Gas Chromatography-electron Capture Detection Research Articles

Rapid analysis of cyclamate in foods and beverages by gas chromatography-electron capture detector (GC-ECD)

A rapid method for determination of sodium cyclamate in foods and beverages was developed. Sodium cyclamate was converted to N,N-dichloridecyclohexylamine by reaction with sodium hypochlorite under acid condition. N,N-dichloridecyclohexylamine was subsequently extracted by n-hexane and determined by gas chromatography. Conditions such as derivatization time, the concentration of sodium hypochlorite and sulphuric acid were optimised. Amino acids, aliphatic amines, and food additives such as preservatives, dyes and sweeteners showed no interference for quantification of cyclamate. The correlation coefficient of calibration curve was 0.9993 in the range of 5.0-250mg/L. The limits of detection (LOD) and limits of quantification (LOQ) were calculated as three or ten times the signal-to-noise ratio (S/N), respectively. The LOD and LOQ for yellow wine and fruit juice were 0.05 and 0.2mg/L, respectively. The LOD and LOQ for cake and preserved fruit were 0.25 and 0.8mg/kg, respectively. The intra-day and inter-day RSD were 0.28% and 1.1% (n=5), respectively. The method was successfully applied for determination of cyclamate in yellow wine, cake, fruit juice and preserved fruit. This method was simple, fast, and sensitive. It was suitable for the determination of cyclamate in foods and beverages for safety and quality control inspections.

Optimization and application of accelerated solvent extraction for rapid quantification of PCBs in food packaging materials using GC-ECD

Polychlorinated biphenyls (PCBs) in food packaging materials which contact with food directly or indirectly can migrate into the food and endanger the health of people. The detection of PCBs in food packaging materials is becoming increasingly essential. The method based on combining accelerated solvent extraction (ASE), sulfuric acid and silica gel column cleanup with gas chromatography-electron capture detector (GC-ECD) was found to be simple and efficient for the determination of the levels of the pollutants in the assays. The calibration curves of PCB28, PCB52, PCB101, PCB118, PCB138, PCB153, PCB180, PCB198 presented a good linearity in the range of 4–200 μg/L, the coefficients of variation and the limits of determination were in the range of 0.9982–0.9999 and 0.1828–0.3798 μg/L, respectively. The overall relative standard derivations were less than 4.11% and the average recoveries were in the range of 77.90%–100.08%. Compared with Soxhlet extraction method, the optimal ASE sample preparation can reduce analysis time and solvent consumption, and enable unequivocal identification and quantification.

Dispersive liquid-liquid microextraction for the determination of organochlorine pesticides residues in honey by gas chromatography-electron capture and ion trap mass spectrometric detection.

A simple dispersive liquid-liquid microextraction (DLLME) protocol for the determination of 15 organochlorine pesticides residues in honey is proposed. The selected pesticides were separated using gas chromatography and detected by electron capture (ECD) or ion trap mass spectrometry (GC-IT/MS). Several parameters affecting the extraction efficiency namely type and volume of organic extraction solvent, type and volume of disperser solvent, sample pH, ionic strength, extraction time and centrifugation speed were systematically investigated. The final DLLME protocol involved the addition of 750 μL acetonitrile (disperser) and 50 μL chloroform (extraction solvent) into a 5 mL aqueous honey solution followed by centrifugation. The sedimented organic phase (chloroform) were analysed directly by GC-IT/MS or evaporated and reconstituted in acetonitrile prior to the GC-ECD analysis. The analytical performance of the GC-ECD and GC-IT/MS methods was compared and discussed. Under the selected experimental conditions, the enrichment factors varied between of 36 and 114. The limits of detection (LOD) were in the range of 0.02-0.15 μg L(-1) (0.4-3 ng g(-1)) for GC-ECD and 0.01-0.2 μg L(-1) (0.2-4 ng g(-1)) for GC-IT/MS which is adequate to verify compliance of products to legal tolerances. The proposed method was applied to the analysis of the selected organochlorine pesticides residues in various honey samples obtained from Greek region. Mean recoveries were ranged from 75% to 119% while the precision was better than 20% in both methodologies.

Low density solvent based dispersive liquid–liquid microextraction with gas chromatography–electron capture detection for the determination of cypermethrin in tissues and blood of cypermethrin treated rats

A simple and rapid method to determine the cypermethrin (CYP) insecticide in rat tissues (kidney, liver and brain) and blood has been developed for the first time using low density solvent-dispersive liquid–liquid microextraction (LDS-DLLME) followed by gas chromatography–electron capture detector (GC–ECD) analysis. Initially, tissue samples containing CYP were homoginized in acetone. Subsequently, homogenate was mixed with n-hexane (extraction solvent) and the mixture was rapidly injected into water. The upper n-hexane layer was collected in a separate microtube and injected into GC–ECD for analysis. Blood samples were diluted with ultrapure water and subjected to DLLME through similar procedure. Parameters such as type and volume of disperser and extraction solvent, salting out effect and extraction time, which can affect the extraction efficiency of DLLME, were optimized. Method was validated by investigating linearity, precision, recovery, limit of detection (LOD) and quantification (LOQ). LODs in tissue were in the range of 0.043–0.314ngmg−1 and for blood it was 8.6ngmL−1 with a signal to noise ratio of 3:1. LOQs in tissue were in the range of 0.143–1.03ngmg−1 and for blood it was 28.3ngmL−1 with a signal to noise ratio of 10:1. Mean recoveries of CYP at three different concentation levels in all the matrices were found to be in the range of 81.6–103.67%. The results show that, LDS-DLLME coupled with GC–ECD offers a simple, rapid and efficient technique for extraction and determination of CYP in rat tissues and blood samples, which in turn would be useful for toxicological studies of CYP.

Rapid analysis of organochlorine and pyrethroid pesticides in tea samples by directly suspended droplet microextraction using a gas chromatography–electron capture detector

A simple and efficient directly suspended droplet microextraction (DSDME) has been developed to extract and pre-concentrate organochlorine and pyrethrin pesticides from tea samples prior to analysis by a gas chromatography–electron capture detector (GC–ECD). The optimal experimental conditions of DSDME were: 100μL isooctane exposed for 15min to 5mL of the tea aqueous sample stirred at 1100rpm. For most of the target analytes, the optimal pretreatment of DSDME processes led to no significant interference of tea matrices. The approach was applied to the determination of organochlorine and pyrethroid pesticides in tea samples, with a linearity range of 0.0005–2μg/mL. The relative recoveries of all the pesticides ranged between 80.0% and 120.8% with relative standard deviations (RSDs) in the range of 0.8–19.9% (n=5). The limits of detections (LODs) ranged between 0.04 and 1μg/L for all the target pesticides.

Assessment and Investigation on the Fate of Organochlorine Pesticides in Water and Sediments of International Amir-kalaye Wetland in North of Iran

A total of 48 water samples and 24 sediment samples were collected at four sampling stations along the wetland during four seasons from 2009 to 2010 and analyzed by gas chromatograph-electron capture detector (GC-ECD). In water the total concentration of OCPs was 0.33, 0.01, 0.1 and 0.07 mg/L in summer, autumn, winter and spring, respectively. The most frequent OCP compounds detected were endrin and chlordane (0.08 and 0.07 mg/L), heaxachlorobenzene and chlordane (0.06, 0.02 mg/L), and chlordane (0.07 mg/L) in summer, winter and spring, respectively. The maximum concentration of ΣOCPs was found in samples collected from station 1 in summer (0.26 mg/L). In sediments the total concentrations of OCPs were 15.84 and 2.62 mg/g-dry weight (dw) in summer and winter, respectively. Chlordane was the most frequently found OCP compound, followed by lindane, 9.92 and 2.47 mg/g-dry weight (dw), respectively, in summer. While, lindane (2.52 mg/g-dw) and endosulfan I (0.1 mg/g-dw) were the highest OCP compounds detected in winter. The results obtained in this study show that there still exist a variety of organochlorine pesticide residues in the water and sediments from the Amir-kalaye wetland in Iran.

Comparison between GC-MS-SIM and GC-ECD for the determination of residues of organochlorine and organophosphorus pesticides in Brazilian citrus essential oils

Brazilian citrus essential oils (mandarin, orange, lemon, bergamot and tangerine) were analyzed for the determination of pesticides residues of organochlorine (aldrin, chlordane, dieldrin and dicofol) and organophosphorus (methyl chlorpyrifos, dimethoate, methidathion and methyl parathion) . A comparative study between gas chromatography-mass spectrometry in selective ion monitoring mode (GC-MS-SIM) and gas chromatography-electron capture detector (GC-ECD) was performed using solid phase extraction (SPE) with Florisil® cartridge for the pre-concentration of pesticides. The average recovery of pesticides was from 71 to 83% as determined by GC-MS-SIM analysis and 99 to 104% by GC-ECD. The limit of quantification was from 0.93 to 3.93 mg L-1 in GC-MS-SIM and 0.09 to 0.12 mg L-1 in GC-ECD. Fifteen samples of commercial Brazilian citrus essential oils were analyzed and six samples showed contamination with values above the maximum residue limit (MRL) allowed by the Codex Alimentarius.

Analysis of haloacetic acids in water and air (aerosols) from indoor swimming pools using HS-SPME/GC/ECD

A solid phase microextraction method was used for the analysis of nine haloacetic acids (HAAs) in water and air (aerosols) from indoor swimming pools (ISPs). The analysis is characterized by derivatization of HAAs to their methyl-esters with dimethyl sulphate, headspace solid phase microextraction (HS-SPME) with a Carboxen–polydimethylsiloxane (CAR-PDMS) fiber and gas chromatography - electron capture detector (GC/ECD). High correlation coefficients were obtained for esters mixture calibration lines and detection limits were found to be at the low ppb level. Repeatability was assessed and coefficients of variation varied from 10 to 20%. Reproducibility was also evaluated and coefficients of variation from 15 to 25% were obtained. Analytical results from four Portuguese ISPs showed that the mean concentration of total HAAs (THAAs) in water ranged from 10 ± 2 to 183 ± 28 μg/L in which 55 ± 20% corresponded to trichloroacetic and dichloroacetic acids (TCAA and DCAA). THAAs highest concentrations were directly related to higher ISPs’ water organic matter content. In the lack of European specific regulation for water from ISPs and taking into consideration that ingestion is a form of exposure, THAAs concentration values were compared with drinking water maximum contamination level (MCL) of 60 μg/L proposed by the US EPA for the sum of five HAAs. In 35% of water sampling campaigns the sum of MBAA (monobromoacetic acid), MCAA (monochloroacetic acid), DCAA and TCAA exceeded that MCL value. The concentrations obtained for THAAs in the ISPs’ atmosphere ranged from 5 ± 1 to 64 ± 10 μg/m3 (T = 28°C at 5 cm above the water surface) and were proportional to the aerosols’ quantity, which was deeply related to indoor air ventilation system.

Group-selective enrichment and determination of pyrethroid insecticides in aquaculture seawater via molecularly imprinted solid phase extraction coupled with gas chromatography-electron capture detection

Two types of molecularly imprinted polymers (MIPs) for the simultaneous determination of six pyrethroid insecticides have been developed using deltamethrin (D-MIPs) and cypermethrin (C-MIPs) as template molecules. A comparison of the performance of D-MIPs, C-MIPs, and the corresponding non-imprinted polymers (NIPs) were conducted. Stronger group-selective interactions between the C-MIPs and the six pyrethroid insecticides were achieved. The MISPE method based on the C-MIPs displayed higher extraction recoveries (86.4–96.0%) with RSD values ranging from 2.4 to 7.8% for the six pyrethroid insecticides in aquaculture seawater. After the C-MIP cartridge procedure, the limits of detection and quantification for fenvalerate, deltamethrin, cypermethrin, cyfluthrin, and bifenthrin were in the 16.6–37.0 and 55.3–109.1ngL−1 ranges, respectively, and 0.68 and 2.26μgL−1 for phenothrin, respectively. The proposed MISPE method coupled with gas chromatography-electron capture detection was successfully used for the determination of the six pyrethroid insecticides in aquaculture seawater.

Identification and determination of chlorinated paraffins using multivariate evaluation of gas chromatographic data

Chlorinated paraffins (CPs) were found in the biodegradable fraction of source separated waste from Uppsala, Sweden. We identified and quantified the CPs by multivariate evaluation of gas chromatography-electron capture detection chromatograms. Using principal component analyses (PCA) we identified different types of CP-formulations and also obtain quantitative data. PCA yielded better identifications of individual CP-formulations than visual comparison of chromatograms. Partial least squares regression gave good calibration curves of the standards, but did not work for the waste samples. No source of CPs could be identified in the waste collection chain, and as the waste samples seemed to contain at least two different CP-formulations the source was probably to be found in the waste material itself. The method was used to determine CPs in additional environmental samples, demonstrating that multivariate methods may be developed into a powerful tool for identification and quantification of complex mixture.

Rapid analysis of pyrethroid insecticides in aquaculture seawater samples viamembrane-assisted solvent extraction coupled with gas chromatography-electron capture detection

A simple, efficient, and environmentally friendly membrane-assisted solvent extraction (MASE) method for the extraction and preconcentration of six pyrethroid insecticides from aquaculture seawater samples followed by gas chromatography-electron capture detection (GC-ECD) was successfully proposed. The operating conditions for MASE, such as the extraction solvent, solvent volume, NaCl concentration, stirring rate, extraction time, and temperature, were optimized. Compared to conventional Florisil-solid phase extraction (SPE), higher extraction recoveries (85.9% to 105.9%) of three spiked levels of the six pyrethroid pesticides in aquaculture seawater were obtained using MASE, and the RSD values were lower than 7.9%. The limits of detection (LOD, signal-to-noise ratio (S/N)=3) and quantification (LOQ, S/N = 10) were in the range of 0.037-0.166 and 0.12-0.55 μg L(-1), respectively. The results demonstrate the excellent applicability of the MASE method in analyzing the six pyrethroid pesticides in aqueous samples. The proposed method exhibited a high potential for routine monitoring analysis of pyrethroid insecticides in seawater samples.

Combination of solid-phase extraction and dispersive liquid–liquid microextraction for detection of cypermethrin and permethrin in environmental water

A new solid-phase extraction and dispersive liquid–liquid microextraction (SPE-DLLME) method coupled with gas chromatography electron capture detection (GC-ECD)was established for the detection of cypermethrin and permethrin in river water. The samples were firstly extracted using a large-volume SPE procedure and the eluents of SPE were further purified and enriched by the following DLLME. Good linearity was observed in the range of 0.015–3.87 μg L−1 for cypermethrin and 0.065–16.2 μg L−1 for permethrin with the correlation coefficient (r) ≥ 0.9995. The limits of detection based on S/N = 3 were 0.48 and 3.81 ng L−1 for cypermethrin and permethrin, respectively. Under optimized conditions, the enrichment factors for cypermethrin and permethrin were 1545- and 2138-fold, respectively. The developed method was successfully used to detect the two pyrethroid residues in river water samples.

Persistence and Dissipation of Dichlorvos and Profenofos on Mulberry Leaves

Persistence and dissipation properties of dichlorvos and profenofos in/on mulberry leaves were studied. Dichlorvos (76 WSC) and profenophos (50 EC) were sprayed at recommended dose (2 mL/L) and double the dose (4 mL/L) and leaf samples were drawn at different intervals in triplicate and residues derived from the QuEChERS method were quantified on gas chromatography-electron capture detector (GC-ECD). The initial deposits of dichlorvos and profenofos were between 1.18 to 2.79 and 2.19 to 4.72 μg g-1 in the recommended and double the dose, respectively. Dichlorvos dissipated to 0.03 (98.8%) and 0.031 μg g-1 (97.38%) in recommended and double the doses, respectively three days after spray. Residues of profenofos dissipated to the extent of 95.8% and 94.30%, respectively, at recommended and double doses in 15 days. Dissipation pattern followed first order kinetics for both the insecticides with half life periods of 0.52 and 4.29 days for dichlorvos and profenophos, respectively. Present studies suggest that the use of dichlorvos and profenofos at the recommended doses does not pose any hazard to silkworm, if waiting periods of 2.89 and 32 days respectively,were observed before harvesting mulberry leaves to feed silkworms.

Stereoselectivity of an enzyme-linked, immunosorbent assay for S-bioallethrin

A sensitive, specific, indirect and competitive enzyme-linked immunosorbent assay (ELISA) based on a polyclonal antibody (PcAb) for S-bioallethrin was developed. The hapten-bovine serum albumin (BSA) conjugate was used as an immunogen while the hapten-ovalbumin (OVA) conjugate was applied as a coating antigen. An anti-S-bioallethrin PcAb was obtained from immunized New Zealand white rabbits, and an ELISA for S-bioallethrin was developed. The inhibition standard curve of S-bioallethrin was obtained using optimized conditions (40% methanol, 0.5 mol L−1Na+, pH 6.5). The half-maximal inhibition concentration (IC50) was 0.089 μg mL−1, and the limit of detection (LOD, IC10) was 0.0025 μg mL−1. Very low cross-reactivity (CR) was found for allethrin and other pyrethroids. The CR for the stereoisomer demonstrates that the antibody has notable stereoselectivity for S-bioallethrin. Water and dust spiked with S-bioallethrin were analyzed by ELISA and acceptable recovery rates, 90.7% to 111.7%, were obtained. The ELISA results were confirmed by gas chromatography/electron capture detection (GC-ECD) and the proposed ELISA could be a highly sensitive, stereoselective, simple, rapid and inexpensive method for the detection of S-bioallethrin in environmental samples.

Microwave assisted extraction combined with dispersive liquid–liquid microextraction as a sensitive sample preparation method for the determination of haloanisoles and halophenols in cork stoppers and oak barrel sawdust

In this paper, a method based on microwave assisted extraction (MAE) in combination with dispersive liquid–liquid microextraction (DLLME) has been proposed as a new approach for the sensitive determination of cork taint responsible compounds in cork stoppers and oak barrel sawdust. For this purpose, haloanisoles and halophenols were extracted from the solid samples using a MAE method. Subsequently, a DLLME-derivatisation procedure was applied on the MAE extracts and the analytes were determined by gas chromatography–electron capture detection (GC–ECD). Parameters affecting the DLLME-derivatisation method were exhaustively investigated by means of experimental design methodology. Once optimised, the proposed method showed satisfactory linearity (correlation coefficients over 0.991), repeatability (below 10.4%) and inter-day precision (below 11.2%). Detection limits obtained were similar or even lower than previously reported. The results obtained proved the suitability of the combination of MAE with DLLME as a sensitive sample preparation methodology for the analysis of haloanisoles and halophenols in solid enological matrices.

Comprehensive Screening Study of Pesticide Degradation via Oxidation and Hydrolysis

This comprehensive study focused on the reactivity of a set of 62 pesticides via oxidization by free chlorine, monochloramine, chlorine dioxide, hydrogen peroxide, ozone, and permanganate; photodegradation with UV(254); and hydrolysis at pH 2, 7, and 12. Samples were analyzed using direct injection liquid chromatography-mass spectrometry detection or gas chromatography-electron capture detection after liquid-liquid extraction. Many pesticides were reactive via hydrolysis and/or chlorination and ozonation mechanisms under typical drinking water treatment conditions, with less reactivity exhibited on average for chlorine dioxide, monochloramine, hydrogen peroxide, and UV(254). The pyrazole and organophosphorous pesticides were most reactive in general, whereas carbamates and others were less reactive. The screening study provides guidance for the pesticide/oxidation systems that are most likely to lead to degradates in water treatment and the environment.

Development and Application of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Detection of p,p′-DDE in Human Milk and Comparison of the Results against GC-ECD

1,1-Dichloro-2,2-bis(p-chlorophenyl) ethylene (p,p'-DDE) is the major metabolite of insecticide 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (p,p'-DDT) and a persistent organic pollutant (POPs) with concerns regarding its bioaccumulation and persistence in the environment and food chain. In the present study, an indirect competitive enzyme-linked immunosorbant assay (ic-ELISA) specific for the detection of p,p'-DDE is described. In hapten synthesis, 2,2'-bis(4-chlorophenyl)ethanol and glutaric anhydride were used as precursor and spacer arm, respectively. The hapten was then conjugated to bovine serum albumin (BSA) as immunogen for mouse immunization and also conjugated to ovalbumin as coating antigen for ELISA. The developed ic-ELISA was used for detecting p,p'-DDE in human milk samples and validated against the results from conventional gas chromatography-electron capture detection (GC-ECD). Coefficients of variation (%CV) of ELISA were 5.7-10.4% for intra-assay and 10.6-19.6% for interassay variations. The Pearson correlation coefficient of p,p'-DDE concentrations between ic-ELISA and GC-ECD was r = 0.766, which was in an acceptable range. The results indicate that the developed assay could be an alternative analytical tool for monitoring p,p'-DDE in lipimic matrices such as human milk.

Hybrid Field-Assisted Solid–Liquid–Solid Dispersive Extraction for the Determination of Organochlorine Pesticides in Tobacco with Gas Chromatography

A novel one-step sample preparation technique termed hybrid field-assisted solid-liquid-solid dispersive extraction (HF-SLSDE) was developed in this study. A simple glass system equipped with a condenser was designed as an extraction vessel. The HF-SLSDE technique was a three-phase dispersive extraction approach. Target analytes were extracted from the sample into the extraction solvent enhanced by the hybrid field. Meanwhile, the interfering components were adsorbed by dispersing sorbent. No cleanup step preceded chromatographic analysis. The efficiency of the HF-SLSDE approach was demonstrated in the determination of organochlorine pesticide (OCP) residues in tobacco with a gas chromatography-electron capture detector (GC-ECD). Various operation conditions were studied systematically. Low detection limits (0.3-1.6 μg/kg) and low quantification limits (1.0-4.5 μg/kg) were achieved under the optimized conditions. The recoveries of OCPs ranged from 70.2% to 118.2%, with relative standard deviations of <9.6%, except for the lowest fortification level. Because of the effect of the hybrid field, HF-SLSDE showed significant predominance compared with other extraction techniques. The dispersing sorbent with good cleanup ability used in this study was also found to be a microwave absorption medium, which could heat the nonpolar extraction solvent under microwave irradiation. Different microstructures of tobacco samples before and after extractions demonstrated the mechanism of HF-SLSDE was based on an explosion at the cell level. According to the results, HF-SLSDE was proved to be a simple and effective sample preparation method for the analysis of pesticide residues in solid samples and could potentially be extended to other nonpolar target analytes in a complex matrix.

Analysis of pesticide residues in drinking water samples using solid-phase micro-extraction (SPME) coupled to a gas chromatography-electron-capture detector (GC-ECD)

The headspace solid-phase micro-extraction (HS-SPME) method with polydimethylsiloxane (PDMS, 100 μm) fiber is a cost-effective technique for the determination of different pesticide residues in environmental and biological samples followed by a gas chromatography-electron-capture detector (GC-ECD). In the present study more than 147 water samples collected in and around Lucknow city were analyzed. The method detection limits and recovery percentages were found in the ranges of 0.05–0.20 μg/L and 87.1–95.3% respectively. The average concentration of organochlorine and organophosphorus pesticides in water samples within the city were ranged between 0.258–0.829 μg/L and 0.143–0.294 μg/L respectively. Organochlorine and organophosphorus pesticides in highway water samples ranged between 0.261–2.80 μg/L and 0.129–0.53 μg/L. Pyrethroid pesticides were not found in any of the water samples tested. The levels of pesticide residues were compared with their MRL values recommended by the Prevention of Food Adulteration Act (PFA), Government of India (1954).

Determination of Diazepam and Nordiazepam in the Plasma of Psychiatric Patients in Long-Term Treatment Employing Liquid-Liquid and Solid Phase Extraction by Gas Chromatography with Electron Capture Detection

A sensitive, rapid, and reproducible gas chromatography-electron capture detection (GC-ECD) method was developed for the determination of diazepam (DZP) and N-desmethyldiazepam (nordiazepam, NDZP) in plasma of psychiatric patients in long-term treatment. Liquid-liquid (LLE) and solid phase (SPE) extractions were employed as techniques of sample preparation. For both techniques, the extraction solvent was hexane: methanol (9: 1) at pH 9.0. The recoveries ranged from 89 to 99% for DZP and from 53 to 69% for NDZ. Some validation parameters were found: linearity in spiked human plasma from 5 to 1200 ng mL−1 for DZP and 10 to 1200 ng mL−1 for NDZP using SPE and LLE techniques. The values for within- and between-day precision (% coefficient of variation) and accuracy (% relative error) were lower than 15%. The methods were applied to the analysis of samples from 54 patients under DZP treatment and proved to be suitable for routine therapeutic drug monitoring. Both studied techniques showed similar results with correlation coefficients higher than 0.98 for the determination of DZP and NDZP in sample plasma of psychiatric patients in continuous use of DZP. The increase in DZP plasma levels was not linear with the increase in administered drug dose, denoting the influence of individual factors, and the magnitude of the therapeutical monitoring to verify the clinical response.