Determination of lipid mediators in breast cancer cells using lyophilization‒supercritical fluid extraction online coupled with supercritical fluid chromatography‒quadrupole tandem mass spectrometry

A lyophilization?supercritical fluid extraction coupled with supercritical fluid chromatography?quadrupole tandem mass spectrometry online method was developed for the determination of lipid mediators in breast cancer cells. Supercritical fluid extraction was applied to the cell samples for the first time due to the use of lyophilization. The conditions of supercritical fluid extraction and supercritical fluid chromatography?quadrupole tandem mass spectrometry were investigated systematically. Under the optimized conditions, all the calibration curves for the lipid mediators showed good linearity (correlation coefficient > 0.99). The limits of detection and the limits of quantification were in the range of 0.190?5.36 pg and 0.560?16.2 pg, respectively. The recoveries were in the range of 70.3?125%. The relative standard deviations of the precision ranged from 1.49?18.7% and the accuracies were higher than 84%. Compared with liquid?liquid extraction coupled with liquid chromatography and tandem mass spectrometry method, the present approach reduced the manual labor and obtained higher sensitivity as well as higher extraction recoveries for all 15 lipid mediators. Finally, the online method was applied to the quantification of lipid mediators in breast cancer cells and normal mammary epithelial cells. On the basis of the results, this lyophilization?supercritical fluid extraction online coupled with supercritical fluid chromatography?quadrupole tandem mass spectrometry method showed great promise in the analysis of lipid mediators in complex biological samples.     

Determination of 14 heterocyclic aromatic amines in meat products using solid‐phase extraction and supercritical fluid chromatography coupled to triple quadrupole mass spectrometry

A novel, simple, and sensitive method has been developed for simultaneous determination of 14 heterocyclic aromatic amines in meat product using solid‐phase extraction combined with ultrahigh‐performance supercritical fluid chromatography coupled to tandem quadrupole mass spectrometry. The analytes could be separated within 7 min and identified using their retention times and mass. The developed method was validated based on the linearity, limits of quantification, precision, and accuracy. The recovery ranged from 52.3 to 97.5% with an acceptable standard deviation, which is not higher than 6%. The limits of quantitation ranged from 0.03 to 0.17 µg/kg. The selectivity and sensitivity were satisfactory in multiple reaction monitoring mode. The method was applied to commercial meat products, and the results demonstrated that the novel method has potential for the analysis of the targets in food matrices. This is the first work reporting the simultaneous quantification of 14 heterocyclic aromatic amines by means of ultrahigh‐performance supercritical fluid chromatography coupled to tandem quadrupole mass spectrometry.     

Analysis of selected designer benzodiazepines by ultra high performance liquid chromatography with high‐resolution time‐of‐flight mass spectrometry and the estimation of their partition coefficients by micellar electrokinetic chromatography

A new ultra high performance liquid chromatography with electrospray ionization time of flight mass spectrometry method for the selective and sensitive separation, identification, and determination of selected designer benzodiazepines (namely, pyrazolam, phenazepam, etizolam, flubromazepam, diclazepam, deschloroetizolam, bentazepam, nimetazepam, and flubromazolam) in human serum was developed. The separation of the studied designer benzodiazepines was achieved on C₁₈ chromatographic column using gradient elution within 6 min without any significant matrix interferences. Liquid–liquid extraction with butyl acetate was applied for serum samples cleanup and preconcentration of studied designer benzodiazepines. The method was validated in terms of linearity, limit of detection, limit of quantification, matrix effects, specificity, precision, accuracy, recovery, and sample stability. The limit of detection values were 0.10–0.15 ng/mL. The method was applied to a spiked serum sample to demonstrate its applicability for systematic toxicology analysis. Furthermore, a capillary chromatographic method with micellar electrokinetic chromatography was used for the estimation of partition coefficients of studied designer benzodiazepines as important parameters to evaluate their pharmacological and toxicological properties.     

Quantification of docetaxel and its main metabolites in human plasma by liquid chromatography/tandem mass spectrometry

Docetaxel is an antineoplastic agent widely used in therapeutics. The objective of this study was to develop and validate a routine assay, using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), for the simultaneous quantification of docetaxel and its main hydroxylated metabolites in human plasma. A structural analogue, paclitaxel, was used as the internal standard. Determination of docetaxel and four metabolites (M1, M2, M3 and M4) was achieved using only 100 microL of plasma. Liquid-liquid extraction was used for sample preparation, with extraction efficiency of at least 90% for all analytes. Detection used positive-mode electrospray ionization in selected reaction monitoring mode. The lower limit of quantification (LLOQ) was 0.5 ng/mL for all analytes. The assay was linear in the calibration curve range 0.5-1000 ng/mL and acceptable precision and accuracy (<15%) were obtained with concentrations above the LLOQ. This method was sufficiently selective and sensitive for quantification of metabolites in plasma from cancer patients receiving docetaxel chemotherapy, and is suitable for routine analyses during pharmacokinetic studies.     

Combined quantification of paclitaxel,docetaxel and ritonavir in human feces and urine using LC‐MS/MS

A combined assay for the determination of paclitaxel, docetaxel and ritonavir in human feces and urine is described. The drugs were extracted from 200 μL urine or 50 mg feces followed by high‐performance liquid chromatography analysis coupled with positive ionization electrospray tandem mass spectrometry. The validation program included calibration model, accuracy and precision, carry‐over, dilution test, specificity and selectivity, matrix effect, recovery and stability. Acceptance criteria were according to US Food and Drug Administration guidelines on bioanalytical method validation. The validated range was 0.5–500 ng/mL for paclitaxel and docetaxel, 2–2000 ng/mL for ritonavir in urine, 2–2000 ng/mg for paclitaxel and docetaxel, and 8–8000 ng/mg for ritonavir in feces. Inter‐assay accuracy and precision were tested for all analytes at four concentration levels and were within 8.5% and <10.2%, respectively, in both matrices. Recovery at three concentration levels was between 77 and 94% in feces samples and between 69 and 85% in urine samples. Method development, including feces homogenization and spiking blank urine samples, are discussed. We demonstrated that each of the applied drugs could be quantified successfully in urine and feces using the described assay. The method was successfully applied for quantification of the analytes in feces and urine samples of patients. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantification of 3‐n‐butylphthalide in beagle plasma samples by supercritical fluid chromatography with triple quadruple mass spectrometry and its application to an oral bioavailability study

A high‐throughput, rapid, sensitive, environmentally friendly, and economical supercritical fluid chromatography with triple quadruple mass spectrometry method was established and validated for the first time to determine a cerebral stroke treatment drug named 3‐n‐butylphthalide in dog plasma. Plasma samples were prepared by protein precipitation with methanol and the analytes were eluted on an ACQUITY UPC^2TM HSS‐C₁₈ SB column (3 × 100 mm, 1.8 μm) maintained at 50°C. The mobile phase comprised supercritical carbon dioxide/methanol (90:10, v/v) at a flow rate of 1.5 mL/min, the compensation solvent was methanol at a flow rate of 0.2 mL/min and the total run time was 1.5 min per sample. The detection was carried out on a tandem mass spectrometer with an electrospray ionization source. Calibration curves were linear over the concentration range of 1.02–1021.00 ng/mL (r² ≥ 0.993) with the lower limit of quantification of 1.02 ng/mL. The intra‐ and inter‐day precision values were below 15% and the accuracy was from 97.90 to 103.70% at all quality control levels. The method was suitable for a pharmacokinetic study of 3‐n‐butylphthalide in beagle dogs.     

A sensitive,high‐throughput,and ecofriendly method for the determination of lumefantrine,artemether, and its active metabolite dihydroartemisinin by supercritical fluid chromatography and tandem mass spectrometry

A quick and sensitive supercritical fluid chromatography with tandem mass spectrometry method for the simultaneous determination of lumefantrine, artemether, and its active metabolite dihydroartemisinin in rat plasma was developed and validated. The chromatographic separation was performed on an ACQUITY UPC²™ BEH 2‐EP column within 2.5 min by gradient elution using compressed CO₂ and methanol containing 2 mM ammonium acetate as the mobile phases. Detection was achieved by multiple reaction monitoring using electrospray ionization in the positive ionization mode. For sample preparation, 50 μL of the sample was processed by modified high‐throughput, one‐step protein precipitation using hydrogen peroxide as a stabilizer to protect the endoperoxide‐containing artemisinin derivatives from degradation. The calibration curves were linear over the concentration range of 2.0–1000 ng/mL for both artemether and dihydroartemisinin, and 1.0–5000 ng/mL for lumefantrine. The values of selectivity, lower limit of quantification, linearity, accuracy, precision, matrix effects, stability, and recovery met the acceptable range according to the Food and Drug Administration guidelines. The developed method enables high resolution and speed as well as low cost, low solvent consumption, and short time and was successfully applied to pharmacokinetic studies through the intravenous administration of an artemether–lumefantrine lipid emulsion in rats.     

Self‐assembly of poly(ionic liquid) functionalized mesoporous magnetic microspheres for the solid‐phase extraction of preservatives from milk samples

In this work, a novel, rapid, and simple analytical method was proposed for the detection of parabens in milk sample by gas chromatography coupled with mass spectrometry. At the same time, milk sample was pretreated by magnetic solid phase extraction, which detected up to five parabens. A series of important parameters of magnetic solid phase extraction were investigated and optimized, such as pH value of loading buffer, amount of material, adsorption time, ionic strength, eluting solvents, and eluting time. Under the optimized conditions, the corresponding values were more than 0.9991, limits of detection and the limit of quantification were 0.1 and 0.5 ng/mL, respectively. In addition, the recoveries were achieved in range of 95–105%, the liner range were within 0.1–600 ng/mL, and the relative standard deviations were even lower than 5%.     

Quantitative determination of acidic hydrolysis products of Chemical Weapons Convention related chemicals from aqueous and soil samples using ion‐pair solid‐phase extraction and in situ butylation

Chemical warfare agents such as organophosphorus nerve agents, mustard agents, and psychotomimetic agent like 3‐quinuclidinylbenzilate degrade in the environment and form acidic degradation products, the analysis of which is difficult under normal analytical conditions. In the present work, a simultaneous extraction and derivatization method in which the analytes are butylated followed by gas chromatography and mass spectrometric identification of the analytes from aqueous and soil samples was carried out. The extraction was carried out using ion‐pair solid‐phase extraction with tetrabutylammonium hydroxide followed by gas chromatography with mass spectrometry in the electron ionization mode. Various parameters such as optimum concentration of the ion‐pair reagent, pH of the sample, extraction solvent, and type of ion‐pair reagent were optimized. The method was validated for various parameters such as linearity, accuracy, precision, and limit of detection and quantification. The method was observed to be linear from 1 to 1000 ng/mL range in selected ion monitoring mode. The extraction recoveries were in the range of 85–110% from the matrixes with the limit of quantification for alkyl phosphonic acids at 1 ng/mL, thiodiglycolic acid at 20 ng/mL, and benzilic acid at 50 ng/mL with intra‐ and interday precisions below 15%. The developed method was applied for the samples prepared in the scenario of challenging inspection.     

l‐Cysteine‐modified magnetic microspheres for extraction and quantification of saxitoxin in rat plasma with liquid chromatography and tandem mass spectrometry

Saxitoxin, which is one of the most typical paralytic shellfish poisoning toxins, ranks the highest intoxication rate of marine biological poisoning cases globally. Efficient clean‐up and extraction of saxitoxin from complex biological matrices are imperative for the analysis and concentration monitoring of the toxin when correlative poisoning cases happen. Herein, l ‐cysteine‐modified magnetic microspheres based on metal‐organic coordination were synthesized by a facile approach and applied for magnetic solid‐phase extraction of saxitoxin from rat plasma samples before liquid chromatography–tandem mass spectrometry detection. Parameters, including adsorbent amount, extraction time, desorption solution, and desorption time that could affect the extraction efficiency, were respectively investigated. The developed method demonstrated good linearity in the range of 5–300 ng/mL (R² = 0.9985) with a limit of quantification of 5 ng/mL and a limit of detection of 0.5 ng/mL, acceptable accuracy. and precision of within‐run and between‐run.     

Ultra‐performance liquid chromatography‐tandem mass spectrometry method for the determination of febuxostat in dog plasma and its application to a pharmacokinetic study

A rapid, sensitive and selective ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the determination of febuxostat in dog plasma. Using paclitaxel as an internal standard (IS), a simple liquid–liquid extraction method with ethyl acetate was adopted for plasma sample pretreatment. Separation was carried out on an Acquity UPLC BEH C₁₈ column with a mobile phase consisting of acetonitrile and water (containing 0.2% formic acid). The assay was linear in the concentration ranged from 5 to 5000 ng/mL with a lower limit of quantification of 5 ng/mL for febuxostat. The single run analysis was as short as 2.0 min. Finally, the developed method was successfully applied to the pharmacokinetic study of febuxostat tablets following oral administration at a single dose of 40 mg in beagle dogs. Copyright © 2012 John Wiley & Sons, Ltd.     

Combination of a sub‐3 μm superficially porous particle packed column with charged aerosol detection for the simple and sensitive measurement of nine macrolides in human urine

Due to the lack of chromophores in many macrolides, analytical methods based on mass spectrometry and electrochemical detection coupled to liquid chromatography have been suggested to be suitable for the quantification of macrolides in complex matrices. In this study, a simple and sensitive analytical method was established for the simultaneous measurement of nine macrolides in human urine by combining a sub‐3 μm superficially porous particle packed column with charged aerosol detection. After thorough investigation of various sample preparation methods, including two liquid–liquid extraction methods and four solid‐phase extraction methods, HLB solid‐phase extraction was selected and further optimized. Absolute recovery of the optimized sample preparation method ranged from 99.5–110.2%, indicating its very high extraction/clean‐up efficiency. For chromatography, parameters influencing macrolide separation were systematically optimized, and the resulting conditions allowed baseline separation of nine macrolides within 24 min using a very simple mobile phase. The established method was validated for linearity, limit of detection, limit of quantification, absolute recovery, and precision. Based on its limit of detection (0.025–0.100 μg/mL), the method had similar or greater sensitivity than most methods based on electrochemical detection. It was found that the current method was appropriate for application to real human urine samples after drug administration.     

A sensitive,high‐throughput,and eco‐friendly analysis of daidzein and its valine carbamate prodrug in rat plasma by supercritical fluid chromatography with tandem mass spectrometry

A valine carbamate prodrug of daidzein was synthesized to improve its bioavailability because of the poor solubility and low permeability of daidzein. To evaluate the pharmacokinetic behavior of the prodrug, a sensitive and high‐throughput method was developed and validated for the simultaneous determination of daidzein and its prodrug in rat plasma. The samples were extracted by ethyl acetate and then analyzed by a supercritical fluid chromatography with electrospray ionization tandem mass spectrometry method. The separation was achieved by an ACQUITY UPC²™ BEH 2‐EP column maintained at 40°C using carbon dioxide (≥99.99%) and methanol within 3.0 min by gradient elution. The mass transition ion pairs were m/z 254.8→136.7, 398.0→254.9, and 271.0→91.07 for daidzein, the prodrug, and genistein, respectively. The calibration curves were linear over the concentration ranges of 2–500 (r > 0.997) and 10.0–5000.0 ng/mL (r > 0.996) with lower limits of quantification of 2 and 10 ng/mL for daidzein and the prodrug, respectively. The intra‐ and interday accuracy and precision were within ±15% for all quality control samples. This developed method enabled high specificity, low cost, low solvent consumption, and a brief analysis time and was successfully applied to a bioavailability evaluation of daidzein and its carbamate prodrug.     

Hydrophilic interaction liquid chromatography tandem mass spectrometry analysis of malonyl‐coenzyme A in breast cancer cell cultures applying online solid‐phase extraction

Cofactors such as coenzyme A and its derivatives acetyl‐coenzyme A and malonyl‐coenzyme A are involved in many metabolic pathways. Due to trace level concentrations in biological samples and the high reactivity of cofactors, a fast, sensitive, and selective method for quantification is mandatory. In this study, online solid‐phase extraction was coupled successfully to hydrophilic interaction liquid chromatography with tandem mass spectrometry for isolation of analytes in complex matrix and quantification by external calibration. Online solid‐phase extraction was carried out by application of a weak anion‐exchange column, whereas hydrophilic interaction liquid chromatography separation was performed on an amide modified stationary phase. Sample preparation of the extracts before the analysis was reduced to a centrifugation and dilution step. Moreover, the applied online solid‐phase extraction significantly reduced matrix effects and increased the signal‐to‐noise ratio. The limit of detection and the limit of quantification were in the lower nanomolar range. Finally, the applicability of this method was demonstrated on MCF‐7 breast cancer cell cultures, a commonly used model system, where acetyl‐coenzyme A and malonyl‐coenzyme A were determined using standard addition procedure in concentrations of 1.98 μM and 41 nM, respectively.     

Simultaneous LC‐MS/MS bioanalysis of etoposide and paclitaxel in mouse tissues and plasma after oral administration of self‐microemulsifying drug‐delivery systems

In this study, a liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated to simultaneously determine the anticancer drugs etoposide and paclitaxel in mouse plasma and tissues including liver, kidney, lung, heart, spleen and brain. The analytes were extracted from the matrices of interest by liquid–liquid extraction using methyl tert‐butyl ether–dichloromethane (1:1, v/v). Chromatographic separation was achieved on an Ultimate XB‐C₁₈ column (100 × 2.1 mm, 3 μm) at 40°C and the total run time was 4 min under a gradient elution. Ionization was conducted using electrospray ionization in the positive mode. Stable isotope etoposide‐d3 and docetaxel were used as the internal standards. The lower limit of quantitation (LLOQ) of etoposide was 1 ng/g tissue for all tissues and 0.5 ng/mL for plasma. The LLOQ of paclitaxel was 0.4 ng/g tissue and 0.2 ng/mL for all tissues and plasma, respectively. The coefficients of correlation for all of the analytes in the tissues and plasma were >0.99. Both intra‐ and inter‐day accuracy and precision were satisfactory. This method was successfully applied to measure plasma and tissue drug concentrations in mice treated with etoposide and paclitaxel‐loaded self‐microemulsifying drug‐delivery systems.     

Analyzing multiple pesticides in tobacco leaf using gas chromatography with quadrupole time‐of‐flight mass spectrometry

A method combining gas chromatography with quadrupole time‐of‐flight mass spectrometry has been developed for the simultaneous analysis of multiple pesticide residues in tobacco leaf. The retention index and high accurate masses of ions from the first‐stage and the second‐stage mass spectra of each pesticide were collected for qualitation and quantification. A total of 115 pesticides were evaluated. The extract from organic tobacco leaf was used as a model matrix. The limit of detection was <10 ng/mL, and the limit of quantification was in the range of 1–20 ng/mL for 95% of the tested pesticides. The correlation coefficients were >0.9900 for all tested pesticides. At three concentrations (10, 50, and 100 ng/mL), most compounds presented satisfactory recoveries ranging from 70 to 120% and good precision <20%. Finally, three tobacco leaf samples collected from a local market were analyzed. A total of three pesticides were found, including dimethachlon, triadimenol, and flumetralin. Each pesticide was confirmed by the presence of three ions at the expected retention index and mass. In conclusion, gas chromatography with quadrupole time‐of‐flight mass spectrometry appears to be one of the most efficient tools for the analysis of pesticide residues in tobacco leaf.     

Supercritical fluid chromatography with tandem mass spectrometry combined with postcolumn compensation and one‐step acetone protein precipitation to evaluate the bioavailability of probucol solid dispersion tablet

Solid dispersion technology was used to improve the bioavailability of probucol due to its low hydrophilicity and high lipophilicity. In this study, a highly rapid and sensitive supercritical fluid chromatography with tandem mass spectrometry method was optimized and validated for the determination of probucol in beagle dog plasma with diazepam as an internal standard. The analyte and internal standard were extracted by acetone and then separated on a polar 2‐ethylpyridine phase column (100 mm × 3 mm, 1.7 μm) at a flow rate of 1.0 mL/min using CO₂ (≥99.99%) and methanol (95:5, v/v) as the mobile phase. The mass transition ion‐pair was m/z 515.6→236.2 and 285.2→193.1 for probucol and internal standard, respectively. Excellent linearity was observed over the concentration range of 5–5000 ng/mL (r² ≥ 0.9999) with a lower limit of quantification of 5 ng/mL. The intra‐ and inter‐day accuracy and precision for all quality control samples were within ±15%. The proposed method was accurate, rapid and reproducible, which was successfully applied to a bioavailabilty evaluation of probucol solid dispersion tablets.     

Determination of phthalates in beverages using multiwalled carbon nanotubes dispersive solid‐phase extraction before HPLC–MS

A microdispersive solid‐phase extraction method has been developed using multiwalled carbon nanotubes of 110–170 nm diameter and 5–9 μm length for the extraction of a group of nine phthalic acid esters (i.e., bis(2‐methoxyethyl) phthalate, bis‐2‐ethoxyethyl phthalate, dipropyl phthalate, butylbenzyl phthalate, bis‐2‐n‐butoxyethyl phthalate, bis‐isopentyl phthalate, bis‐n‐pentyl phthalate, dicyclohexyl phthalate, and di‐n‐octyl phthalate) from tap water as well as from different beverages commercialized in plastic bottles (mineral water, lemon‐ and apple‐flavored mineral water, and an isotonic drink). Determination was carried out by high‐performance liquid chromatography coupled to mass spectrometry. The extraction procedure was optimized following a step‐by‐step approach, being the optimum extraction conditions: 50 mL of each sample at pH 6.0, 80 mg of sorbent, and 25 mL of acetonitrile as elution solvent. To validate the methodology, matrix‐matched calibration and a recovery study were developed, obtaining determination coefficients >0.9906 and absolute recovery values between 70 and 117% (with relative standard deviations < 17%) in all cases. The limits of quantification of the method were between 0.173 and 1.45 μg/L. After the evaluation of the matrix effects, real samples were also analyzed, finding butylbenzyl phthalate in all samples (except in apple‐flavored mineral water), though at concentrations below its limit of quantification of the method.     

Simultaneous determination of selected estrogenic endocrine disrupting chemicals and bisphenol A residues in whole milk using fabric phase sorptive extraction coupled to HPLC‐UV detection and LC‐MS/MS

A simple and sensitive analytical methodology is developed for rapid screening and quantification of selected estrogenic endocrine disrupting chemicals and bisphenol A from intact milk using fabric phase sorptive extraction in combination with high‐performance liquid chromatography coupled to ultraviolet detection/tandem mass spectrometry. The new approach eliminates protein precipitation and defatting step from the sample preparation workflow. In addition, the error prone and time‐consuming solvent evaporation and sample reconstitution step used as the sample post‐treatment has been eliminated. Parameters with most significant impact on the extraction efficiency of fabric phase sorptive extraction including sorbent chemistry, sample volume, extraction time have been thoroughly studied and optimized. Separation of the selected estrogenic endocrine disrupting chemicals including α‐estradiol, hexestrol, estrone, 17α‐ethinyl estradiol, diethylstilboestrol, and bisphenol A were achieved using a Zorbax Extend‐C18 high‐performance liquid chromatography column (15 cm × 4.6 mm, 5 μm particle size). The limit of detection values obtained in fabric phase sorptive extraction with high‐performance liquid chromatography with ultraviolet detection ranged from 25.0 to 50.0 ng/mL. The method repeatability values were 3.6–13.9 (relative standard deviation, %) and intermediate precision values were 4.6–12.7 (relative standard deviation, %). The fabric phase sorptive extraction method was also coupled to liquid chromatography with tandem mass spectrometry for identifying each endocrine disrupting chemical at 10 ng/mL.     

Dispersive solid‐phase extraction adsorbent of methamphetamine using in‐situ synthesized carbon‐based conductive polypyrrole nanocomposite: focus on clinical applications in human urine

Determination of methamphetamine is of great importance in clinical and forensic laboratories because there are low dosages of drugs in the biological media and social problems created due to the methamphetamine consumption. Polymeric carbon based‐nano composites are reasonable candidates for dispersive solid phase extraction method due to facial and affordable synthesis process and high selectivity and sensitivity. Nano graphene oxide polypyrolle composite was synthesized and employed as dispersive solid‐phase extraction adsorbent for methamphetamine extraction from complex urine matrix. Full characterization of the prepared nano graphene oxide polypyrolle composite was completed and the influential extraction parameters were investigated through one‐parameter‐at‐a‐time method. High‐performance liquid chromatography detectors were applied as detection and quantification instrument. The optimized extraction parameters included 300 µL of methanol, 10 min of extraction and desorption time, 6000 stirring rate, urine pH value of 10, 60 mg of adsorbent, and 6 mL of urine volume. After outlining the calibration curve, the linear range of the method was considered as 30–800 ng/mL. The detection limit for the suggested method was 9 ng/mL. The analysis of addicted subjects with the proposed method confirmed the utility of the method in different analytical and clinical laboratories.