Enantioselective analysis of mirtazapine and its two major metabolites in human plasma by liquid chromatography-mass spectrometry after three-phase liquid-phase microextraction

A three-phase liquid-phase microextraction (LPME) method using porous polypropylene hollow fibre membrane with a sealed end was developed for the extraction of mirtazapine (MRT) and its two major metabolites, 8-hydroxymirtazapine (8-OHM) and demethylmirtazapine (DMR), from human plasma. The analytes were extracted from 1.0 mL of plasma, previously diluted and alkalinized with 3.0 mL 0.5 mol L⁻¹ pH 8 phosphate buffer solution and supplemented with 15% sodium chloride (NaCl), using n-hexyl ether as organic solvent and 0.01 moL L⁻¹ acetic acid solution as the acceptor phase. Haloperidol was used as internal standard. The chromatographic analyses were carried out on a chiral column, using acetonitrile-methanol-ethanol (98:1:1, v/v/v) plus 0.2% diethylamine as mobile phase, at a flow rate of 1.0 mL min⁻¹. Multi-reaction monitoring (MRM) detection was performed by mass spectrometry (MS-MS) using a triple-stage quadrupole and electrospray ionization interface operating in the positive ion mode. The mean recoveries were in 18.3-45.5% range with linear responses over the 1.25-125 ng mL⁻¹ concentration range for all enantiomers evaluated. The quantification limit (LOQ) was 1.25 ng mL⁻¹. Within-day and between-day assay precision and accuracy (2.5, 50 and 100 ng mL⁻¹) showed relative standard deviation and the relative error lower than 11.9% for all enantiomers evaluated. Finally, the method was successfully used for the determination of mirtazapine and its metabolite enantiomers in plasma samples obtained after single drug administration of mirtazapine to a healthy volunteer.     

Stereoselective determination of hydroxychloroquine and its metabolites in human urine by liquid-phase microextraction and CE

Liquid-phase microextraction based on polypropylene hollow fibers and CE were applied for the chiral determination of hydroxychloroquine (HCQ) and its metabolites (desethylchloroquine, DCQ; desethylhydroxychloroquine, DHCQ; bisdesethylchloroquine, BDCQ) in human urine. The analytes were extracted from 3 mL of urine spiked with the internal standard (metoprolol) and alkalinized with 250 muL of 2 M NaOH. The analytes were extracted into 1-octanol impregnated in the pores of the hollow fiber, and into an acid acceptor solution inside the hollow fiber. The electrophoretic separations were carried out in 100 mmol/L Tris buffer (pH adjusted to 9.0 with phosphoric acid) containing 1% w/v S-beta-CD and 30 mg/mL HP-beta-CD with a constant voltage of +18 kV. The method was linear over the concentration range of 10-1000 ng/mL for each HCQ stereoisomer and 21-333 ng/mL for each metabolite stereoisomer. Within-day and between-day assay precision and accuracy for the analytes were studied at three concentration levels for each stereoisomer and were lower than 15%. The developed method was applied for the determination of the cumulative urinary excretion of HCQ, DCQ, and DHCQ after oral administration of rac-HCQ to a health volunteer. The results obtained are in agreement with previous literature data.     

Dispersive liquid-phase microextraction using ionic liquid as extractant for the enrichment and determination of DDT and its metabolites in environmental water samples

Ionic liquids are a kind of environmentally friendly solvents which have drawn great attention in many fields. The potential of ionic liquid as dispersive liquid-phase microextraction (DLPME) solvent for the enrichment of typical persistent organic pollutants, dichlorodiphenyltrichloroethane (DDT), and its metabolites including 1,1-dichloro-2,2-bis-(4′-chlorophenyl)ethane and 1,1-dichloro-2,2-bis-(4′-chlorophenyl)ethylene has been investigated. Parameters that may influence the extraction efficiency, such as the type and volume of ionic liquid, the type and volume of disperser solvent, extraction time, and sample pH, were investigated and optimized in detail. The experimental results showed the excellent linear relationship between peak area and the concentration of DDT and its metabolites over the range of 1–50 μg L⁻¹, and the precisions (RSDs) were 5.27–6.73% under the optimal conditions. The limits of detection could reach 0.33–0.63 μg L⁻¹. Satisfied results were achieved when the proposed method was applied to determine the target compounds in real-world water samples with spiked recoveries over the range 94.4–115.3%. All these facts indicated that ionic liquid DLPME coupled to HPLC was an environmentally friendly alternative for the rapid analysis of DDT and its metabolites at trace level in environmental water samples.     

可忽略损耗液相微萃取法同时测定辛醇-水分配系数和电离常数

以4-氯酚(4-CP)、2,4-二氯酚(2,4-DCP)和2,4,6-三氯酚(2,4,6-TCP)为模型化合物,建立了中空纤维膜支载-可忽略损耗液相微萃取法(HF-nd-LPME)和高效液相色谱(HPLC)联用,同时测定辛醇-水分配系数(Kow)和电离常数(pKa)的方法。在60 mL正辛醇饱和的样品溶液(1mmol/L NaH2PO4,2μg/mL4-CP、2,4-DCP和2,4,6-TCP)中放入2个支载有0.16μL正辛醇的中空纤维膜萃取装置,静置96 h确保达到萃取平衡。然后将萃取装置取出并用甲醇解吸,经HPLC测定得到萃取到正辛醇中的目标物浓度coctanol。coctanol与样品中目标物原始浓度cwater的比值即为Kow。通过测定模型化合物在不同pH3.0~13条件下的Kow值,并依据化合物的Kow、pKa与pH的关系模型进行非线性拟合,即可计算得出模型化合物pKa值。实验表明,本方法测定的三种模型化合物的Kow和pKa值与文献报道值一致。     

Hollow-fiber liquid-phase microextraction for the determination of pesticides and metabolites in soils and water samples using HPLC and fluorescence detection

A new and simple method has been developed for the determination of a group of four benzimidazole pesticides (carbendazim/benomyl, thiabendazole, and fuberidazole), a carbamate (carbaryl), and an organophosphate (triazophos), together with two of their main metabolites (2-aminobenzimidazole, metabolite of carbendazim/benomyl, and 1-naphthol, metabolite of carbaryl) in soils. First, an ultrasound-assisted extraction (UAE) was performed, followed by evaporation and reconstitution in water. Then, extraction and preconcentration of the analytes was accomplished by two-phase hollow-fiber liquid-phase microextraction (HF-LPME) using 1-octanol as extraction solvent. Parameters that affect the extraction efficiency in HF-LPME technique (organic solvent, pH of the sample, extraction time, stirring speed, temperature, and ionic strength) were deeply investigated. Optimum HF-LPME conditions involved the use of a 2.0 cm polypropylene fiber filled with 1-octanol to extract 10 mL of an aqueous soil extract at pH 9.0 containing 20% (v/v) of NaCl for 30 min at 1440 rpm. Separation and quantification was achieved by HPLC with fluorescence detection (FD). The proposed optimum UAE-HF-LPME-HPLC-FD methodology provided good calibration, precision, and accuracy results for two soils of different physicochemical properties. LODs were in the range 0.001-6.94 ng/g (S/N = 3). With the aim of extending the validation, the HF-LPME method was also applied to different types of waters (Milli-Q, mineral and run-off), obtaining LODs in the range 0.0002-0.57 μg/L.     

Enantioselective assay of chloroquine and its main metabolite deethyl chloroquine in human plasma by capillary electrophoresis

A sensitive assay for the determination of chloroquine (Clq) and its pharmacologically active metabolite deethyl chloroquine in plasma by capillary electrophoresis (CE) is developed. Plasma levels of drug and metabolite are measured using HeCd laser-induced fluorescence (LIF) detection over a range of three orders of magnitude from 2 to 1000 ng/mL after liquid-liquid extraction. A limit of detection of 0.5 ng/mL is achieved. Validation of the method yields intra- and interday precision data within the limits of 10% (20% at limit of quantitation) and intra- and interday accuracy data greater than 6% throughout the whole working range. The method is applied for the drug monitoring of patients treated with Clq. Based upon this assay, two enantioselective CE-LIF methods for Clq and its main metabolite are developed. Mixtures of substituted gamma-cyclodextrins are used as chiral selectors. A baseline separation of the enantiomers of both analytes in one run is achieved in less than 11 min (method A) and less than 9 min (method B), respectively. Hydroxychloroquine is used as the internal standard for both methods.     

Analysis of quinolones by voltage-assisted liquid-phase microextraction combined with LC-MS

The method of liquid-phase microextraction assisted with voltage was developed and applied on determination of quinolones in water sample in this study. Both of the reproducibility and extraction time were improved with the aid of applying voltage. Four analytes in neutral state such as cinoxacin, oxolinic acid, nalidixic acid, and flumequine were extracted from a sample solution at pH 2.0, through a polypropylene hollow fiber which was immobilized with 2-octanone, and then into a 25 μL of the acceptor phase of 40 mM borate buffer at pH 10.0 by applying voltage of 100 V. Subsequently, the acceptor solution was directly subjected to analysis by LC-MS. The performance of the method for four quinolones was also evaluated. Linearity was obtained in the range of 1.0-25.0 ng/mL with R(2) > 0.996. Limits of detection were below 0.6 ng/mL, and recoveries of water sample were ranged from 90.8 to 109.6%.     

Application of liquid-phase microextraction and gas chromatography-mass spectrometry for the determination of polychlorinated biphenyls in blood plasma

This study investigated the feasibility of applying liquid-phase microextraction combined with gas chromatography-mass spectrometry (GC-MS) to determine polychlorinated biphenyls (PCBs) in blood plasma. An efficient and simple extraction technique has been developed for the enrichment of PCBs from human blood plasma samples using single-step liquid-phase microextraction (LPME) in conjunction with a hollow fibre membrane (HFM). An eight PCB congener mixture was spiked into 2.5 ml of blood plasma, and the solution was then adjusted to pH 10.5 with a salinity of 20% (w/v) prior to making the total volume to 5 ml with ultrapure water. The porous HFM, filled with 3 microl of organic solvent, was then immersed into the solution, which was continuously agitated at 700 rpm for 30 min. Extract (1 microl) containing the pre-concentrated analytes was then injected into a GC-MS without further pre-treatment. Using an optimised extraction procedure, a large enrichment factor of the analytes, i.e. up to 241-fold was achieved in 30 min. The procedure resulted in a relative standard deviation of < 11% (n = 6), and a linear calibration range from 2.5 to 150 microg/l (r > 0.999), and detection limits between 0.07 and 0.94 microg/l, respectively. To demonstrate the feasibility of the procedure, PCB concentrations were determined in actual blood samples collected from the local population in Singapore using the optimised LPME technique.     

Headspace liquid-phase microextraction using ionic liquid as extractant for the preconcentration of dichlorodiphenyltrichloroethane and its metabolites at trace levels in water samples

A novel technique, high temperature headspace liquid-phase microextraction (HS-LPME) with room temperature ionic liquid (RTIL), 1-butyl-3-methylimidazolium hexafluorophosphate ([C₄MIM][PF₆]) as extractant, was developed for the analysis of dichlorodiphenyltrichloroethane (p,p′-DDT and o,p′-DDT) and its metabolites including 4,4′-dichlorodiphenyldichloroethylene (p,p′-DDE) and 4,4′-dichlorodiphenyldichloroethane (p,p′-DDD) in water samples by high performance liquid chromatography with ultraviolet detection. The parameters such as salt content, sample pH and temperature, stirring rate, extraction time, microdrop volume, and sample volume, were found to have significant influence on the HS-LPME. The conditions optimized for extraction of target compounds were as follows: 35% NaCl (w/v), neutral pH condition, 70 °C, 800 rpm, 30 min, 10 μL [C₄MIM][PF₆], and 25 mL sample solutions. Under the optimized conditions, the linear range, detection limit (S/N = 3), and precision (R.S.D., n = 6) were 0.3-30 μg L⁻¹, 0.07 μg L⁻¹, and 8.0% for p,p′-DDD, 0.3-30 μg L⁻¹, 0.08 μg L⁻¹, and 7.1% for p,p′-DDT, 0.3-30 μg L⁻¹, 0.08 μg L⁻¹, and 7.2% for o,p′-DDT, and 0.2-30 μg L⁻¹, 0.05 μg L⁻¹, and 6.8% for p,p′-DDE, respectively. Water samples including tap water, well water, snow water, reservoir water, and wastewater were analyzed by the proposed procedure and the recoveries at 5 μg L⁻¹ spiked level were in the range of 86.8-102.6%.     

Simultaneous determination of metacavir and its metabolites in rat plasma using high-performance liquid chromatography with tandem mass spectrometric detection (LC-MS/MS)

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the simultaneous determination of metacavir and its two metabolites in rat plasma was developed and validated. Tinidazole was used as an internal standard and plasma samples were pretreated with one‐step liquid–liquid extraction. In addition, these analytes were separated using an isocratic mobile phase on a reverse‐phase C₁₈ column and analyzed by MS in the selected reaction monitoring mode. The monitored precursor to product‐ion transitions for metacavir, 2′,3′‐dideoxyguanosine, O‐methylguanine and the internal standard were m/z 266.0 → 166.0, m/z 252.0 → 152.0, m/z 166.0 → 149.0 and m/z 248.0 → 202.0, respectively. The standard curves were found to be linear in the range of 1–1000 ng/mL for metacavir, 5–5000 ng/mL for 2′,3′‐dideoxyguanosine and 1–1000 ng/mL for O‐methylguanine in rat plasma. The precision and accuracy for both within‐ and between‐batch determination of all analytes ranged from 2.83 to 9.19% and from 95.86 to 111.27%, respectively. No significant matrix effect was observed. This developed method was successfully applied to an in vivo pharmacokinetic study after a single intravenous dose of 20 mg/kg metacavir in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Comparison of continuous-flow microextraction and static liquid-phase microextraction for the determination of p-toluidine in Chlamydomonas reinhardtii

In this study, two microextraction methods, viz. continuous-flow microextraction (CFME) and static liquid-phase microextraction (s-LPME), were optimized and compared for the determination of p-toluidine in water and Chlamydomonas reinhardtii samples. The calibration curve for p-toluidine was linear in the concentration range of 0.01-5 microg/mL, and the squared regression coefficients (r(2)) for the lines were up to 0.999 for both CFME and s-LPME treatments. Detection limits in CFME and s-LPME were 8.2 ng/mL and 4.9 ng/mL, based on a signal-to-noise (S/N) ratio of 3, respectively. The precision was tested, in five replicates, by analysis of a 100-ng/mL standard solution of p-toluidine and the relative standard deviations were 5.43 and 3.08% for CFME and s-LPME, respectively. The concentration factors were 5.5 and 14.4 for CFME and s-LPME, respectively. s-LPME has a higher extraction efficiency, lower detection limit, and higher concentration factor than that of CFME. Additionally, the s-LPME method is precise and reproducible, and requires only a 3.0-microL microdrop of extraction solvent. Therefore, this procedure is more convenient in use, and viable for qualitative and quantitative analysis of p-toluidine in water and biota samples.     

Temperature-controlled headspace liquid-phase microextraction device using volatile solvents

A novel temperature-controlled headspace liquid-phase microextraction (TC-HS-LPME) device was established in which volatile solvents could be used as extractant. In this device, a PTFE vial cap with a cylindrical cavity was used as the holder of the extraction solvent. Up to 40 μl of extraction solvent could be suspended in the cavity over the headspace of aqueous sample in the vial. A cooling system based on thermoelectric cooler (TEC) was used to lower the temperature of extractant in PTFE vial cap to reduce the loss of volatile solvent during extraction process and increase the extraction efficiency. The selection of solvents for HS-LPME was then extended to volatile solvents, such as dichloromethane, ethyl acetate and acetone. The use of volatile extraction solvents instead of semi-volatile solvent reduced the interference of the large solvent peak to the analytes peaks, and enhanced the compatibility of HS-LPME with gas chromatograph (GC). Moreover, the use of larger volume of extractant solvent increases the extraction capacity and the injection volume of GC after extraction, thus improving detection limits. Several critical parameters of this technique were investigated by using chlorobenzenes (CBs) as the model analytes. High enrichment factors (498–915), low limits of detection (0.004–0.008 μg/L) and precision (3.93–5.27%) were obtained by using TC-HS-LPME/GC-FID. Relative recoveries for real samples were more than 83%.     

Advances in sample preparation using membrane-based liquid-phase microextraction techniques

The article highlights some of the most important developments in membrane-based liquid-phase microextraction techniques and applications. We discuss the evolution of different configurations from the flat type of module through the hollow-fiber module to the latest membrane combination with other sorbents and coating of the hollow fiber. We also discuss the basic principles and important parameters that control the extraction process in two-phase and three-phase systems. Finally, we highlight future trends in module configuration and applications.     

Enantioselective HPLC-UV method for determination of eslicarbazepine acetate (BIA 2-093) and its metabolites in human plasma

Eslicarbazepine acetate (BIA 2-093) is a novel central nervous system drug undergoing clinical phase III trials for epilepsy and phase II trials for bipolar disorder. A simple and reliable chiral reversed-phase HPLC-UV method was developed and validated for the simultaneous determination of eslicarbazepine acetate, oxcarbazepine, S-licarbazepine and R-licarbazepine in human plasma. The analytes and internal standard were extracted from plasma by a solid-phase extraction using Waters Oasis HLB cartridges. Chromatographic separation was achieved by isocratic elution with water-methanol (88:12, v/v), at a flow rate of 0.7 mL/min, on a LichroCART 250-4 ChiraDex (beta-cyclodextrin, 5 microm) column at 30 degrees C. All compounds were detected at 225 nm. Calibration curves were linear over the range 0.4-8 microg/mL for eslicarbazepine acetate and oxcarbazepine, and 0.4-80 microg/mL for each licarbazepine enantiomer. The overall intra- and interday precision and accuracy did not exceed 15%. Mean relative recoveries varied from 94.00 to 102.23% and the limit of quantification of the assay was 0.4 microg/mL for all compounds. This method seems to be a useful tool for clinical research and therapeutic drug monitoring of eslicarbazepine acetate and its metabolites S-licarbazepine, R-licarbazepine and oxcarbazepine.     

Simultaneous determination of lidocaine and its metabolites in plasma and myocardium

No validated method exists for measuring lidocaine and its metabolites in myocardial tissue. We modified a previously described high-performance liquid chromatographic assay and applied it to plasma and to homogenized myocardial samples obtained from dogs that had received lidocaine by a double-infusion technique. Recovery of lidocaine, monoethylglycylxylidide and glycylxylidide after homogenization and extraction is reported. Assay variability, sensitivity and linearity over a wide range of sample sizes are also described. The results obtained with high-performance liquid chromatographic analysis are compared to quantitation of 14C-labeled lidocaine plus metabolites measured by an oxidation-scintillation technique. Myocardium to plasma partition coefficients for lidocaine, monoethylglycylxylidide and glycylxylidide were 2.16, 4.27, and 2.91, respectively.     

Headspace liquid-phase microextraction of trihalomethanes in drinking water and their gas chromatographic determination

In the present work, a novel method for the determination of trihalomethanes (THMs) such as chloroform, dichlorobromomethane, chlorodibromomethane and bromoform in drinking water has been described. It is based on coupling headspace liquid-phase microextraction (HS-LPME) with gas chromatography-electron capture detector (GC-ECD). A microdrop of organic solvent at the tip of a commercial microsyringe was used to extract analytes from aqueous samples. Three organic solvents—xylene, ethylene glycol and 1-octanol—were compared and 1-octanol was the most sensitive solvent for the analytes. Extraction conditions such as headspace volume, extraction time, stirring rate, content of NaCl and extraction temperature were found to have significant influence on extraction efficiency. The optimized conditions were 15 ml headspace volume in a 40 ml vial, 10 min extraction time and 800 rpm stirring rate at 20 °C with 0.3 g ml⁻¹ NaCl. The linear range was 1-100 μg l⁻¹ for THMs. The limits of detection (LODs) ranged from 0.15 μg l⁻¹ (for dichlorobromomethane and chlorodibromomethane) to 0.4 μg l⁻¹ (for chloroform); and relative standard deviations (RSD) for most of THMs at the 10 μg l⁻¹ level were below 10%. Real samples collected from tap water and well water were successfully analyzed using the proposed method. The recovery of spiked water samples was from 101 to 112%.     

Chemical reactions in liquid-phase microextraction

In recent years, liquid-phase microextraction (LPME), a microscale implementation of liquid-liquid extraction, has become a very popular sample pretreatment technique because it combines extraction and enrichment, and is inexpensive, easy to operate and nearly solvent-free. Especially so in hollow fiber-protected LPME, sample cleanup is also effected. Essentially, owing to its high sample-to-extracting solvent volume ratio, LPME can achieve high analyte enrichment. Since its advent, the technique has been widely used, and applied to environmental, pharmaceutical, biological and forensic analyses. This review focuses on developments relating to chemical reactions associated with LPME applications, in contrast to conventional, straightforward extractions in which analytes remain as they are during the extraction process. Chemical reactions brought about during LPME serve to promote the extractability of the analytes (thus expanding the scope of applicability of the technique), facilitate their (analyte) compatibility with the analytical system and/or improve detection sensitivity. The reactions that are usually enabled during LPME include ion-pair extraction (carrier-mediated membrane transport), complexation, chemical (pre-extraction, in situ, and post-extraction) derivatization, phase-transfer catalysis and other "special affinity" reactions. Strategies on chemical reactions in LPME are overviewed in this report.     

Enantioselective fungal biotransformation of risperidone in liquid culture medium by capillary electrophoresis and hollow fiber liquid-phase microextraction

Knowing that microbial transformations of compounds play vital roles in the preparation of new derivatives with biological activities, risperidone and its chiral metabolites were determined by capillary electrophoresis and hollow fiber liquid-phase microextraction after a fungal biotransformation study in liquid culture medium. The analytes were extracted from 1 mL liquid culture medium into 1-octanol impregnated in the pores of the hollow fiber, and into an acid acceptor solution inside the polypropylene hollow fiber. The electrophoretic separations were carried out in 100 mmol/L sodium phosphate buffer pH 3.0 containing 2.0% w/v sulfated-α-CD and carboxymethyl-β-CD 0.5% w/v with a constant voltage of -10 kV. The method was linear over the concentration range of 100-5000 ng/mL for risperidone and 50-5000 ng/mL for each metabolite enantiomer. Within-day and between-day assay precisions and accuracies for all the analytes were studied at three concentration levels, and the values of relative standard deviation and relative error were lower than 15%. The developed method was applied in a pilot biotransformation study employing risperidone as the substrate and the filamentous fungus Mucor rouxii. This study showed that the filamentous fungus was able to metabolize risperidone enantioselectively into its chiral active metabolite, (-)-9-hydroxyrisperidone.     

Specific spectrofluorimetric determination of chloroquine in blood plasma

Chloroquine (? 150 ng ml^?1 is separated from its dealkylated metabolites by reacting the latter with ethylchloroformate followed by extraction of unchanged chloroquine into dilute acid. The aqueous extract is made alkaline and its fluorescence intensity (λ(ex) = 335 nm, λ(em) = 390 nm) is measured.     

Determination of organochlorine pesticides in water using dynamic hook-type liquid-phase microextraction

We developed a simple and efficient headspace liquid-phase microextraction (LPME) technique named dynamic hook-type liquid-phase microextraction (DHT-LPME) and used it in combination with gas chromatography-mass spectrometry (GC-MS) and an electron capture detector (ECD). Aqueous specimens of organochlorine pesticides (OCPs) were used as model compounds to demonstrate the effectiveness of the technique. In the present study, the calibration curves were linear over at least 2 orders of magnitude with R² values of 0.997. The method detection limits (MDLs) varied from 2 to 44.0 ng L⁻¹. The precision of DHT-LPME ranged from 6.5 to 14.4%. The relative recoveries of OCPs in rainwater were more than 84.2%. Enrichment factors (EF) in the range 275-1127 were obtained using DHT-LPME.