, 0.015–0.020 (n = 3); benzoylecgonine, 0.028 (n = 1); methadone, 0.83–0.85 (n = 2); diazepam, 0.0040–0.080 (n = 6); N-desmethyldiazepam, 0.010–0.15 (n = 4); 3-OH-diazepam, 0.010 (n = 1); oxazepam, 0.010 (n = 1); alprazolam, 0.010–0.20 (n = 5); zopiclone, 0.010–0.24 (n = 6); clonazepam, 0.0022–0.74 (n = 4); 7-aminoclonazepam, 0.0080–1.1 (n = 4); flunitrazepam, 0.0040–1.6 (n = 5); 7-aminoflunitrazepam, 0.0010–0.61 (n = 6); nitrazepam, 0.0030–0.012 (n = 4); and 7-aminonitrazepam, 0.0050–0.13 (n = 3). MDA was not routinely analyzed in blood, but 4 samples were positive in oral fluid (range, 0.27–3.2 µmol/L).


Figure 2
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  		Figure 2. Positive components from a real sample.

Measured concentrations (in µmol/L): morphine, 0.4; amphetamine, 4.6; codeine, 0.09; 6-MAM, 0.01; 7-aminoflunitrazepam, 0.004; cocaine, 0.008 (below cutoff); alprazolam, 0.2; THC, 0.02.

Differentiation between heroin and legal prescription opiate use can be readily determined from oral fluid testing because of high concentrations of the heroin metabolite 6-MAM in oral fluid (40). Because of the low concentrations of 6-MAM in whole blood, confirmations are usually performed at our institution with urine specimens. Of the 11 6-MAM positive oral fluid samples in our study, 8 were confirmed in urine. Measured values in oral fluid were 0.0099–0.91 µmol/L.

In conclusion, the presented method can be used to analyze a large number of drugs of abuse, including low-dose benzodiazepines, with easy sample preparation, good sensitivity, and short run time, thus facilitating high-throughput screening of road-side samples. If quantification of positive results is needed, samples should be reanalyzed with analytical conditions designated for the specific drug or drug group, using 2 MRM-transitions.


   Acknowledgments
 
We thank Inge Frydenlund, Oslo Police District, for collection of oral fluid samples during the ROSITA project and Åse Marit Leere Øiestad, Solfrid Hegstad, Jean-Paul Bernard, and Jørg Mørland for advice and critical reading of the manuscript.


   Footnotes
 
1 Nonstandard abbreviations: ROSITA, European Union Roadside Testing Assessment; LC-MS/MS, liquid chromatography–tandem mass spectrometry; THC, tetrahydrocannabinol; MDA, 3,4-methylenedioxyamphetamine; LSD, lysergic acid diethylamide; 6-MAM, 6-monoacetylmorphine; MDMA, 3,4-methylenedioxymethamphetamine; MDEA, 3,4-methylenedioxyethylamphetamine; ME, matrix effect; MRM, multiple reaction monitoring.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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