Extraction and determination of opium alkaloids in urine samples using dispersive liquid-liquid microextraction followed by high-performance liquid chromatography

A simple, rapid and sensitive method based on dispersive liquid-liquid microextraction (DLLME) combined with high-performance liquid chromatography-ultraviolet detection (HPLC-UV) was used to determine opium alkaloids in urine samples. Some effective parameters on extraction were studied and optimized. Under the optimum conditions, enrichment factors and recoveries for different opiates are in the range of 63.0-104.5 and 31.5-52.2%, respectively. The calibration graphs are linear in the range of 0.50-500 μg L(-1) and limit of detections (LODs) are in the range of 0.2-10 μg L(-1). The relative standard deviations (RSDs) for 200 μg L(-1) of morphine, codeine and thebaine, 5.0 μg L(-1) of papaverine and 10.0 μg L(-1) of noscapine in diluted urine sample are in the range of 2.8-6.1% (n=7). The relative recoveries of urine samples spiked with alkaloids are 84.3-106.0%. The obtained results show that DLLME combined with HPLC-UV is a fast and simple method for the determination of opium alkaloids in urine samples.     

Simultaneous determination of six phthalate esters in bottled milks using ultrasound-assisted dispersive liquid-liquid microextraction coupled with gas chromatography

A new method was developed for the simultaneous determination of six phthalate esters in bottled milks using ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME) followed by gas chromatography-flame ionization detection (GC-FID). 0.8 mL of methanol (dispersant) and 40 μL of CCl(4) (extractant) were injected into 8.0 mL of milk solution and then emulsified the mixture by ultrasound for 2.0 min to form the cloudy solution. Under the optimum condition, the enrichment factors of the analytes ranged from 220 to 270 fold and the recovery ranged from 93.2% to 105.7%. Good linearity was observed for all analytes in a range of 0.8-51 ngg(-1) with the correlation coefficient (r(2)) ≥ 0.9992. The limits of detection (LODs) based on signal to noise of 3 were 0.64-0.79 ngg(-1). The repeatability evaluated as intra-day and inter-day precision (relative standard deviation, RSD) were less than 4.0% (n=5). The presented UA-DLLME-GC-FID method was successfully applied to determine the six phthalate esters in different bottled milk products.     

Determination of phenothiazine derivatives in human urine by using ionic liquid-based dynamic liquid-phase microextraction coupled with liquid chromatography

A simple and rapid method for the determination of seven phenothiazines derivatives (chlorpromazine, promethazine, levomepromazine, prochlorperazine, trifluoperazine, fluphenazine and thioridazine) in human urine samples is presented. The analytes are extracted from the sample in 50 μL of the ionic liquid 1-butyl-3-methyl-imidazolium hexafluorophosphate working in an automatic flow system under dynamic conditions. The chemical affinity between the extractant and the analytes allows a good isolation of the drugs from the sample matrix achieving at the same time their preconcentration. The separation and detection of the extracted compounds is accomplished by liquid chromatography and UV detection. The proposed method is a valuable alternative for the analysis of these drugs in urine within the concentration range 0.07–10 μg mL^?1. Limits of detection were in the range from 21 ng mL^?1 (thioridazine) to 60 ng mL^?1 (levomepromazine). The repeatability of the proposed method expressed as RSD (n = 5) varied between 2.2% (levomepromazine) and 3.9% (chlorpromazine).     

Determination of trace lead in biological and water samples with dispersive liquid-liquid microextraction preconcentration

A new method for the determination of trace lead was developed by dispersive liquid–liquid microextraction preconcentration and graphite furnace atomic absorption spectrometry. In the proposed approach, 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP) was used as a chelating agent, and carbon tetrachloride and ethanol were selected as extraction and dispersive solvents. Some factors influencing the extraction efficiency of lead and its subsequent determination, including extraction and dispersive solvent type and volume, pH of sample solution, concentration of the chelating agent, and extraction time, were studied and optimized. Under the optimum conditions, the enrichment factor of this method for lead was reached at 78. The detection limit for lead was 39 ng L⁻¹ (3σ), and the relative standard deviation (RSD) was 3.2% (n = 7, c = 10 ng mL⁻¹). The method was successfully applied to the determination of trace amounts of lead in human urine and water samples.     

Determination of tramadol in human plasma and urine samples using liquid phase microextraction with back extraction combined with high performance liquid chromatography

Liquid phase microextraction by back extraction (LPME-BE) combined with high performance liquid chromatography (HPLC)-fluorescence detection was developed for the determination of tramadol in human plasma. Tramadol was extracted from 2 mL of basic sample solution (donor phase) with pH 11.5 through a micro liter-size organic solvent phase (100 microL n-octane) for 25 min and finally into a 3.5 microL acidic aqueous acceptor microdrop with pH 2.5 suspended in the organic phase from the tip of a HPLC microsyringe needle for 15 min with the stirring rate of 1250 rpm. After extraction for a period of time, the microdrop was taken back into the syringe and injected into HPLC. Effected the experimental parameters such as the nature of the extracting solvent and its volume, sample temperature, stirring rate, volume of the acceptor phase, pH and extraction time on LPME-BE efficiency was investigated. At the optimized condition, enrichment factor of 366 and detection limit (LOD) of 0.12 microg L(-1) were obtained. The calibration curve was linear (r=0.999) in the concentration range of 0.3-130 microg L(-1). Within-day relative standard deviation RSD (S/N=3) and between-day RSD were 3.16% and 6.29%, respectively. The method was successfully applied to determine the concentration of tramadol in the plasma and urine samples and satisfactory results were obtained.     

Determination of epitestosterone in human urine by off-line immunoaffinity solid-phase extraction coupled with high performance liquid chromatography

Epitestosterone (ET) has been used as a masking agent and prohibited by the World Anti-Doping Agency (WADA) because its administration will decrease the urinary T/ET ratio, a marker of testosterone (T) administration. In this study, an off-line immunoaffinity extraction coupled with high performance liquid chromatography (HPLC) was developed to quantify the endogenous steroid ET in human urine. The immunoaffinity column (IAC) was prepared by immobilizing the anti-ET monoclonal antibodies on CNBr-activated Sepharose 4B, which can remove the contaminations and non-target compounds from matrix to enrich the target analyte ET. The mobile phase was ammonium acetate (10 mM, pH 4.0)/acetonitrile (45/55, v/v) at an isocratic flow of 1.0 mL/min and the UV absorbance detection wavelength was 244 nm for the detection of ET. The IAC showed good reliability and durability since it had been used for more than 100 runs in a year. The limit of quantification (LOQ) was 1 ng/mL. Satisfied repeatability and precision of the day-to-day and within-day were obtained with the RSD values less than 10%. Results of the recovery of the urine samples were ranged from 98% to 102% with repeatability less than 9%, indicating that the method developed can be used for the real urine sample analysis.     

Automated determination of rifampicin in plasma samples by in-tube solid-phase microextraction coupled with liquid chromatography

A sensitive and automated method is described for determination of rifampicin in plasma samples for therapeutic drug monitoring by in-tube solid-phase microextraction coupled with liquid chromatography (in-tube SPME/LC). Important factors in the optimization of in-tube SPME are discussed, such as coating type, sample pH, sample draw/eject volume, number of draw/eject cycles, and draw/eject flow rate. Analyte pre-concentrated in the polyethylene glycol phase was directly transferred to the liquid chromatographic column by percolation of the mobile phase, without carryover. The method was linear over the 0.1-100 μg/mL range, with a linear coefficient value (r(2)) of 0.998. The inter-assay precision presented coefficient of variation ≤ 1.7%. The effectiveness and practicability of the proposed method are proven by analysis of plasma samples from ageing patients undergoing therapy with rifampicin.     

Simultaneous determination of 5-hydroxyindoles and catechols from urine using polymer monolith microextraction coupled to high-performance liquid chromatography with fluorescence detection

To make analytes amenable for fluorescence (FL) detection, polymer monolith microextraction (PMME) coupled to high-performance liquid chromatography with FL detection was developed for the simultaneous determination of catechols and 5-hydroxyindoleamines (5-HIAs) from urine samples. In this method, a two-step pre-column derivatization method was employed to derivatize the analytes and a poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolithic capillary column was used as the extraction medium for PMME. The conditions for the derivatization and subsequent extraction of 5-HIAs and catechols derivatives were optimized. Using our optimum conditions, the detection limit of the target analytes were 0.11–21 nM. Reproducibility of the method was obtained with intra-day and inter-day relative standard deviations less than 12% and a recovery of higher than 82%. In this study, we show how our proposed method can be used as a rapid sensitive technique for the determination of catechols and 5-HIAs from urine samples.     

Rapid determination of trace thiabendazole in apple juice utilizing dispersive liquid–liquid microextraction combined with fluorescence spectrophotometry

Food safety has become a large concern and prompts an urgent need for the development of rapid, simple and sensitive analytical methods that can monitor pesticide residues in foods. This study aimed to provide a method for quantitative determination of trace thiabendazole in apple juice. Due to its high sensitivity and selectivity, fluorescence spectrophotometry was utilized as a front end to dispersive liquid–liquid microextraction (DLLME). The experimental parameters that influenced the extraction were systematically investigated. Under optimum conditions, the whole procedure, including DLLME and analysis of one sample, was carried out within 5 min, and linearity was found in the 5–50 µg/L range with a correlation coefficient (r) of 0.9987. The limit of detection value was 2.2 µg/L. Good reproducibility was achieved based with a less than 4.5% relative standard deviation (RSD) for five replicates at different sample concentrations. This method was shown to be suitable for rapid and sensitive quantification of thiabendazole in apple juice. Copyright © 2015 John Wiley & Sons, Ltd.     

Improvement and validation the method using dispersive liquid-liquid microextraction with in situ derivatization followed by gas chromatography-mass spectrometry for determination of tricyclic antidepressants in human urine samples

A simple, rapid and sensitive method termed dispersive liquid-liquid microextraction (DLLME) combined with gas chromatography-mass spectrometry (GC/MS) was developed for the determination of tricyclic antidepressants (TCAs) in human urine sample. An appropriate mixture of methanol (disperser solvent), carbon tetrachloride (extraction solvent), and acetic anhydride (derivatization reagent) was injected rapidly into human urine sample. After extraction, the sedimented phase was analyzed by GC/MS. The calibration curves obtained with human urine were linear with a correlation coefficient of over 0.99 in the range of 2.0/5.0-100 ng mL(-1). Under the optimum conditions (carbon tetrachloride: 10 μL, methanol: 150 μL), the detection limits and the quantification limits of the tricyclic antidepressants were 0.5-2.0 ng mL(-1) and 2.0-5.0 ng mL(-1), respectively. The average recoveries of TCAs were 88.2-104.3%. Moreover, the inter- and intra-day precision and accuracy was acceptable at all concentrations. The results showed that DLLME is applicable to the determination of trace amounts of TCAs in human urine sample.     

Hollow fiber liquid phase microextraction followed by high performance liquid chromatography for determination of ultra-trace levels of Se(IV) after derivatization in urine,plasma and natural water samples

In the present work, a simple and high sensitive method based on hollow fiber liquid phase microextraction (HF-LPME) was developed followed by high performance liquid chromatography (HPLC) for determination of ultra-trace amounts of Se(IV) after derivatization in biological and natural water samples. Se(IV) was complexed with o-phenylenediamine to form piazselenol. The formed piazselenol was extracted into 20 μL of 1-octanol located in the lumen of a hollow fiber and the solution was injected into HPLC-UV for analysis. Using the Taguchi method, an orthogonal array design (OAD), OA₁₆ (4⁵) was employed to optimize the HF-LPME of piazselenol. The effect of five experimental factors (each factor at four levels) including the volume of the organic phase, extraction time, pH of the solution, stirring rate and ionic strength on the extraction efficiency of piazselenol was studied and optimized. The maximum extraction efficiency of piazselenol was obtained at 20 μL of 1-octanol as the extracting solvent, 30 min extraction time, pH 2, stirring rate of 500 rpm and 30% (w/v) NaCl. Under the optimum conditions, preconcentration factors up to 130 were achieved and the relative standard deviation (%RSD) of the method was <3.7% for different concentrations of Se(IV). The calibration curves were obtained in the ranges of 0.2–100 and 0.05–10 μg L^?1 for the 11 and 50 mL of the sample volumes with reasonable linearity, respectively (r² > 0.995). The limits of detection (LOD) were 0.1 and 0.02 μg L^?1 for the 11 and 50 mL sample volumes, respectively (S/N = 3). Finally, the applicability of the proposed method was evaluated by the extraction and determination of Se(IV) in the plasma, urine and water samples.     

Application of poly(butyl methacrylate-co-ethylene glycol dimethacrylate) monolith microextraction coupled with high performance liquid chromatography to the determination of polycyclic aromatic hydrocarbons in smoked meat products

This paper presents a study of the synthesis of a polymer monolith column and its application to the analysis of PAHs in smoked meat products. A poly(butyl methacrylate-co-ethylene glycol dimethacrylate) monolith capillary has been successfully prepared with in situ polymerization method. The polymer monolith microextraction combined with HPLC determinations is employed for the analysis of naphthalene, biphenyl, phenanthrene, and anthracene. Various parameters affecting the extraction efficiency have been investigated and optimized. Under the optimum experimental conditions, the method provides an acceptable linearity (2-10,000 μg/L), low limits of detection (1.4-2.0 μg/L), and good precision (intraday relative standard deviations<4.1%, interday relative standard deviations<5.7%). When applied to the determination of the four PAHs in smoked meat samples, recoveries are obtained in the range of 86.6-101.5%.     

Simple and sensitive high-performance liquid chromatography method for the determination of docetaxel in human plasma or urine

Several methods for quantification of docetaxel have been described mainly using HPLC. We have developed a new isocratic HPLC method that is as sensitive and simpler than previous methods, and applicable to use in clinical pharmacokinetic analysis. Plasma samples are spiked with paclitaxel as internal standard and extracted manually on activated cyanopropyl end-capped solid-phase extraction columns followed by isocratic reversed-phase HPLC and UV detection at 227 nm. Using this system, the retention times for docetaxel and paclitaxel are 8.5 min and 10.5 min, respectively, with good resolution and without any interference from endogenous plasma constituents or docetaxel metabolites at these retention times. The total run time needed is only 13 min. The lower limit of quantification is 5 ng/ml using 1 ml of plasma. The validated quantitation range of the method is 5–1000 ng/ml with RSDs≤10%, but plasma concentrations up to 5000 ng/ml can be accurately measured using smaller aliquots. This method is also suitable for the determination of docetaxel in urine samples under the same conditions. The method has been used to assess the pharmacokinetics of docetaxel during a phase I/II study of docetaxel in combination with epirubicin and cyclophosphamide in patients with advanced cancer.     

Tetrahydrocurcumin in plasma and urine: quantitation by high performance liquid chromatography

Tetrahydrocurcumin (THC), one of the major metabolites of curcumin, exhibits many of the same physiologic and pharmacological activities as curcumin and in some systems may exert greater antioxidant activity than curcumin. However, evaluation of clinical efficacy is limited by lack of sensitive methods for quantifying intake/absorption in blood or urine. We have developed a sensitive high performance liquid chromatography (HPLC) analytical method for detection of THC in plasma and urine. The method involves extracting the THC from 0.2 mL samples with 95% ethyl acetate/5% methanol, and beta-17-estradiol acetate as an internal standard. Analysis with a reversed-phase C18 column and UV detection at 280 nm demonstrates linear performance from 0.050 to 6.0 microg/mL in plasma, and 0.060 to 6.0 microg/mL in urine. The coefficients of variation for intra- and inter-assays were each<8.6%. The average recovery of THC from plasma and urine was greater than 98.5%. These data demonstrate a rapid, sensitive and accurate method for HPLC quantification of THC in plasma and urine.     

Liquid-phase and dispersive liquid-liquid microextraction techniques with derivatization: recent applications in bioanalysis

Liquid phase microextraction (LPME), especially hollow fiber liquid-phase microextraction (HF-LPME), and dispersive liquid-liquid microextraction (DLLME) offer high enrichments of target analytes in a single step. The analytical usefulness of these techniques is significantly enhanced by coupling them with suitable derivatization methods. Due to their simplicity, diverse bioanalytical applications have recently been reported. This review focuses on the recent developments of the combined LPME (mainly HF-LPME and single drop microextraction (SDME)) and DLLME techniques with derivatization for the analysis of biological samples. A broad range of sample matrices such as urine, blood, plasma and human hair samples with various derivatization methods for polar or ionizable organic compounds will be considered. These techniques can also be extended to the determination of trace metal ions, such as the heavy metal ions (Hg, Pb, and Co) and Se. Future trends of the techniques will also be discussed.     

Development of a dispersive liquid-liquid microextraction method for spectrophotometric determination of barbituric acid in pharmaceutical formulation and biological samples

In this paper, a novel and simple method for the determination of trace amounts of barbituric acid in water and biological samples was developed by using dispersive liquid–liquid microextraction (DLLME) techniques combined with spectrophotometric analysis. The procedure is based on color reaction of barbituric acid with p-dimethylaminobenzaldehyde and extraction of the color product using the DLLME technique. Some important parameters such as reaction conditions and the type and volume of extraction and dispersive solvents as well as the extraction time were investigated and optimized in detail. Under the optimum conditions, the calibration graphs were linear over the range of 5.0 to 200 ng ml⁻¹ with limit of detection of 2.0 ng ml⁻¹. Relative standard deviation for five replicate determinations of barbituric acid at 50 ng ml⁻¹ concentration level was calculated to be 1.64%. Average recoveries for spiked samples were determined to be between 94% and 105%. The proposed method was applied for the determination of barbituric acid in pharmaceutical formulation and biological samples.     

Determination of lectin-sugar binding constants by microequilibrium dialysis coupled with high performance liquid chromatography

We used high performance liquid chromatography to determine the concentrations of free ligands after equilibrium dialysis to examine lectin-sugar interactions. The binding of p-nitrophenyl 1-thio-alpha-mannoside, p-nitrophenyl N-acetyl-1-thio-beta-glucosaminide, and the pyridylamino derivative of Man6GlcNAc2 to concanavalin A and Triticum vulgaris lectin was examined. The binding constant, Ka, and the concentration of total binding sites, [L]t, were calculated from the trace amounts of sugars and lectins using the equation, [S]/[LS] = 1/Ka[L]t + [S]/[L]t, where [S] is the concentration of free ligand and [LS] the concentration of bound ligand.     

Hollow fiber-liquid phase microextraction combined with gas chromatography for the determination of phenothiazine drugs in urine

A simple method of hollow fiber-liquid phase microextraction (HF-LPME) combined with gas chromatography (GC) was developed for the analysis of four phenothiazine drugs (promethazine, promazine, chlorpromazine and trifluoperazine) in human urine samples. All variables affecting the extraction of target analytes including organic solvent type, stirring rate, extraction time, extraction temperature, pH of sample solution and ionic strength were carefully studied and optimized. Under the optimal conditions, the analytical performance of HF-LPME-GC-flame photometric detector (FPD) and HF-LPME-GC-flame ionization detector (FID) were evaluated and compared. The results showed that the HF-LPME-GC-FID was more sensitive than HF-LPME-GC-FPD for the determination of four target phenothiazine drugs, while the signal peak shape and resolution obtained by HF-LPME-GC-FPD was better than that obtained by HF-LPME-GC-FID. HF-LPME-GC-FPD/FID was successfully applied for the assay of the interested phenothiazine drugs in urine sample, and the excretion of the drugs was also investigated by monitoring the variation of the concentration of chlorpromazine in urine of a psychopath within 8 h after drug-taking. The proposed method provided an effective and fast way for the therapeutic drug monitoring (TDM) of phenothiazine.     

A method for the determination of betaine in tissues using high performance liquid chromatography

Betaine is a major metabolite of choline in liver and kidney and may be an important product of choline metabolism in other tissues. The available methods for assay of betaine, however, are time consuming and relatively insensitive. We describe a modification of published methods that provides a sensitive and specific assay for betaine by derivatization and HPLC separation with UV quantitation. Betaine and other water-soluble choline metabolites are extracted from biological samples and separated by HPLC based on mobility of 14C-labeled internal standards. The betaine fraction is collected and derivatized with 4'-bromo-phenacyl triflate. The betaine-triflate derivative is quantitated by UV absorbance and the result is corrected for possible losses due to incomplete extraction recovery and incomplete derivatization by simultaneous measurement of radioactivity from the derivatized 14C-betaine internal standard. Betaine concentrations determined with this procedure are reported for several adult and fetal rat tissues.