Selective extraction based on poly(MAA‐VB‐EGMDA) monolith followed by HPLC for determination of hordenine in plasma and urine samples

Hordenine is an active compound found in several foods, herbs and beer. In this work, a novel sorbent was fabricated for selective solid‐phase extraction (SPE) of hordenine in biological samples. The organic polymer sorbent was synthesized in one step in the plastic barrel of a syringe by a pre‐polymerization solution consisting of methacrylic acid (MAA), 4‐vinylphenylboronic acid (VB) and ethylene glycol dimethacrylate (EGDMA). The conditions for preparation were optimized to generate a poly(MAA‐VB‐EGMDA) monolith with good permeability. The monolith exhibited good enrichment efficiency towards hordenine. By using tyramine as the internal standard, a poly(MAA‐VB‐EGMDA)‐based SPE‐HPLC method was established for analysis of hordenine. Conditions for SPE, including volume of eluting solvent, pH of sample solution, sampling rate and sample volume, were optimized. The proposed SPE‐HPLC method presented good linearity (R² = 0.9992) within 10–2000 ng/mL and the detection limits was 3 ng/mL, which is significantly more sensitive than reported methods. The method was also applied in plasma and urine samples; good capability of removing matrices was observed, while hordenine in low content was well extracted and enriched. The recoveries were from 90.6 to 94.7% and from 89.3 to 91.5% for the spiked plasma and urine samples, respectively, with the relative standard deviations <4.7%. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of UV filters in river water samples by in‐line SPE‐CE‐MS

The use of SPE coupled in‐line to CE using electrospray MS detection (in‐line SPE‐CE‐ESI‐MS) was investigated for the preconcentration and separation of four UV filters: benzophenone‐3, 2,2‐dihydroxy‐4‐methoxybenzophenone, 2,4‐dihydroxybenzophenone and 2‐phenylbenzimidazole‐5‐sulphonic acid. First, a CE‐ESI‐MS method was developed and validated using standard samples, obtaining LODs between 0.06 μg/mL and 0.40 μg/mL. For the in‐line SPE‐CE‐ESI‐MS method, three different sorbents were evaluated and compared: Oasis HLB, Oasis MCX, and Oasis MAX. For each sorbent, the main parameters affecting the preconcentration performance, such as sample pH, volume, and composition of the elution plug, and sample injection time were studied. The Oasis MCX sorbent showed the best performance and was used to validate the in‐line SPE‐CE‐ESI‐MS methodology. The LODs reached for standard samples were in the range between 0.01 and 0.05 ng/mL with good reproducibility and the developed strategy provided sensitivity enhancement factors between 3400‐fold and 34 000‐fold. The applicability of the developed methodology was demonstrated by the analysis of UV filters in river water samples.     

An in‐line SPE strategy to enhance sensitivity in CE for the determination of pharmaceutical compounds in river water samples

In this study, the suitability of solid‐phase extraction (SPE) coupled in‐line to CE with UV–Vis detection was evaluated for the preconcentration and separation of diluted solutions of five pharmaceuticals compounds: benzafibrate, piroxicam, diclofenac sodium, naproxen and clofibric acid. An SPE analyte concentrator containing Oasis^® HLB sorbent was constructed without frits and placed near the inlet end of the separation capillary. Different parameters such as sample pH, composition and volume of the elution plug and sample loading time were studied in order to obtain the maximum preconcentration factors. The LODs reached for standard samples were in the range 0.06–0.5 ng/mL with good reproducibility, and the developed strategy provides sensitivity enhancement factors around 14 000‐fold in peak area and 5900‐fold in peak height compared with the normal hydrodynamic injection. Finally, river water samples fortified with the pharmaceutical compounds were analyzed by the developed in‐line SPE‐CE‐UV method in order to show the potential of the methodology for the analysis of environmental aquatic samples. For these samples, high values of relative recoveries, between 73–107% and 79–103% for two concentration levels, 5 and 25 ng/mL, respectively, were obtained and LODs ranged between 0.19 and 1 ng/mL.     

Liquid chromatographic high-throughput analysis of ketamine and its metabolites in human plasma using a monolithic silica column and solid phase extraction

A rapid and sensitive liquid chromatographic (LC) assay was developed for the simultaneous determination of ketamine (KE) and its two main metabolites, namely, norketamine (NK) and dehydronorketamine (DHNK) in human plasma. Each compound together with an internal standard (Labetalol) was extracted from the plasma matrix using solid phase extraction (SPE). The applicability of monolithic LC phases in the field of quantitative bioanalysis has been evaluated. The existing method with UV detection set at 220 nm was successfully transferred from a conventional reversed phase column to a 10 cm × 4.6 mm i.d. monolithic silica column. By simply increasing the mobile phase flow-rate, run times were about six-fold reduced and consumption of mobile phase were about two-fold decreased, while the chromatographic resolution of the analytes remain unaffected. The method was validated over the range 25-2000 ng/mL for KE, 25-1500 ng/mL for NK, and 15-750 ng/mL for DHNK. The method proved to be precise (within-run precision ranges from 2.2 to 7.2% and between-run precision ranges from 3.7 to 8.2%) and accurate (within-run accuracies ranged from 1.3 to 7.2% and between-run accuracies ranged from 1.5 to 8.7%). The mean absolute recoveries were 95.3, 96.9, and 103.9% for KE, NK and DHNK, respectively. The limit of quantitation (LOQ) and limit of detection (LOD) for KE and NK in human plasma were 25 and 12.5 ng/mL, respectively, and for DHNK were 15 and 7.5 ng/mL (S/N = 3). The assay should be suitable for use in routine determination of KE and its metabolites in human plasma.     

On‐line flow‐through extraction–preconcentration‐large volume injection‐RP LC for trace determination of pyrethroids in Slovak soil

A rapid micro‐analytical multiresidue method was developed for analysis of pyrethroids (kadethrin K, cypermethrin C and permethrin P) in soil micro‐sample (200 mg). It uses on‐line flow‐through extraction of soil micro‐samples (packed into a short glass column) with a methanol‐aqueous citric acid buffer mixture, successive on‐line SPE preconcentration of analytes from the extract and on‐line RP‐HPLC analysis with UV photometric detection. The separation of pyrethroids is performed on a Purospher RP‐18e column with methanol/water as mobile phase. Effects of sorbent placed at the bottom of a short column holding the soil sample and different kinds of on‐line SPE columns were tested. Besides, the influence of volume of the effluent on the pyrethroids recovery was also studied. Calibration curves were linear over the range assayed from 0.01 to 0.2 μg/mL with correlation coefficients of linear regression (least‐squares method) in the range 0.998–0.999. Recovery studies were carried out at 0.25–1.00 μg/g dry soil fortification level and obtained recoveries were for K 81–84%, C 56–59% and for P 58–63%. Achieved LOD (confidence band) of studied pyrethroids were for large‐volume injection (1 mL) 4.5 ng K, 3.7 ng C, 3.6 ng P or 27 ng/g K, 32 ng/g C and 29 ng/g P in dry soil “solid sampling HPLC”.     

Assay of urinary 8‐hydroxy‐2′‐deoxyguanosine by capillary electrophoresis with spectrophotometric detection using a high‐sensitivity detection cell and solid‐phase extraction

A sensitive capillary electrophoretic method featuring spectrophotometric detection using a commercial Z‐cell was devised for the assay of 8‐hydroxy‐2′‐deoxyguanosine (8OHdG) in human urine. Solid‐phase extraction (SPE) based on hydrophilic‐lipophilic‐balanced RP sorbent was utilized for urine sample pretreatment and analyte preconcentration. The separation was carried out in conventional fused‐silica capillaries employing a Z‐cell with hydrodynamic sample injection (at 50 mbar for 12 s). The BGE (pH* 9.2, adjusted with 1 M NaOH) contained 0.15 M boric acid and 10% v/v ACN. The detection wavelength was 282 nm. The calibration curve for 8OHdG (measured in spiked urine) was linear in the range 10–1000 ng/mL; R² = 0.9993. The LOD was 3 ng/mL (11 nmol/L) of 8OHdG. Determination of the 8OHdG urinary levels was possible even in healthy individuals.     

Determination of rimantadine in human urine by HPLC using a monolithic stationary phase and on‐line post‐column derivatization

In the present study, we propose the first HPLC method coupled to postcolumn derivatization for the determination of rimantadine in human urine samples. The analyte and amantadine (internal standard) were isocratically separated using an RP monolithic stationary phase (100 × 4.6 mm id) with a mobile phase consisting of CH₃OH/phosphate buffer (25 mmol/L, pH 3.0) at a volume ratio of 50:50. Postcolumn derivatization involved on‐line reaction with o‐phthalaldehyde (20 mmol/L) and N‐acetyl‐cysteine (5 mmol/L) at alkaline medium (100 mmol/L borate pH 11.0). Spectrofluorimetric detection at λ_ex/λ_em = 340/455 nm enabled the selective and sensitive determination of rimantadine in urine samples at a range of 50–500 ng/mL with an LOD of 5 ng/mL. Human urine samples were analyzed successfully after SPE using hydrophilic‐lipophilic balanced RP cartridges (30 mg/mL, Oasis HLB). Recoveries ranged between 89.7 and 102.7%.     

Zeolitic imidazolate framework 8 (ZIF‐8) reinforced macroporous resin D101 for selective solid‐phase extraction of 1‐naphthol and 2‐naphthol from phenol compounds

Macroporous resin has been attracting intensive attention due to its critical role in separation and purification of natural products. Herein, a zeolitic imidazolate framework 8 reinforced macroporous resin D101 was prepared via a room temperature growth method and used for dispersive SPE of 1‐naphthol and 2‐naphthol. The parameters affecting the adsorption and desorption efficiency such as the sample pH, adsorbent amount, extraction time, desorption solvent, and desorption time were investigated. The as‐prepared adsorbent showed selectivity for 1‐naphthol and 2‐naphthol compared to other phenols. Under the optimum dispersive SPE conditions, the detection of 1‐naphthol and 2‐naphthol coupled with a CZE method was conducted and the LODs for 1‐naphthol and 2‐naphthol were 1.37 and 1.43 ng/mL, respectively. Moreover, the results of urine sample analysis showed the spiked recoveries to be in the range of 96.2–106.9%. This study indicated that D101@ZIF‐8 (where ZIF is zeolitic imidazolate framework) is a promising selective adsorbent for the analysis of 1‐naphthol and 2‐naphthol in urine samples.     

Molecularly imprinted polymer‐based solid phase clean‐up for analysis of ochratoxin A in beer,red wine,and grape juice

A simple, reliable, and low‐cost method based on molecularly imprinted polymer as a selective sorbent of SPE was proposed for the determination of ochratoxin A (OTA) in beer, red wine, and grape juice by HPLC coupled with fluorescence detection (HPLC‐FLD). Samples were diluted with water and cleaned up with an AFFINIMIP® SPE OTA column. After washing and eluting, the analyte was analyzed by HPLC‐FLD. Under the optimized conditions, LOD and LOQ for OTA were 0.025 and 0.08 ng/mL, respectively. The recoveries of OTA from beer, red wine, and grape spiked at 0.1, 2, and 5 ng/mL ranged from 91.6 to 101.7%. Furthermore, after a simple regenerated procedure, the molecularly imprinted polymer based SPE column could be reused at least 14 times to achieve more than 80% recoveries of OTA in real samples. The developed method was applied to the detection of 30 beer, red wine, and grape juice samples and only four samples were contaminated by OTA with levels below the legal limits.     

Development and validation of a HPLC method for quantification of rivastigmine in rat urine and identification of a novel metabolite in urine by LC‐MS/MS

A sensitive, specific and accurate HPLC method for the quantification of rivastigmine (RSM) in rat urine was developed and validated. The method involves the simple liquid–liquid extraction of RSM and pyridostigmine as an internal standard (IS) from rat urine with tertiary methyl butyl ether. The chromatographic separation of RSM and IS was achieved with 20 mm ammonium acetate buffer (pH 6.5) and acetonitrile (65:35, v/v) delivered at flow‐rate of 1 mL/min on a Kromasil KR‐100. The method was in linear range from 50 to 5000 ng/mL. The validation was done as per FDA guidelines and the results met the acceptance criteria. The method was successfully applied for the quantification of RSM in rat urine. Besides method validation, we have identified two metabolites of RSM in urine. Both the metabolites were characterized by HPLC‐PDA and LC‐MS/MS and it was found that one metabolite is novel. Copyright © 2010 John Wiley & Sons, Ltd.     

Study of immobilized metal affinity chromatography sorbents for the analysis of peptides by on‐line solid‐phase extraction capillary electrophoresis‐mass spectrometry

Several commercial immobilized metal affinity chromatography sorbents were evaluated in this study for the analysis of two small peptide fragments of the amyloid β‐protein (Aβ) (Aβ(1–15) and Aβ(10–20) peptides) by on‐line immobilized metal affinity SPE‐CE (IMA‐SPE‐CE). The performance of a nickel metal ion (Ni(II)) sorbent based on nitrilotriacetic acid as a chelating agent was significantly better than two copper metal ion (Cu(II)) sorbents based on iminodiacetic acid. A BGE of 25 mM phosphate (pH 7.4) and an eluent of 50 mM imidazole (in BGE) yielded a 25‐fold and 5‐fold decrease in the LODs by IMA‐SPE‐CE‐UV for Aβ(1–15) and Aβ(10–20) peptides (0.1 and 0.5 μg/mL, respectively) with regard to CE‐UV (2.5 μg/mL for both peptides). The phosphate BGE was also used in IMA‐SPE‐CE‐MS, but the eluent needed to be substituted by a 0.5% HAc v/v solution. Under optimum preconcentration and detection conditions, reproducibility of peak areas and migration times was acceptable (23.2 and 12.0%RSD, respectively). The method was more sensitive for Aβ(10–20) peptide, which could be detected until 0.25 μg/mL. Linearity for Aβ(10–20) peptide was good in a narrow concentration range (0.25–2.5 μg/mL, R² = 0.93). Lastly, the potential of the optimized Ni(II)‐IMA‐SPE‐CE‐MS method for the analysis of amyloid peptides in biological fluids was evaluated by analyzing spiked plasma and serum samples.     

Highly sensitive determination of tetrabromobisphenol A and bisphenol A in environmental water samples by solid‐phase extraction and liquid chromatography‐tandem mass spectrometry

Using bamboo‐activated charcoal as SPE adsorbent, a novel SPE method was developed for the sensitive determination of tetrabromobisphenol A and bisphenol A in environmental water samples by rapid‐resolution LC‐ESI‐MS/MS. Important parameters influencing extraction efficiency, including type of eluent, eluent volume, sample pH, volume and flow rate, were investigated and optimized. Under the optimal extraction conditions (eluent: 8 mL methanol, pH: 7; flow rate: 4 mL/min; sample volume: 100 mL), low LODs (0.01–0.02 ng/mL), good repeatability (6.2–8.3%) and wide linearity range (0.10–10 ng/mL) were obtained. Satisfied results were achieved when the proposed method was applied to determine the two target compounds in real‐world environmental water samples with spiked recoveries over the range of 80.5–119.8%. All these facts indicate that trace determination of tetrabromobisphenol A and bisphenol A in real‐world environmental water samples can be realized by bamboo‐activated charcoal SPE‐rapid resolution‐LC‐ESI‐MS/MS.     

Automated online pretreatment and cleanup recycle coupled with high‐performance liquid chromatography‐mass spectrometry for determination of deca‐bromodiphenyl ether in human serum

Automated online SPE‐HPLC‐MS was established for the determination of deca‐bromodiphenyl ether in human serum. The online SPE with large volume injection was utilized to enhance the sensitivity. Online SPE with dilution line greatly decreased matrices effect, which enabled serum samples to be injected directly into pre‐column. Washing line was designed for the system to solve the serious residual phenomenon and reduce the risk of sample wastage and contamination. Under the optimized conditions, the linear of the method was in the range 0.1–10 ng/mL with the LOD of 0.026 ng/mL. The recoveries of serum samples spiked with deca‐bromodiphenyl ether at 0.5 ng/mL was in the range from 83.30 to 102.7% with RSD in interday less than 8.67%. The satisfactory results demonstrated that the method of online sample pretreatment and cleanup recycle were reliable for human serum analysis.     

Enantioselective determination of cathinones in urine by high pressure in‐line SPE–CE

This work presents a strategy based on the in‐line coupling of SPE and CE for the chiral determination of cathinones (R,S‐mephedrone, R,S‐4‐methylephedrine, and R,S‐ methylenedioxypyrovalerone) in urine samples, using a sample pretreatment based on liquid‐liquid extraction. The chiral separation of the compounds is achieved by adding a mixture of 8 mM 2‐hydroxypropil β‐CD and 5 mM β‐CD to the BGE, which consists of 70 mM of monosodium phosphate aqueous solution at pH 2.5. Oasis HLB was the selected sorbent for the in‐line SPE device, and to reduce analysis time and LODs, several parameters affecting the in‐line SPE system were evaluated, such as pressure and time of sample injection and dimensions of the SPE device. The highest preconcentration factors were achieved by using 3 bar of injection pressure for 20 min with an in‐line SPE device of 2 mm length and 150 µm of i.d. The developed method was applied to determine the presence of the compounds in spiked urine samples. The LODs obtained were between 3 and 8 ng/mL, and these levels were below the usual concentrations at which these drugs are present in urine from cathinone abusers. Thus, the optimized method has the potential to be applied for toxicological and forensic purposes.     

Gas chromatography‐mass spectrometric method for the screening and quantification of illicit drugs and their metabolites in human urine using solid‐phase extraction and trimethylsilyl derivatization

A simple and rapid GC‐MS method has been developed for the screening and quantification of many illicit drugs and their metabolites in human urine by using automatic SPE and trimethylsilylation. Sixty illicit drugs, including parent drugs and their metabolites that are possibly abused in Korea, can be monitored by this method. Among them, 24 popularly abused illicit drugs were selected for quantification. Very delicate optimizations were carried out in SPE, trimethylsilylation derivatization, and GC/MS to enable such remarkable achievements. Trimethylsilylated analytes were well separated within 21 min by GC‐MS. In the validation results, the LOD of all the analytes were in the range of 2–75 ng/mL. The LOQ of the quantified analytes were in the range of 5–98 ng/mL. The linearity (r²) of the quantified analytes ranged 0.990–1.000 in each concentration range between 10 and 1000 ng/mL. The mean recoveries ranged from 62 to 126% at three different concentrations of each analyte. The inter‐day and inter‐person accuracies were within ?13.3～14.9%, and ?10.1～13.0%, respectively, and the inter‐day and inter‐person precisions were less than 12.9%. The method was reliable and efficient for the screening and quantification of abused illicit drugs in routine urine analysis.     

Characterization and tentative identification of new flunitrazepam metabolites in authentic human urine specimens using liquid chromatography‐Q exactive‐HF hybrid quadrupole‐Orbitrap‐mass spectrometry (LC‐QE‐HF‐MS)

Flunitrazepam (FNZ) is a potent hypnotic, sedative, and amnestic drug used to treat severe insomnia. In our recent study, FNZ metabolic profiles were investigated carefully. Six authentic human urine samples were purified using solid phase extraction (SPE) without enzymatic hydrolysis, and urine extracts were then analyzed by liquid chromatography‐Q exactive‐HF hybrid quadrupole‐Orbitrap‐mass spectrometry (LC‐QE‐HF‐MS), using the full scan positive ion mode and targeted MS/MS (ddms2) technique to make accurate mass measurements. There were 25 metabolites, including 13 phase I and 12 phase II metabolites, which were detected and tentatively identified by LC‐QE‐HF‐MS. In addition, nine previously unreported phase II glucuronide conjugates and four phase I metabolites are reported here for the first time. Eight metabolic pathways, including N‐reduction and O‐reduction, N‐glucuronidation, O‐glucuronidation, mono‐hydroxylation and di‐hydroxylation, demethylation, acetylation, and combinations, were implicated in this work, and 2‐O‐reduction together with dihydroxylation were two novel metabolic pathways for FNZ that were identified tentatively. Although 7‐amino FNZ is widely considered to be the primary metabolite, a previously unreported metabolites (M12) can also serve as a potential biomarker for FNZ misuse.     

Simultaneous quantitation of seven pyrethroid metabolites in human urine by capillary gas chromatography–mass spectrometry

Pyrethroid insecticides are applied in the residential environment, as well as in agricultural crops, for insect control purpose. We developed and validated an accurate, sensitive, and reproducible analytical method to simultaneously detect seven pyrethroid metabolites, namely, 3‐(2,2‐dichlorovinyl)‐2,2‐dimethyl‐(1‐cyclopropane) carboxylic acid, 3‐(2,2‐dibromovinyl)‐2,2‐dimethyl‐(1‐cyclopropane) carboxylic acid, 3‐phenoxybenzoic acid, 4‐fluoro‐3‐phenoxybenzoic acid, 2‐methyl‐3‐phenylbenzoic acid, 4‐chloro‐α‐isoproply benzeneacetic acid, and 3‐(2‐chloro‐3,3,3‐trifluoroprop‐1‐enyl)‐2,2‐dimethylcyclopropanecarboxylic acid, in human urine. This method employs deconjugation with enzyme, SPE using Agilent C18 cartridges on a RapidTrace SPE workstation, derivatization using hexafluoro isopropanol and N,N′‐diisopropylcarbodiimide, and compounds separation and identification on GC–MS. The detection limits of seven metabolites were 0.02–0.08 ng/mL in urine. The recoveries of seven metabolites were 81–104%, 85–99%, and 83–99% in urine specimens fortified at 0.1, 0.4, and 3.2 ng/mL concentrations, respectively. The overall coefficient of variation was 4.3–10.8% in two quality control specimens which were repeatedly measured during a period of 2 months. This method was applied to urine samples collected from children living in Boston, MA. The median concentrations of six detected pyrethroid metabolites ranged from 0.06 to 0.86 ng/mL in urine.     

Trace Determination of Rhodamine B and Rhodamine 6G Dyes in Aqueous Samples by Solid‐phase Extraction and High‐performance Liquid Chromatography Coupled with Fluorescence Detection

A simple and reliable method was developed to detect two basic synthetic dyes, rhodamine B (RB) and rhodamine 6G (R6G), in wastewater and surface water samples by high performance liquid chromatography with fluorescence detection (HPLC‐FLD). These dyes have been reported to be both mutagenic and carcinogenic in various organisms. The contents of these two dyes in water samples were extracted by Oasis HLB solid‐phase extraction (HLB‐SPE), and were then determined by an isocratic HPLC using an Atlantis® T3‐C18 column. Water samples at various pH conditions and the compositions of eluents for SPE were evaluated. The results indicate that the proposed method is precise and sensitive in analyzing these two basic synthetic dyes, and the limits of quantitation were 1.5 ng/L for RB and 0.3 ng/L for R6G in 100 mL of water samples. The recovery of analytes in spiked surface water and municipal wastewater treatment plant (WWTP) effluent samples ranged from 61 to 90% with the precision (RSD) ranging from 2 to 12%. The concentrations of analytes were detected in various water samples ranging from 0.7 to 81 ng/L.     

Comparison of C18 silica and multi‐walled carbon nanotubes as the adsorbents for the solid‐phase extraction of Chlorpyrifos and Phosalone in water samples using HPLC

A comparison between C₁₈ silica and multi‐walled carbon nanotubes (MWCNTs) in the extraction of Chlorpyrifos and Phosalone in environmental water samples was carried out using HPLC. Parameters affecting the extraction were type and volume of elution solvent, pH and flow rate of sample through the adsorbent. The optimum conditions obtained by C₁₈ cartridge for adsorption of these pesticides were 4 mL dichloromethane as elution solvent, sample pH of 5, flow rate of 1 mL/min, and those for MWCNT cartridge were 3 mL dichloromethane, pH of 5 and flow rate of 10 mL/min, respectively. Optimized mobile phase for separation and determination of these compounds by HPLC was methanol/water (80:20 v/v) with pH=5 (adjusted with phosphate buffer). Under optimal chromatographic and SPE conditions, LOD, linear range and precision (RSD n=8) were 3.03×10^?3, 0.01–5.00 μg/mL and 2.7% for Chlorpyrifos and 4.03×10^?4, 0.01–5.00 μg/mL and 2.3% for Phosalone, in C₁₈ cartridge, respectively. These values for MWCNT were 4.02×10^?6, 0.001–0.500 μg/mL and 1.8% for Chlorpyrifos and 1.02×10^?6, 0.001–0.500 μg/mL and 1.5% for Phosalone, respectively.     

Ionic‐liquid‐based dispersive liquid–liquid microextraction coupled with high‐performance liquid chromatography for the forensic determination of methamphetamine in human urine

Determination of methamphetamine in forensic laboratories is a major issue due to its health and social harm. In this work, a simple, sensitive, and environmentally friendly method based on ionic liquid dispersive liquid–liquid microextraction combined with high‐performance liquid chromatography was established for the analysis of methamphetamine in human urine. 1‐Octyl‐3‐methylimidazolium hexafluorophosphate with the help of disperser solvent methanol was selected as the microextraction solvent in this process. Various parameters affecting the extraction efficiency of methamphetamine were investigated systemically, including extraction solvent and its volume, disperser solvent and its volume, sample pH, extraction temperature, and centrifugal time. Under the optimized conditions, a good linearity was obtained in the concentration range of 10–1000 ng/mL with determination coefficient >0.99. The limit of detection calculated at a signal‐to‐noise ratio of 3 was 1.7 ng/mL and the relative standard deviations for six replicate experiments at three different concentration levels of 100, 500, and 1000 ng/mL were 6.4, 4.5, and 4.7%, respectively. Meanwhile, up to 220‐fold enrichment factor of methamphetamine and acceptable extraction recovery (>80.0%) could be achieved. Furthermore, this method has been successfully employed for the sensitive detection of a urine sample from a suspected drug abuser.