Cocaine Samples Research Articles

Cocaine classification using alkaloid and residual solvent profiling

Many different groups of chemical compounds can be used in statistical impurity-profile comparison in order to establish links between different seizures of illicit drugs. For cocaine, some of these compounds descent directly from the coca leaf while others are remnants from the manufacturing process; and each of the compound groups exhibit different degrees of stability and discrimination power. Information obtained from the different groups can be handled in numerous ways and selecting the right method using a balanced combination of the compound groups is highly important in order to provide investigators and courtrooms with accurate conclusions.By using logistic regression or discriminant analysis (linear and quadratic), cocaine alkaloid and residual solvent distances can be combined in order to obtain probabilities for the two possibilities: linked or unlinked. We examined different data transformations and distance methods and ranked the different models using cross validation. Validation in an unrelated data set proved the consistency of the results. Data consisted of five large groups of linked samples exposed to different storage conditions during 12 months, 124 different cocaine sample seizures and 15 smaller groups of linked samples stored at room temperature for up to 15 months. The alkaloid and residual solvent impurity profiles of the samples were analysed using gas chromatography–mass spectrometry (GC–MS) and headspace GC–MS, respectively.Residual solvent profiles exhibited considerable higher discrimination power than cocaine alkaloid profiles. Thus, a residual-solvent-weighted model (log10 transformation and cosine distance) was found superior at distinguishing correctly between linked and unlinked seizures compared to models using alkaloid distance alone. The model only gives weight to the residual solvents when the alkaloid profiles are very similar. This finding demonstrates the possibility to combine information from the highly stable, non-coca leaf-descent residual solvent profiles and the less stable cocaine alkaloid profiles for statistical comparative analysis of cocaine seizures in a simple and easy-to-implement way.

The impact of compliance with a compulsory model of drug diversion on treatment engagement and reoffending

Aim: Over a 10-year period from 2003, around 1.7 million arrests in England and Wales resulted in the suspect being exposed to mandatory drug testing and assessment processes. These provisions formed part of a wider drug interventions programme costing £1.3 billion. This study sought to assess the impact of compliance with these measures on treatment uptake and reoffending. Recidivism risk factors were also investigated. Methods: The use of linked administrative data relating to matched samples of recent heroin and/or cocaine (H/C) users identified in one English police force area over a 12-month period (N = 1630). Findings: There was no association between compliance with a compulsory model of drug diversion and subsequent engagement with structured treatment services, rates of treatment retention and successful discharge. Compliance was also not found to be associated with reductions in the rate and volume of reoffending after 12 months. The factor with the largest effect on risk of recidivism was poly use of H/C. Main offence, engagement with structured treatment, number of prior convictions and (younger) age were also identified as recidivism risk factors. Conclusions: These results are discussed in the context of subsequent legislation and policy which further expands the reach of mandatory testing and assessment measures as a form of constraints-based drug diversion.

Drugged Drivers Blood Concentrations in England and Wales Prior to the Introduction of Per Se Limits.

This report details the blood concentration of drugs found in motorists suspected of driving under the influence of drugs from 2010 to 2012 in England and Wales. This study was carried out as new legislation has come into place, setting fixed blood concentration limits for drugs in motorists. These include a cannabis (Δ9-THC) blood concentration of 2 µg/L, amphetamine 250 µg/L, benzoylecgonine (BZE) 50 µg/L, cocaine 10 µg/L,6-monoacetylmorphine 5 µg/L, morphine 80 µg/L, diazepam 550 µg/L and methadone 500 µg/L. Samples were screened for opiates, methadone, benzodiazepines, cannabinoids, cocaine, amphetamines and methamphetamine. Cannabinoids were the most prevalent drug group (29.7%) followed by benzodiazepines (22.7%), opiates (18.8%), cocaine (16.3%), amphetamine (7%) and methadone (5.6%). The analytical results are compared with the new per se limits to give a reference of drug concentrations prior to this legislation coming into effect. Our studies show that 64.9% of the cannabis samples, 59.1% of the cocaine samples and 94.6% of the BZE samples would be above the new per se limits set under Section 5a of the Road Traffic Act. In contrast, the medicinal drugs such as benzodiazepines and opiates (morphine) were predominantly detected at concentrations below the new per se limit. Given its medical applications, amphetamines appear to have been grouped with the medicinal type drugs, with our data showing that 25.2% of the amphetamine positive samples would exceed the new specified limit. These data show that samples containing medicinal and prescription drugs are likely to be detected below the new legal limits, while illicit drugs were typically found at concentrations above the new specified limits.

Single injection quantification of cocaine using multiple isotopically labeled internal standards

The purity of street cocaine is an important parameter in drug research and for forensic purposes, as it can be used to group samples, determining their origin, and to assess the monetary value of a drug sample. Current methods require batch processing of samples, as calibration curves need to be run. In this paper, a method is presented for the quantification of cocaine by single injection, avoiding the need for a calibration curve by using multiple, differently isotopically labeled internal standards. Using this fast method, results can be reported immediately after analysis and fully open-access mass spectrometric analysis becomes possible. The method was fully validated, with recoveries compared to certified standards of 99–101 % and to an accredited method from an independent laboratory of 86–117 %. Precision was tested both interday and intraday on three levels and all relative standard deviations were lower than 6.1 %. A linear response was found down to a purity of 1.3 %. The total analysis time for a single sample was approximately 30 min. The method was applied to 106 cocaine samples collected from a large UK music festival. Cocaine purity ranged from 1.3 to 78.8 %, with a mean of 43.1 %. This was comparable to other studies of UK cocaine samples. Our new approach has the potential to be applied to simple quantification of a variety of analytes in biological and non-biological samples.

The persistence of illicit drug smoke residues and their recovery from common household surfaces.

Third-hand smoke is the residue remaining on surfaces during smoking events. It is composed of particles and vapours that form upon heating. The phrase 'third-hand smoke' is primarily used to describe nicotine and other chemicals from cigarettes, but any residues formed from the smoking of various substances could be classified similarly. There has been an increasing body of research on third-hand smoke from cigarettes in the last decade, but little has been done in regards to understanding the persistence of particles and vapours from illicit drugs. In this work, small samples of cocaine and methamphetamine were volatilized to produce an illicit drug smoke that was collected onto various surface materials and left exposed to ambient conditions over 672 h (four weeks). Chemical analyses by electrospray ionization-mass spectrometry of residues on silicon, plastic, laminate, and artificial leather surfaces indicated a rapid decrease in recovery of the parent molecule, with varied formation of decomposition products over the first 168 h of exposure. Measurable amounts of the parent molecule were still present after 672 h, exhibiting a strong persistence of these drugs on various household materials. This is important in a forensic science context, as third-hand smoke residues could provide a viable source of trace evidence previously not utilized. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Crack cocaine addiction, early life stress and accelerated cellular aging among women

BackgroundEarly life stress (ELS) and addiction are related to age-related diseases and telomere shortening. However, the role of telomere length (TL) in crack cocaine addiction remains unknown. The purpose of this study was to investigate the TL in a sample of crack cocaine dependent-women who reported an ELS history and in a community-based sample of elderly women as a reference group for senescence. MethodsThis study included treatment seeking crack cocaine dependents women (n=127) and elderly women without a psychiatric diagnosis (ELD, n=49). The crack cocaine sample was divided in two groups according to their Childhood Trauma Questionnaire (CTQ) scores: presence of history of childhood abuse and neglect (CRACK-ELS) and absence of ELS history (CRACK). TL was assessed by T/S ratio obtained from peripheral blood DNA using quantitative PCR assay. ResultsCRACK and CRACK-ELS subjects exhibited shortened TL in comparison to the ELD group, despite their younger age. Among crack cocaine sample, CRACK-ELS group had significantly shorter telomeres than the CRACK group. Correlation analysis within crack cocaine group indicated that TL was negatively correlated with emotional abuse scores. ConclusionsThese results support previous findings associating telomere shortening with both ELS and drug addiction. This study suggests new evidence of a distinct biological phenotype for drug-dependent women with ELS. The results support the biological senescence hypothesis underpinning ELS experience.

A case story, involving the use of maltitol, a sugar alcohol, as a cutting agent in amphetamine and cocaine powders

Abstract In a criminal case involving cutting and resale of amphetamine and cocaine in the Copenhagen area of Denmark, maltitol was used as a cutting agent. The analysis of maltitol in seizures of pure diluents as well as in amphetamine and cocaine powders was carried out using reversed-phase high performance liquid chromatography (HPLC) with high-resolution (HR) mass spectrometric detection. Maltitol was identified in four out of nine amphetamine samples and in five out of six cocaine samples from the case in question. The use of maltitol as a cutting agent was considered by the police as a specific marker of the particular criminal group under investigation. To support or reject this hypothesis, cocaine and amphetamine samples from a four month period after the involved persons had been arrested were evaluated, also as part of the police investigation. None of these samples contained maltitol. The work described covers the part of the case involving the department of forensic chemistry, and not the whole police investigation, but everything was done within the frames given by the police. To the best of our knowledge, this is the first report of a disaccharide polyol being used as a cutting agent for illicit drugs.

Results of an international drug testing service for cryptomarket users

IntroductionUser surveys indicate that expectations of higher drug purity are a key reason for cryptomarket use. In 2014–2015, Spain's NGO Energy Control conducted a 1-year pilot project to provide a testing service to cryptomarket drug users using the Transnational European Drug Information (TEDI) guidelines. In this paper, we present content and purity data from the trial. Methods219 samples were analyzed by gas chromatography associated with mass spectrometry (GC/MS). Users were asked to report what substance they allegedly purchased. Results40 different advertised substances were reported, although 77.6% were common recreational drugs (cocaine, MDMA, amphetamines, LSD, ketamine, cannabis). In 200 samples (91.3%), the main result of analysis matched the advertised substance. Where the advertised compound was detected, purity levels (m±SD) were: cocaine 71.6±19.4%; MDMA (crystal) 88.3±1.4%; MDMA (pills) 133.3±38.4mg; Amphetamine (speed) 51.3±33.9%; LSD 123.6±40.5μg; Cannabis resin THC: 16.5±7.5% CBD: 3.4±1.5%; Ketamine 71.3±38.4%. 39.8% of cocaine samples contained the adulterant levamisole (11.6±8%). No adulterants were found in MDMA and LSD samples. DiscussionThe largest collection of test results from drug samples delivered from cryptomarkets are reported in this study. Most substances contained the advertised ingredient and most samples were of high purity. The representativeness of these results is unknown.

Simple and Sensitive Paper-Based Device Coupling Electrochemical Sample Pretreatment and Colorimetric Detection.

We report the development of a simple, portable, low-cost, high-throughput visual colorimetric paper-based analytical device for the detection of procaine in seized cocaine samples. The interference of most common cutting agents found in cocaine samples was verified, and a novel electrochemical approach was used for sample pretreatment in order to increase the selectivity. Under the optimized experimental conditions, a linear analytical curve was obtained for procaine concentrations ranging from 5 to 60 μmol L(-1), with a detection limit of 0.9 μmol L(-1). The accuracy of the proposed method was evaluated using seized cocaine samples and an addition and recovery protocol.

Analysis of street cocaine samples in nasal fluid by Raman spectroscopy

The principal objective of this work was to demonstrate the capability of Raman spectroscopy to detect small amounts of cocaine in nasal fluid, and to identify the main drug and the most widely used cutting agents. Initially, standard samples were analysed and sampling conditions were studied by comparing different swabs used for the sample collection. Once the most appropriate swab was selected, which permitted a relatively simple detection of the standard cocaine hydrochloride, qualitative analyses of real samples were carried out. Three street cocaine samples were analysed, and the presence of cutting substances was highlighted by the appearance of different bands not corresponding to the ones of the standard cocaine. To identify the substances present in each sample, the spectra of the street cocaine samples were collected and compared with a digital library created on purpose with the spectra of the most common cutting agents. In this case, correlation coefficients permitted to recognize the most important substances presumably present in the samples, and gave an estimation of the purity of the cocaine. However, when nasal fluid was present, its strong signal could overlap or interfere with the smaller signal of the cutting substances, hindering their identification.

Scott test evaluation by multivariate image analysis in cocaine samples

The Scott test is a preliminary colorimetric method to analyze cocaine. A blue color result in the final step denotes a positive indication for cocaine; however, some pharmacological products may lead to false positives when concentration is higher than 1mg. In order to eliminate these false positives, digital images derived from the Scott test for pure cocaine, adulterants, diluents, and their binary and ternary mixtures were acquired with a commercial scanner, and the histogram of such images were assessed through Principal Component Analysis (PCA), Partial Least Squares Discriminant Analysis (PLS-DA) and Support Vector Machine Discriminant Analysis (SVM-DA). PCA scores indicated a clear separation between a true positive and a false positive group, with exception for the higher concentrations of the adulterants assessed and their mixtures. In addition, we applied two supervised techniques, PLS-DA and SVM-DA, to the histograms of the digital images derived from the Scott test aimed at categorizing samples into true positive and true negative classes. PLS-DA yielded highly satisfactory results as only two samples were not correctly classified; the SVM-DA correctly classified all samples, yielding sensitivity and specificity equal to 1.

Combined Effects of Simultaneous Exposure to Caffeine and Cocaine in the Mouse Striatum.

Caffeine is the world's most popular psychoactive drug and is also an active adulterant found in many drugs of abuse, including seized cocaine samples. Despite several studies which examine the effects of caffeine or cocaine administered as single agents, little data are available for these agents when given in combination. The purpose of the present study was to determine if combined intake of both psychostimulants can lead to maladaptive changes in striatal function. Mice were injected with abinge regimen (intermittent treatment for 13days) of caffeine (3×5mg/kg), cocaine (3×10mg/kg), or combined administration. We found that chronic caffeine potentiated locomotion induced by cocaine and that both caffeine-treated groups showed sensitization. Striatal tissue was obtained 24h and 7days after last injection (withdrawal) for immunohistochemistry and mRNA expression. Our results show that combined intake of both psychostimulants can increase GFAP immunoreactivity in the striatum at both times post treatment. Gene expression analysis, targeted at dopamine, adenosine, and glutamate receptor subunit genes, revealed significant transcript down-regulation in the dorsal striatum of AMPA, NMDA, D1 and D2 receptor subunit mRNA expression in the group that received combined treatment, but not after individual administration. At withdrawal, we found increased D1 receptor mRNA expression along with increased A1, AMPA, NMDA, and metabotropic subunit expression. A2A mRNA showed decreased expression after both times in all experimental groups. Our study provides evidence that there are striatal alterations mediated by combined caffeine and cocaine administration, and highlights negative outcomes of chronic intake of both psychostimulants.

Levamisole-adulterated cocaine: Two fatal case reports and evaluation of possible cocaine toxicity potentiation

Levamisole has been identified as a cocaine adulterant in the United States since 2002. Although there is a variation in the percentage of levamisole in cocaine samples between European countries, measurement of levamisole in human samples of cocaine users has become increasingly important. To our best knowledge, only five deaths are reported (one twice) as a result of complications secondary to levamisole-tainted cocaine and none of these cases reports the post-mortem levamisole concentration. In this article, we present the post-mortem levamisole concentrations in fluids and tissues in two young cocaine users, dead after levamisole-adulterated cocaine intake. With the dearth of levamisole reported concentrations in literature, this particular report is of interest to the forensic toxicological and pathological communities.This article aims to be a supplementary alert to aware the risk that may occur using levamisole-adulterated cocaine and an incentive to publication of toxicity reports and new researches involving the combination of levamisole and cocaine.

Electrochemical fingerprint of street samples for fast on-site screening of cocaine in seized drug powders.

We report on a wearable fingertip sensor for on-the-spot identification of cocaine and its cutting agents in street samples. Traditionally, on-site screening is performed by means of colour tests which are difficult to interpret and lack selectivity. By presenting the distinct voltammetric response of cocaine, cutting agents, binary mixtures of cocaine and street samples in solution and powder street samples, we were able to elucidate the electrochemical fingerprint of all these compounds. The new electrochemical concept holds considerable promise as an on-site screening method.

A survey of adulterants used to cut cocaine in samples seized in the Espírito Santo State by GC–MS allied to chemometric tools

Cocaine is a stimulant drug of the central nervous system (CNS) extracted from the leaves of Erytroxylum coca. It is defined as a tropane alkaloid containing 1R-(exo,exo)-3-(benzoyloxy)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid methyl esther. However, despite its defined composition, a wide variety of chemical additives are present in cocaine found in the illicit market, such as benzocaine, lidocaine, caffeine, procaine and phenacetin. In this work, 512 cocaine samples seized by the Civil Police of Espirito Santo state (PC-ES, Brazil) were analyzed by gas chromatography mass spectrometry (GC–MS) allied to principal component analysis (PCA) in order to classify the samples as a function of seizure year (2008, 2009, 2010, 2011 and 2012) and location (metropolitan, north, south and central). The cocaine content (wt.%) and its adulterants were also estimated. Analyzing the samples seized between 2008 and 2011, three sample sets are clearly grouped according to the degree of adulteration with caffeine and lidocaine: 100–50wt.% of cocaine; 50–20wt.% of cocaine; and 20–80wt.% of lidocaine and 60–80wt.% of caffeine, simultaneously. The last group is formed by samples seized between 2008 and 2009, which proves the higher degree of adulteration during this period. In 2012, higher cocaine content was observed for the 191 analyzed samples than in samples from previous years. The PCA data also suggests that the metropolitan region samples had a higher degree of adulteration than the state countryside samples.

The relationship between chemical concentration and odor activity value explains the inconsistency in making a comprehensive surrogate scent training tool representative of illicit drugs

This report highlights the importance of an individual chemical's odor impact in the olfactory identification of marijuana, cocaine, and heroin. There are small amounts of highly odorous compounds present in headspace of these drugs, with very low odor detection thresholds, that are more likely responsible for contributing to the overall odor of these drugs. Previous reports of the most abundant compounds in headspace can mislead researchers when dealing with whole odor of these drugs. Surrogate scent formulations, therefore, must match the odor impact of key compounds and not just the chemical abundance of compounds. The objective of this study was to compare odorous volatile organic compounds (VOCs) emitted from illicit drug samples of marijuana, cocaine, and heroin to surrogate smell formulations using simultaneous sensory (via human olfaction) and chemical analyses. Use of solid phase microextraction (SPME) allowed VOCs in drug headspace to be extracted and pre-concentrated on site, and analyzed by multidimensional gas chromatography–mass spectrometry–olfactometry (MDGC–MS-O). Use of MDGC–MS-O allowed for further separation of odorous compounds and simultaneous detection by the human nose of the separate odor parts that make up the total aroma of these drugs. The compounds most abundant in headspace were not the most odor impactful when ranked by odor activity values (OAVs) (defined as ratio of concentration to odor detection threshold, ODT). There were no apparent correlations between concentrations and OAVs. A 1g marijuana surrogate lacked in odor active acids, aldehydes, ethers, hydrocarbons, N-containing, and S-containing VOCs and was overabundant in odor active alcohols and aromatics compared with real marijuana. A 1g cocaine surrogate was overabundant in odor active alcohols, aldehydes, aromatics, esters, ethers, halogenates, hydrocarbons, ketones and N-containing compounds compared with real. A 1g heroin surrogate should contain less odor active acids, alcohols, aromatics, esters, ketones, and N-containing compounds. Drug quantity, age and adulterants can affect VOC emissions and their odor impact. The concept of odor activity value, then, is useful to researchers without access to more sophisticated instrumentation. Odor activity values can be calculated from published odor detection thresholds. More research is warranted to expand the database, and determine odor detection thresholds for compounds of interest. Additional information could be obtained from establishing ODTs of key odorants for canines.

Multicriteria wavenumber selection in cocaine classification

Cocaine ATR-FTIR spectra consist of a large number of wavenumbers that typically decreases the performance of exploratory and predictive multivariate techniques. This paper proposes a framework for selecting the most relevant wavenumbers to classify cocaine samples into two categories regarding chemical composition, i.e. salt and base. The proposed framework builds a wavenumber importance index based on the Bhattacharyya distance (BD) followed by a procedure that removes wavenumbers from the spectra according to the order suggested by the BD index. The recommended wavenumber subset is chosen based on multiple criteria assessing classification performance, which are recalculated after each wavenumber is eliminated. The method was applied to ATR-FTIR spectra from 513 samples of cocaine, remarkably reducing the percent of retained wavenumbers and yielding near to perfect classifications in the testing set. In addition, we compared our propositions with other methods tailored to wavenumber selection; we found that the proposed framework, which relies on simple mathematical fundamentals, yielded competitive results.

Detection of Illicit Drugs by Trained Honeybees (Apis mellifera).

Illegal drugs exacerbate global social challenges such as substance addiction, mental health issues and violent crime. Police and customs officials often rely on specially-trained sniffer dogs, which act as sensitive biological detectors to find concealed illegal drugs. However, the dog “alert” is no longer sufficient evidence to allow a search without a warrant or additional probable cause because cannabis has been legalized in two US states and is decriminalized in many others. Retraining dogs to recognize a narrower spectrum of drugs is difficult and training new dogs is time consuming, yet there are no analytical devices with the portability and sensitivity necessary to detect substance-specific chemical signatures. This means there is currently no substitute for sniffer dogs. Here we describe an insect screening procedure showing that the western honeybee (Apis mellifera) can sense volatiles associated with pure samples of heroin and cocaine. We developed a portable electroantennographic device for the on-site measurement of volatile perception by these insects, and found a positive correlation between honeybee antennal responses and the concentration of specific drugs in test samples. Furthermore, we tested the ability of honeybees to learn the scent of heroin and trained them to show a reliable behavioral response in the presence of a highly-diluted scent of pure heroin. Trained honeybees could therefore be used to complement or replace the role of sniffer dogs as part of an automated drug detection system. Insects are highly sensitive to volatile compounds and provide an untapped resource for the development of biosensors. Automated conditioning as presented in this study could be developed as a platform for the practical detection of illicit drugs using insect-based sensors.

An assessment of detection canine alerts using flowers that release methyl benzoate, the cocaine odorant, and an evaluation of their behavior in terms of the VOCs produced

In recent years, the high frequency of illicit substance abuse reported in the United States has made the development of efficient and rapid detection methods important. Biological detectors, such as canines (Canis familiaris), are valuable tools for rapid, on-site identification of illicit substances. However, research indicates that in many cases canines do not alert to the contraband, but rather to the volatile organic compounds (VOCs) that are released from the contraband, referred to as the “active odor.” In 2013, canine accuracy and reliability were challenged in the Supreme Court case, State of Florida v. Jardines. In this case, it was stated that if a canine alerts to the active odor, and not the contraband, the canine's accuracy and selectivity could be questioned, since many of these compounds have been found in common household products. Specifically, methyl benzoate, the active odor of cocaine, has been found to be the most abundant compound produced by snapdragon flowers. Therefore, the purpose of this study is to evaluate the odor profiles of various species of snapdragon flowers to assess how significantly methyl benzoate contributes to the total VOC profile or fragrance that is produced. Particularly, this study examines the VOCs released from newly grown snapdragon flowers and determines its potential at eliciting a false alert from specially trained detection canines. The ability of detection canines to differentiate between cocaine and snapdragon flowers was determined in order to validate the field accuracy and discrimination power of these detectors. An optimized method using headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS–SPME/GC–MS) was used to test the different types and abundances of compounds generated from snapdragon flowers at various stages throughout the plants’ life cycle. The results indicate that although methyl benzoate is present in the odor profile of snapdragon flowers, other compounds are present that contribute significantly, if not more, than that of methyl benzoate. Canine teams, from various police departments throughout South Florida, certified for narcotics detection, took part in this study. Two canine trials involving 21 canines teams were performed by exposing the teams to 4 different species of snapdragon flowers. Of the 21 canine teams tested, none alerted to the snapdragon flowers presented, while all (100%) alerted to real cocaine samples, the positive control. Notably, the results revealed that although methyl benzoate is produced by snapdragon flowers, certified narcotics detection canines can distinguish cocaine's odor profile from that of snapdragon flowers.

Development of an electroanalytical method for the quantification of aminopyrine in seized cocaine samples

We report the development of a simple and accurate analytical method to detect aminopyrine in seized cocaine samples using a platinum electrode. The quantification of aminopyrine was performed using square wave voltammetry (SWV), where the peak current response was found to increase linearly with aminopyrine concentration over the range 100–1000μmolL−1. The repeatability of the electrode response was determined to be 2.4% (n=15), and the detection limit of the proposed method was estimated to be 22μmolL−1 (3σ/slope). The accuracy of the proposed method was evaluated comparing the measured aminopyrine concentration in seized cocaine sample to the value obtained by gas chromatography coupled to flame ionization detector (GC-FID). An addition and recovery protocol in seized cocaine samples was also performed.