Psychoactive substances belonging to the amphetamine class potently activate brain carbonic anhydrase isoforms VA, VB, VII, and XII.

Identifying possible new biological activities of psychoactive substances belonging to various chemical classes may lead to a better understanding of their mode of action and side effects. We report here that amines structurally related to amphetamine, a widely used psychoactive substance, such as amphetamine, methamphetamine, phentermine, mephentermine, and chlorphenteramine, potently activate several carbonic anhydrase (CA, EC 4.2.1.1) isoforms involved in important physiological functions. Of the 11 investigated human (h) isoforms, the widespread hCA I and II, the secreted hCA VI, as well as the cytosolic hCA XIII, and membrane-bound hCA IX and XIV were poorly activated by these amines, whereas the extracellular hCA IV, the mitochondrial enzymes hCA VA/VB, the cytosolic hCA VII, and the transmembrane isoform hCA XII were potently activated. Some of these enzymes are abundant in the brain, raising the possibility that some of the cognitive effects of such psychoactive substances might be related to their activation of these enzymes.

Advances in new psychoactive substances identification: the U.R.I.To.N. Consortium.

Identification of new psychoactive substances (NPS) in biological and non-biological samples represents a hard challenge for forensic toxicologists. Their great chemical variety and the speed with which new NPS are synthesised and spread make stringent the need of advanced tools for their detection based on multidisciplinary approaches. For this reason, in August 2016, the "Unit of Research and Innovation in Forensic Toxicology and Neuroscience of Addiction" (U.R.I.To.N.) was founded by the Forensic Toxicology Division of the University of Florence. In this Research Unit, various professionals (i.e. forensic toxicologists, chemists, physicians) collaborate to study all the aspects of drugs of abuse, especially NPS. Herein, we describe the multidisciplinary approach comprising liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), gas chromatography hyphenated to mass spectrometry (GC-MS) and solution nuclear magnetic resonance analysis that allowed the identification of three NPS such as 1-(benzofuran-5-yl)-N-methylpropan-2-amine, 2-amino-1-(4-bromo-2,5-dimethoxyphenyl)ethan-1-one (bk-2C-B), and 3-(2-aminopropyl)indole (α-methyltryptamine) in seized materials.

Alcohol, caffeine, and nicotine consumption in adolescents: hair analysis versus self-report.

BACKGROUND: Clinical reliability of self-reported data for alcohol, caffeine, and nicotine consumptions is lacking, particularly in adolescents. OBJECTIVES: To compare a self-report questionnaire and hair analysis to assess the reliability and effectiveness of the self-report. METHODS: A cross-sectional study on 14-15-year-old Italian students (n = 874, 38% males, 62% females) was performed comparing self-reported data to hair analysis. The latter quantified hair concentrations of caffeine, nicotine, cotinine, ethyl glucuronide (EtG), and fatty acid ethyl esters (FAEEs) using mass spectrometry. RESULTS: Concordance between self-report and hair testing ranged from good to poor across substances and levels of use: poor for heavy alcohol intake (EtG: k = 0.36, 20 positive cases by hair analysis, false negative by self-report, 2.3% of total sample; FAEE k = 0.31, 25 positive cases, 2.9% of total sample); fair to poor for active smokers (k = 0.40, 125 positive cases, 14.3% of total sample); and moderate for caffeine (k = 0.57, 56 positive cases, 6.4% of total sample). CONCLUSIONS: Epidemiological studies on alcohol, caffeine, and nicotine consumption in adolescents may benefit from the inclusion of toxicological analysis on hair samples to overcome the under-reporting phenomenon of questionnaires and detect more cases of problematic substance use.

A novel, simultaneous extraction of FAEE and EtG from meconium and analysis by LC-MS/MS.

Fatty acid ethyl esters (FAEEs) and ethyl-glucuronide (EtG) in meconium have been widely studied as biomarkers of maternal alcohol consumption during pregnancy. Many analytical approaches have been proposed for their analysis, mostly consisting of separated extraction procedures requiring the use of two meconium aliquots. This study aimed to validate a new analytical procedure for the simultaneous extraction of FAEEs and EtG from a meconium aliquot through a single solid-phase extraction (SPE) applied to 242 anonymized samples of meconium. Targeted FAEEs were: ethyl-myristate (Myr), ethyl-palmitate (Pal), ethyl-oleate (Ole) and ethyl-stearate (Ste). Two hundred milligrams of meconium was sonicated with acetonitrile, and a single SPE performed by means of aminopropyl columns. FAEEs were eluted with hexane, followed by EtG elution with water. Both the mixtures were dried, recovered, and analyzed by liquid chromatography-tandem mass spectrometry using C8 (FAEEs) and C18 (EtG) columns. Transitions were: m/z 257 → 57,88, Myr; m/z 262 → 57,88, Myr-d5; m/z 285 → 57, 72, Pal; m/z 290 → 57,258, Pal-d5; m/z 311 → 72,114, Ole; m/z 316 → 72,265, Ole-d5; m/z 257 → 57,72 Ste; m/z 318 → 57,286, Ste-d5; m/z 221 → 75,85, EtG; m/z 226 → 75,85, EtG-d5. Lower limit of quantification range was 10-15 ng/g for FAEEs and 10 ng/g for EtG. Linearity was evaluated for different concentration ranges; the mean coefficients of determination (R (2)) were above 0.9961. Precision and accuracy for FAEEs and EtG were consistently ≤20 % and ±20 %, respectively. Ion suppression was observed for all the analytes. Matrix effect did not significantly affect the analyses. Recovery efficiency was 93 % for EtG and 75-85 % for FAEEs.

The successful binomium of multivariate strategies and electrophoresis for the Quality by Design separation of a class of drugs: the case of triptans.

Quality by Design (QbD) approach was followed having as analytical target profile the determination of different antimigraine drugs (seven triptans, TRP) available on the market. Multivariate strategies were used for defining the design space and solvent-modified MEKC was the selected analytical method. The versatility of electrophoretic technique, allowing a fine modulation of experimental parameters, made it possible to define the design space (DS). The DS limited a suitable range of experimental parameters in which all possible combinations of variables led to an electrokinetic method able to determine all the considered analytes with a predefined quality. Design of experiments and risk analysis fully assisted the method development from the initial investigation of MEKC knowledge space to the DS definition and finally to the control strategy. Applying the working operative conditions, the baseline separation of TRP was obtained in less than 9 min. The method was finally used for the quantification of three TRP in different pharmaceutical products.

Enhancing the sensitivity of the LC-MS/MS detection of propofol in urine and blood by azo-coupling derivatization.

Propofol is a low-polarity, volatile molecule that is difficult for an electrospray ion source (ESI) to ionize in either negative ion mode (NIM) or positive ion mode (PIM), which hampers its detection via liquid chromatography-mass spectrometry. The aim of the present study was to use a new derivatization agent to improve ionization efficiency and to develop an efficient liquid chromatography-multiple mass spectrometry (LC-MS/MS) determination of propofol in urine and blood, taking advantage of an electrophilic aromatic substitution. An azo-coupling reaction with a diazonium salt from aniline was performed to introduce a protonation site into the molecule. The diazonium salt was generated by aniline in water solution by HCl and sodium nitrite; derivatization was achieved by stirring a mixture of the diazonium salt and propofol in sodium hydroxide solution for 30 min below 5 °C. A liquid-liquid extraction with dichloromethane and ethyl acetate was performed to obtain the azo derivative (molecular composition: C18H22ON2; molecular weight: 282 Da) in high yield. The compound provided very high ionization yields in both PIM and NIM ESI, and the protonated or deprotonated molecule gave intense signals. The transitions m/z 283 → 77, 241 and m/z 281 → 176, 161 were chosen for the PIM and NIM, respectively, in order to develop quantitative methods of detecting propofol in urine and blood via triple-quadrupole LC-MS/MS. These methods proved to be highly sensitive, with limits of quantification of 0.4 pg/mL and 0.1 ng/mL obtained in the NIM when analyzing 1 mL of urine and 100 µL of blood, respectively.

Comparison of two methods for the extraction of ethylglucuronide from hair.

The aim was the comparison between the Society of Hair Testing (SoHT) consensus for the use of alcohol markers which powdering hair for the extraction of ethylglucuronide (EtG) in water and extraction using the patented M3 Reagent Test kit on cut hair. Hair samples were cut into small segments and washed twice with methanol and diethyl ether. The SoHT-Consensus entails the extraction of pulverised hair in water. This is obtained by incubation of 25 mg of hair at room temperature overnight and 2 h sonication, even if the overnight incubation is not mandatory. The M3 method entails incubation of 25 mg of cut hair with the M3-Reagent at 100°C for 60 min. After centrifugation, the supernatant is injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples (191) were collected in the APSS laboratory in Trento, Italy, between 2021 and 2022. The limit of quantification (LOQ) was set at 5 pg/mg for the pulverised and M3-Reagent methods. Assays showed good linearity above the range of LOQ-300 pg/mg. Precision (within 20%) values were also obtained using both methods. In the Passing-Bablock linear regression, the final regression curve between M3 (y) and the pulverising method (x) showed good agreement; the Bland-Altman analysis did not show any significant bias between the two methods. The M3-Reagent method, due to cut hair use, is easy to perform, saves time and allows for a smaller sample quantity loss with use of nondisposable grinding jars for the ball mill to obtain the extraction of EtG.

THC and THC-COOH hair concentrations: Influence of age, gender, consumption habits, cosmetics treatment, and hair features.

Evaluation of Cannabis consumption is required for many purposes (i.e., workplace drug testing and driving license renewal). Hair analysis represents the most adopted and reliable approach for the investigation of repeated or chronic exposure to Cannabis. The main markers are the Δ9-tetrahydrocannabinol (THC) and its main metabolite, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), as stated by the Society of Hair Testing (SoHT) and the European Workplace Drug Testing Society (EWDTS). In this paper we presented an observational study on the hair concentrations of THC and THC-COOH and influences due to age, gender, consumption habits, and hair features. Data were collected from analysis of scalp hair samples (3-cm proximal segment) provided by subjects tested for THC consumption for personal purposes (i.e., workplace drug testing, personal use proving). The subjects provided an informed consent and a short questionnaire. A new analytical method was previously developed and then adopted. It consisted in a hydrolysis (1 mL of 1 M NaOH at 65 °C, 20 min) and a liquid-liquid extraction (with hexane/ethyl acetate,90/10, v/v in presence of 1.5 mL of H2SO4 1 M) of 25 mg of hair. A liquid chromatograph - tandem mass spectrometer (LC-MS/MS) equipped with a C18 column was used. The acquisition was in multiple reaction monitoring for the following transitions: 315→259, 193 m/z, for THC; 318→196, 123 m/z, for THC-d3; 345→299, 193 m/z for THC-COOH; 348→196, 302 m/z for THC-COOH-d3. Correlation between THC and THC-COOH hair concentrations was analyzed by Spearman's rank correlation coefficient. In order to study the influences of several variables, a new value, Sqrt(THC*THCCOOH), was adopted. Its effectiveness and reliability were proved by the Principal Component Analysis. Relationships between the Sqrt(THC*THCCOOH) and the variables were studied through the Stepwise regression (p = 0.05). The normality of data distribution was tested by the Shapiro-Wilk test. The Lower limits of quantification were 10.0 (THC) and 0.2 (THC-COOH) pg/mg. Accuracy and precision always met the acceptable criteria. Recoveries were > 78% and ion suppression was observed for both the compounds. Data from 126 hair samples were included in this study: 54 subjects(42.9%) were positive both for THC and THC-COOH; none of the samples was positive for a single substance. Concentrations ranged from 0.18 to 1.75 ng/mg (median: 0.78 ng/mg) for THC and from 0.04 to 0.85 ng/mg (median: 0.31 ng/mg) for THC-COOH. Cannabinoids levels seemed to decrease with the age, with lower amounts in the subjects aged > 40 years (p < 0.05). Also years of consumption seemed to have a significant impact on hair concentrations, as higher levels were observed in consumers from > 10 years (p = 0.013). Moreover, this study further provided evidences of a significant reduction of THC and THC-COOH in bleached hair (p = 0.042).

Poisoning Caused by Medicines and Drugs of Abuse.

We believe it is necessary to state a premise on the framing of poison and poisoning in the context of Forensic Toxicology as an important contribution to this Special Issue, which is composed of articles about this discipline-the discipline of "poison" [...].

A Forensic Diagnostic Algorithm for Drug-Related Deaths: A Case Series.

The best evidence provided in the literature worldwide suggests the importance of harmonizing the investigation in drug-related fatalities. In this study, the application of a multidisciplinary approach in eight cases of drug-related deaths is presented. Although death scene findings could be highly suggestive of drug intoxication, external examination and toxicological screening test alone are insufficient. There are several variables, and it is not always easy to give the proper interpretation of the drug detection. A complete autopsy is necessary to correctly complete organ and tissues sampling for further histological and toxicological studies and obtain body fluids. The use of peripheral blood is recommended to avoid artifacts. The collection of many specimens is warranted to get more responses. The sampling aims to provide a picture of the distribution of the substance in the body. The sample and the selection of the drugs and the matrices to investigate are case-dependent. The presented diagnostic algorithm provides the coroner with all the elements to investigate drug-related deaths and cooperate with toxicologists. Toxicological forensic diagnosis is still extremely heterogeneous in regional and national contexts. Funding for method development, research, networking, facilities, and technologies improvement is mandatory to standardize the toxicological investigation.

Toxicological Findings of Self-Poisoning Suicidal Deaths: A Systematic Review by Countries.

The use of illicit and non-illicit substances is widespread in suicides. The toxicological data may help in understanding the mechanism of death. This systematic review aimed to analyze autopsies related to suicides by consuming poison, focusing on the correlation between substance use and the country of origin to create an alarm bell to indicate that suicide maybe attempted and prevent it. The systematic review was conducted according to the PRISMA guidelines, with the primary objective of identifying autopsies conducted in cases of suicide by consuming poison in specific geographic areas. Significant differences in substances were observed between low-income and Western countries that confirm previous literature data. In rural areas and Asian countries, most suicides by consuming poison involve the use of pesticides, such as organophosphates and carbamates. In Western countries, illicit drugs and medically prescribed drugs are the leading cause of suicide by self-poisoning. Future research should shed light on the correlation between social, medical, and demographic characteristics and the autopsy findings in suicides by self-poisoning to highlight the risk factors and implement tailored prevention programs worldwide. Performing a complete autopsy on a suspected suicide by self-poisoning could be essential in supporting worldwide public health measures and policy makers. Therefore, complete autopsies in such cases must be vigorously promoted.

EtG Quantification in Hair and Different Reference Cut-Offs in Relation to Various Pathologies: A Scoping Review.

Ethyl glucuronide (EtG) is a non-volatile, non-oxidative, hydrophilic, and stable ethanol phase II metabolite. EtG is produced through ethanol glucuronidation by UDP-glucuronosyltransferase (UGT), a phase II enzyme. EtG can be extracted from different biological matrices, including keratin ones, such as hair or nails. The purpose of this scoping review is to describe the relationship between EtG levels in hair and some of the most common and frequent pathological conditions and verify whether different reference cut-offs in relation to various pathologies have been identified in the scientific literature. In fact, in-depth knowledge of the influence of pathologies, such as diabetes mellitus, hepatic and renal dysfunction, on EtG production and its storage in keratin matrices would allow a more appropriate interpretation of obtained data and rule out false positives or false negatives. This scoping review is based on bibliographic research carried out on PubMed regarding the quantification of EtG in hair of subjects affected by different pathological conditions. According to the scientific literature, the main and most common pathologies that can affect the concentration of EtG in hair are liver and kidney diseases and diabetes. The EtG quantification analytical data should be interpreted carefully as they may have a great impact in both forensic and clinical contexts.

Determination of endogenous GHB levels in chest and pubic hair.

Endogenous nature of GHB represents a critical issue for forensic toxicologists, especially in alleged sexual assaults. Therefore, discrimination between physiologically and additional amounts from exogenous sources of such a substance must be effective and reliable in order to avoid severe misinterpretation. This study aimed to quantify the GHB baseline concentrations in chest and pubic hairs collected from 105 healthy volunteers, non-consumers of any drugs of abuse. The final scope was to investigate if these keratin matrices could represent valid alternative to scalp hair when not available. Moreover, we also evaluated the age and gender influences on the GHB baseline levels. 25 mg of hair were incubated overnight with NaOH at 56 °C. After acidification with H2SO4, the solution was liquid-liquid extracted with ethyl acetate and a trimethylsilyl derivatization was then achieved. Analysis was performed in gas chromatography-mass spectrometry in single ion monitoring mode (m/z 233, 234, 147 for GHB; m/z 239, 240 and 147 for GHB-d6). The endogenous amount in "blank" hair was estimated by the standard addition method (0.301 for chest hair and 0.235 ng/mg for pubic hair). GHB concentration ranged from 0.205 to 1.511 ng/mg for chest hair and from 0.310 to 1.913 ng/mg for pubic hair. These values were consistent with previous studies on scalp hair and on pubic hair. Unfortunately, research on chest hair is not available in literature. T-Test and Linear Regression highlighted no statistically significant differences for the two matrices and for all age/gender sub-groups. However, further studies are required to estimate a reliable cut-off value for these keratin matrices. For the first time, we demonstrated the suitability of chest and pubic hair to detect endogenous levels of GHB.

Overdose of Quetiapine-A Case Report with QT Prolongation.

Quetiapine is an atypical antipsychotic drug used to treat bipolar disorder, schizophrenia, and major depressive disorder. Although several studies describe the adverse effects of intoxication with Quetiapine, only a few report an extreme overdose without comedications that lead to a life threat. We present a case of a 75-year-old male who tried to attempt suicide by ingesting 28 g of Quetiapine. During the management in the emergency department, both serum and urine samples were collected, allowing a complete pharmacokinetic analysis to be conducted, from the admission to the discharge.

Ethyl Glucuronide Concentration in Hair of Detainees: A Preliminary Study.

Through the measurement of ethyl glucuronide in hair (hETG), it is possible to assess chronic alcohol abuse over time. In this paper, we present a study on hETG in Italian prison inmates. Analyses were performed by LC-MS according to a previously published method. Results were evaluated using the cut-offs established by the Society of Hair Testing. Positives samples (ETG > 30 pg/mg) accounted for 6% of all subjects, with concentrations ranging from 42 pg/mg up to 270 pg/mg, abstinent subjects (ETG < 7 pg/mg) accounted for 88%, and moderate alcohol consumption (7 < ETG < 30 pg/mg) for 6% of the subjects. No females displayed ETG values above 30 pg/mg. Among positive samples, only two subjects did not declare heavy alcohol consumption and were found strongly positive at 210 and 270 pg/mg. To the best of our knowledge, this represents the first study on ETG hair concentration on prison inmates.

A novel LC-MS/MS analytical method for detection of articaine and mepivacaine in blood and its application to a preliminary pharmacokinetic study.

Local anaesthetics (LAs) are commonly used in surgery, especially in dentistry. They cause a transitory inhibition of nerve signal due to the blockade of the voltage-gated sodium channels. LAs are administrated alone or with vasoconstriction agents, such as adrenaline. Toxicity of LAs is associated to neurological and cardiovascular alterations. Tachycardia, arrhythmia, tremors, tonic-clonic seizure and respiratory depression (at high doses) are the main symptoms of intoxication by LAs. Lidocaine, articaine and mepivacaine are among the most used anaesthetics. This study aimed to fully validated a new method for the simultaneous detection of articaine and mepivacaine in whole blood. Sample treatment consisted in a liquid-liquid extraction with phosphate buffer (pH 8, 0.1 M) and ethyl-acetate. Analysis was performed by liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode (transitions: articaine, 285→8658 m/z; mepivacaine, 247→9870 m/z; lidocaine - internal standard -, 235→8658 m/z). The method proved to be highly sensitive with limit of quantifications for articaine and mepivacaine of 0.8 and 0.1 ng/mL, respectively. Accuracy and precision were always within the acceptance criteria. The new procedure was also successfully applied to a preliminary pharmacokinetics study.

New psychoactive substances: An actual problem or an overestimated phenomenon?

In last years, international and national Institutions have been completely focused on the new psychoactive substances (NPS) phenomenon. Many contrast policies have been planned in order to control their spread. Even scientific entities, such as our Forensic Toxicology Division, have spent time and resources for NPS identification in biological (from clinical and forensic caseworks) and non-biological (seized material) samples. Last reports show a low prevalence of NPS across the Europe and Italy, while the classical drugs are still the main cause of drug-related deaths. In particular, a worrisome datum is represented by the increasing number of deaths due to heroin. Seen these statistics, is the NPS phenomenon overestimated? Is the interest in classical drugs decreased? Were we diverted by NPS?

Zwitterionic HILIC stationary phase as a valuable alternative in separative techniques: Application to the analysis of gamma-hydroxybutyric acid and its metabolite in hair.

In this work, the physical and chemical properties of a novel zwitterionic LC stationary phase are applied to the development, validation and application of a new fast and reliable method devoted to the analysis of GHB (gamma-hydroxybutyric acid) and its relatively new discovered glucuronide metabolite in hair. The obtained sensitivity, expressed as limit of detection (LOD) and quantification (LOQ), were 0.033 and 0.10 ng/mg for GHB and 0.11 and 0.37 ng/mg, for GHB-glucuronide respectively. Linearity was assessed between LOQ and 50 ng/mg for both compounds. GHB and GHB-glucuronide extraction from hair matrix was maintained simple and consisted in an acidified-solvent incubation. No samples purification was required before LC-MS/MS analysis. The method was finally applied to 65 real hair sample, 60 adults and 5 children below 2 years old. The obtained results highlighted that GHB concentrations were in the range 0.11-0.96 ng/mg (average 0.38 ±â€¯0.25 ng/mg) in 44 cases (68%) while in 21 samples GHB concentrations were in the range between LOD and LOQ (0.033-0.1 ng/mg). GHB-glucuronide was detected in few samples (n. 3) at levels below LOQ. The interest on these molecules relies on the fact that GHB is both a naturally occurring inhibitory neurotransmitter in the central nervous system and an illicit drug often experienced by victims of drug-facilitated sexual assault. GHB-glucuronide was firstly identified in urine by the group of Petersen in 2013 and, as per analogy to ethyl glucuronide, it was proposed as a longer biomarker for GHB intoxication.

Psychotropic substance abuse and fitness to hold a driving license in Italy.

Objective: Driving under the influence (DUI) of psychotropic substances is a serious and widespread problem in road safety. All countries try to reduce the impact with legislative controls over the criteria to regain a driver's license after suspension. In many European countries there are mandatory clinical and toxicological examinations required before a license is regranted. In Italy, individuals convicted of driving under the influence of drugs and/or alcohol must undergo a mandatory medico-legal and forensic toxicological examination prior to regranting of a license. This article reports on the prevalence, trends, and implications of psychotropic substances detected in more than 5,000 subjects submitted to driving license reissuance in the period 2011-2016. Methods: The study involved taking a clinical history, medical examination, and toxicological analysis of both urine and hair samples. Results: There was no change in the prevalence of psychoactive substances in the period 2011-2016. Cocaine was found most often (60%), followed by cannabinoids (15%) and opiates (9%). Methadone and amphetamine stimulants accounted for less than 5% each. Benzodiazepines were present in 15% of samples throughout the period. Conclusion: Cocaine and cannabinoids were the most used substances in the analyzed population, alone and in combination. Benzodiazepines were the most commonly detected prescription medication, raising questions about prescribed medication and driving risk that are not addressed by current legislation.

Application of HRAM screening and LC-MS/MS confirmation of active pharmaceutical ingredient in "natural" herbal supplements.

The growing market of herbal remedies worldwide could pose severe problems to consumers' health due to the possible presence of potentially harmful, undeclared synthetic substances or analogues of prescription drugs. The present work shows a simple but effective approach to unequivocally identify synthetic anorectic compounds in allegedly 'natural' herbal extracts, by exploiting liquid chromatography/time of flight (Q-TOF LC/MS) technology coupled to liquid chromatography/triple quadrupole (LC-MS/MS) confirmation and quantitation. The procedure was applied to five tea herbal extracts and pills sold as coadjutant for weigh loss. The method exploited liquid-liquid sample extraction (LLE) and separation in a C18 (2.1mm×150mm, 1.8µm) column. QTOF acquisitions were carried out both in scan mode and all ion MS/MS mode and results were obtained after search against ad hoc prepared library. Sibutramine, 4-hydroxyamphetamine, caffeine and theophylline were preliminary identified samples. Confirmation and quantitation of the preliminary identified compounds were obtained in LC-MS/MS after preparation of appropriated standards. Sibutramine, caffeine and theophylline were finally confirmed and quantitate.