Microextraction sample preparation techniques in forensic analytical toxicology

Sample preparation is a critical step in forensic analytical toxicology. Different extraction techniques are employed with the goals of removing interferences from the biological samples, such as blood, tissues and hair, reducing matrix effects and concentrating the target analytes, among others. With the objective of developing faster and more ecological procedures, microextraction techniques have been expanding their applications in the recent years. This article reviews various microextraction methods, which include solid‐based microextraction, such as solid‐phase microextraction, microextraction by packed sorbent and stir‐bar sorptive extraction, and liquid‐based microextraction, such as single drop/hollow fiber‐based liquid‐phase microextraction and dispersive liquid–liquid microextraction, as well as their applications to forensic toxicology analysis. The development trend in future microextraction sample preparation is discussed.     

填充吸附剂微萃取技术及其在微小体积样品萃取应用中的研究进展

微萃取技术是分析化学领域发展迅速,且已经得到广泛应用的样品前处理技术。填充吸附剂微萃取(MEPS)是一种微量固相萃取技术,使用微量的吸附剂填充于微量注射器,通过反复抽推方式使样品多次流经吸附剂以完成样品吸附萃取过程,萃取后的样品可直接用于色谱分析。典型的MEPS萃取设备包括MEPS注射器和MEPS吸附床(BIN)。MEPS优化的主要因素为MEPS处理过程的参数,包括样品流速、样品量与样品萃取循环次数,吸附剂及淋洗、洗脱溶剂的种类和体积,还需要考虑样品基质对MEPS性能的影响和样品残留和重复使用问题。MEPS中最重要的部分是吸附剂,主要有商品化的MEPS吸附剂,包括硅基的Silica、C18、C8等,碳材料的Hypercarb和聚苯乙烯聚合物类的SDVB、HDVB吸附剂等。研究用的吸附剂包括分子印迹材料、限进分子印迹材料、碳基材料、导电聚合物类材料、改性硅基材料及共价-有机骨架材料等。MEPS结合多种分析仪器已经成功应用于从不同基质中提取单一或多种分析物,所涵盖基质包括生物样品(尿液、唾液、血浆或血液)、河流水体或生活污水以及几种食品和饮料。MEPS处理复杂生物基质样品时,通常需要稀释样品、除蛋白质等预处理。MEPS具有需要样品体积小、操作快速等特点,在生物基质样品分析中有望得到更广泛的使用。在环境样品中,该技术可与现场便携仪器联用,未来将有望在现场进行快速检测,并于易分解样品等方面发挥作用。     

Recent advances in microextraction by packed sorbent for bioanalysis

Microextraction by packed sorbent (MEPS) is a new format for solid-phase extraction (SPE) that has been miniaturized to work with sample volumes as small as 10 μL. The commercially available presentation of MEPS uses the same sorbents as conventional SPE columns and so is suitable for use with most existing methods by scaling the reagent and sample volumes. Unlike conventional SPE columns, the MEPS sorbent bed is integrated into a liquid handling syringe that allows for low void volume sample manipulations either manually or in combination with laboratory robotics. The key aspect of MEPS is that the solvent volume used for the elution of the analytes is of a suitable order of magnitude to be injected directly into GC or LC systems. This new technique is very promising because it is fast, simple and it requires very small volume of samples to produce comparable results to conventional SPE technique. Furthermore, this technique can be easily interfaced to LC/MS and GC/MS to provide a completely automated MEPS/LC/MS or MEPS/GC/MS system. This extraction technique (MEPS) could be of interest in clinical, forensic toxicology and environmental analysis areas. This review provides a short overview of recent applications of MEPS in clinical and pre-clinical studies for quantification of drugs and metabolites in blood, plasma and urine. The extraction of anti-cancer drugs, β-blockers drugs, local anaesthetics, neurotransmitters and antibiotics from biological samples using MEPS technique will be illustrated.     

The chromatographic assay of 4‐hydroxynonenal as a biomarker of diseases by means of MEPS and HPLC technique

The aim of this study was to develop and validate a new analytical method for the determination of 4‐hydroxy‐2‐nonenal (4‐HNE) in biological samples while applying microextraction by packed sorbent as a sample preparation method and HPLC with UV–vis detection. Various microextraction by packed sorbent (MEPS) sorbents like C₂, C₈, C₁₈, M1 (80% C₈ and 20% SCX) and silica were used to separate 4‐HNE from biological samples. The highest affinity of 4‐HNE was observed for sorbents like C₁₈. The extraction efficiency was in the range from 47.4 to 89.2% dependent on the concentration of 4‐HNE. Lower efficiency of 4‐HNE extraction was obtained with use of MEPS packings such as C₈ and M1. The extraction efficiency was in the range from 35.2 to 66.3% for packing C8 and from 34.2 to 64.3% for packing M1, respectively. The limit of detection and lower limit of quantitation for UV–vis detection were respectively 4.5 and 9.0 nmol/mL. The proposed method can be used for the evaluation of extraction efficiency of 4‐HNE in biological sample because the values of lower limit of quantitation are lower than the determined amounts of the analyte in samples. The method yields excellent performance of quantification and identification in analysis of inflammation biomarkers. Copyright © 2014 John Wiley & Sons, Ltd.     

Application of microextraction by packed sorbent to isolation of psychotropic drugs from human serum

A method of microextraction by packed sorbent (MEPS) followed by liquid chromatography with diode array detection has been developed and optimized for the extraction of six tricyclic antidepressants (amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin) from human serum. The optimal parameters of MEPS extraction (type of sorbent, volume of sample, composition, and volume of washing and elution solutions) for these drugs in spiked samples were defined. The developed MEPS procedure was validated and then successfully applied to the analysis of serum reference material. The limit of detection (0.02–0.05 μg/mL), intraday (2.7–8.8%) and interday (4.4–11.6%) precision (RSD), and the accuracy of the assay (94.5–108.8%) at three concentration levels—0.2, 0.5, and 0.8 μg/mL—were estimated. The accuracy of the method was evaluated by the analysis of certified reference material. Moreover, the validated procedure was compared with the solid-phase extraction technique. Finally, microextraction by packed sorbent was assessed as a suitable tool in forensic and clinical methods for serum sample preparations.     

Simultaneous Quantification of 25 Fentanyl Derivatives and Metabolites in Oral Fluid by Means of Microextraction on Packed Sorbent and LC–HRMS/MS Analysis

Fentanyl and fentalogs’ intake as drugs of abuse is experiencing a great increase in recent years. For this reason, there are more and more cases in which it is important to recognize and quantify these molecules and related metabolites in biological matrices. Oral fluid (OF) is often used to find out if a subject has recently used a psychoactive substance and if, therefore, the person is still under the effect of psychotropics. Given its difficulty in handling, good sample preparation and the development of instrumental methods for analysis are essential. In this work, an analytical method is proposed for the simultaneous determination of 25 analytes, including fentanyl, several derivatives and metabolites. OF was collected by means of passive drool; sample pretreatment was developed in order to be fast, simple and possibly semi-automated by exploiting microextraction on packed sorbent (MEPS). The analysis was performed by means of LC–HRMS/MS obtaining good identification and quantification of all the analytes in less than 10 min. The proposed method was fully validated according to the Scientific Working Group for Forensic Toxicology (SWGTOX) international guidelines. Good results were obtained in terms of recoveries, matrix effect and sensitivity, showing that this method could represent a useful tool in forensic toxicology. The presented method was successfully applied to the analysis of proficiency test samples.     

Two new techniques for sample preparation in bioanalysis: Microextraction in packed sorbent (MEPS) and use of a bonded monolith as sorbent for sample preparation in polypropylene tips for 96-well plates

Analytical methods providing high throughput are required for the ever increasing number of samples in bioanalysis. Currently, the method of choice in bioanalysis is LC-MS-MS. This method is quite rapid and thereby the focus has been directed to sample preparation as being a bottleneck in total analysis systems. It has become necessary to develop sample preparation techniques to a new improved level. This development has been based on a systematic and scientific approach. The key factors in this development have been miniaturization, integration, and automation of the techniques. This review provides a short overview of recent developments. Special emphasis is on two techniques: microextraction in packed syringe (MEPS) and use of a monolithic acrylamide plug as sorbent in polypropylene tips primarily intended for use with 96-well plate systems.     

Determination of selected β-receptor antagonists in biological samples by solid-phase extraction with cholesterolic phase and LC/MS

A new method is presented for the determination of five selected β-receptor antagonists by HPLC, which emphasizes sample preparation via retention on a new type of silica gel sorbent used for solid-phase extraction (SPE). Sorbents of this type were obtained by the chemical modification of silica gels of various porosities by cholesterol ligands. The cholesterol-based packing material was investigated by spectroscopic methods and elemental analysis. The recoveries obtained with the extraction procedure were optimum over a relatively broad sample pH range (3.08–7.50). Analytical factors such as the sample loading, the washing step and elution conditions, the concentration of β-receptor antagonists to be extracted, and the type of sorbent were found to play significant roles in the sample preparation procedure and would therefore need to be controlled to achieve optimum recoveries of the analytes. Under optimum conditions, the recoveries of nadolol, acebutolol, esmolol, oxprenolol and propranolol from spiked buffers, blood and urine were reproducible and dependent on the polarity or hydrophilicity of the compounds. The above analytes were determined by reverse-phase high-performance liquid chromatography (HPLC) with UV and ESI-ion trap mass spectrometry (MS) detection. The described method was found to be suitable for the routine measurement of compounds that are both polar and basic, and can be applied for the analysis of biological samples such as urine and blood in clinical, toxicological or forensic laboratories. The recovery measurements were performed on spiked human urine and serum, and on real samples of mouse blood serum.     

Novel sample preparation technique with needle-type micro-extraction device for volatile organic compounds in indoor air samples

A novel needle-type sample preparation device was developed for the effective preconcentration of volatile organic compounds (VOCs) in indoor air before gas chromatography–mass spectrometry (GC–MS) analysis. To develop a device for extracting a wide range of VOCs typically found in indoor air, several types of particulate sorbents were tested as the extraction medium in the needle-type extraction device. To determine the content of these VOCs, air samples were collected for 30 min with the packed sorbent(s) in the extraction needle, and the extracted VOCs were thermally desorbed in a GC injection port by the direct insertion of the needle. A double-bed sorbent consisting of a needle packed with divinylbenzene and activated carbon particles exhibited excellent extraction and desorption performance and adequate extraction capacity for all the investigated VOCs. The results also clearly demonstrated that the proposed sample preparation method is a more rapid, simpler extraction/desorption technique than traditional sample preparation methods.     

Microextraction by packed sorbent followed by ultra high performance liquid chromatography for the fast extraction and determination of six antidepressants in urine

The growing use of antidepressants in recent years has led to their increasing presence in forensic analyses. In this work, microextraction by packed sorbent followed by ultra‐performance liquid chromatography with photodiode array detection provided a fast method for determining the antidepressants mirtazapine, venlafaxine, escitalopram, fluoxetine, fluvoxamine, and sertraline in human urine. The microextraction conditions (viz., type of sorbent, number of draw–eject extraction cycles or strokes, sample volume and pH, and type and volume of washing solution and eluent) were optimized by using an experimental design. The ensuing analytical method was validated in terms of linearity (25–1000 ng/mL urine), limit of detection (lower than 7.1 ng/mL), limit of quantification (25 ng/mL), precision (4.7–15.1% as relative standard deviation), and accuracy (80.4–126.1% as mean recovery for four replicate determinations). The proposed method allowed the six target antidepressants to be determined at concentrations from therapeutic to toxic levels. The application to small volumes (300 μL) of urine afforded fast extraction of the analytes and provided results on a par with those of existing clinical and forensic alternatives.     

A fast method for screening and/or quantitation of tetrahydrocannabinol and metabolites in urine by automated SPE/LC/MS/MS

Marijuana is one of the most commonly used illicit substances. The high usage of this substance results in it being commonly encountered in clinical samples throughout the USA and Europe. Due to its wide availability and use, marijuana is also commonly encountered in forensic toxicology laboratories. The proposed method utilized an automated solid phase extraction (SPE) coupled to liquid chromatography/mass spectrometry (LC/MS). The automated SPE procedure was developed using Hysphere C8-EC sorbent, and the high performance liquid chromatography (HPLC) separation was performed using an Xterra MS C₁₈ column with a total runtime of 10 min. The standard curves linearity generally fell between 6 and 500 ng/mL. The limits of detection ranged from 2 to 4 ng/mL, and the limits of quantitation ranged from 8 to 12 ng/mL. The bias and imprecision were determined using a simple analysis of variance (single factor). The results demonstrate bias as <11% and percent imprecision as <12% for all components at four quality control levels. This method has been in use for over 2 years and has been applied to numerous forensic samples. When compared to other published methods, it exceeds others in its simplicity and speed of analysis. This method takes advantage of robotics and automation for a total analysis time of 10 min, including sample preparation, separation, and detection.     

Development of a microextraction by packed sorbent with gas chromatography‐mass spectrometry method for quantification of nitroexplosives in aqueous and fluidic biological samples

A new method for quantification of 12 nitroaromatic compounds including 2,4,6‐trinitrotoluene, its metabolites and 2,4,6‐trinitrophenyl‐N‐methylnitramine with microextraction by packed sorbent followed by gas chromatography and mass spectrometric detection in environmental and biological samples is developed. The microextraction device employs 4 mg of C₁₈ silica sorbent inserted into a microvolume syringe for sample preparation. Several parameters capable of influencing the microextraction procedure, namely, number of extraction cycles, washing solvent, volume of washing solvent, elution solvent, volume of eluting solvent and pH of matrix, were optimized. The developed method produced satisfactory results with excellent values of coefficient of determination (R² > 0.9804) within the established calibration range. The extraction yields were satisfactory for all analytes (> 89.32%) for aqueous samples and (> 87.45%) for fluidic biological samples. The limits of detection values lie in the range 14–828 pg/mL.     

Simple and rapid determination of phthalates using microextraction by packed sorbent and gas chromatography with mass spectrometry quantification in cold drink and cosmetic samples

A simple and rapid method using microextraction by packed sorbent coupled with gas chromatography and mass spectrometry has been developed for the analysis of five phthalates, namely, diethyl phthalate, benzyl‐n‐butyl phthalate, dicyclohexyl phthalate, di‐n‐butyl phthalate, and di‐n‐propyl phthalate, in cold drink and cosmetic samples. The various parameters that influence the microextraction by packed sorbent performance such as extraction cycle (extract–discard), type and amount of solvent, washing solvent, and pH have been studied. The optimal conditions of microextraction using C₁₈ as the packed sorbent were 15 extraction cycles with water as washing solvent and 3 × 10 μL of ethyl acetate as the eluting solvent. Chromatographic separation was also optimized for injection temperature, flow rate, ion source, interface temperature, column temperature gradient and mass spectrometry was evaluated using the scan and selected ion monitoring data acquisition mode. Satisfactory results were obtained in terms of linearity with R² >0.9992 within the established concentration range. The limit of detection was 0.003–0.015 ng/mL, and the limit of quantification was 0.009–0.049 ng/mL. The recoveries were in the range of 92.35–98.90% for cold drink, 88.23–169.20% for perfume, and 88.90–184.40% for cream. Analysis by microextraction by packed sorbent promises to be a rapid method for the determination of these phthalates in cold drink and cosmetic samples, reducing the amount of sample, solvent, time and cost.     

Liquid chromatographic analysis of oxcarbazepine and its metabolites in plasma and saliva after a novel microextraction by packed sorbent procedure

A rapid and reliable analytical method suitable for the simultaneous determination of the antiepileptic drug, oxcarbazepine and its metabolites in human plasma and saliva by means of liquid chromatography with diode array detection (DAD) has been developed. Oxcarbazepine and its metabolites (10,11-dihydro-10-hydroxycarbamazepine, trans-10,11-dihydro-10,11-dihydroxycarbamazepine and 3-hydroxycarbamazepine) were baseline separated within 6.5 min on a reversed-phase C18 column with a phosphate buffer-acetonitrile-triethylamine mixture as the mobile phase. The DAD detector was set at 240 nm. A sample preparation method for biological samples using a microextraction by packed sorbent technique has been implemented, employing a C18 sorbent inserted into a microvolume syringe and using only a small volume (25 μL) of plasma or saliva. The extraction yield values were satisfactory for all analytes (>86.5%) as well as the precision data, which were always in the low percentage of relative standard deviation values (<4.6%). The method was successfully applied to both plasma and saliva samples drawn from psychiatric and neurological patients undergoing treatment with oxcarbazepine (Tolep^®) tablets.     

Disposable pipette extraction for gas chromatographic determination of codeine, morphine, and 6-monoacetylmorphine in vitreous humor

The availability of a sensitive and rapid analytical method for the determination of opiates, and other substances of forensic interest, in a variety of biological specimens is of utmost importance to forensic laboratories. Solid-phase extraction is very popular in the pre-treatment of forensic samples. Nevertheless, a new approach, disposable pipette extraction (DPX), is gaining increasing interest in sample preparation. DPX has already been applied to the analysis of drugs of abuse in common biological matrices, such as urine and blood, but has not yet been evaluated on alternative biological samples, such as vitreous humor. The objective of this study was to evaluate the applicability of DPX on the analysis of opiates in vitreous humor. The currently developed method is fast, reliable, and easy to perform. The sensitivity, precision, and accuracy are satisfactory. Recoveries obtained are within the range of 72-91%, whereas the sample volume of vitreous humor required is only 100 μL.     

Metabolic Profiling and Quantitative Analysis of Cerebrospinal Fluid Using Gas Chromatography–Mass Spectrometry: Current Methods and Future Perspectives

Cerebrospinal fluid is a key biological fluid for the investigation of new potential biomarkers of central nervous system diseases. Gas chromatography coupled to mass-selective detectors can be used for this investigation at the stages of metabolic profiling and method development. Different sample preparation conditions, including extraction and derivatization, can be applied for the analysis of the most of low-molecular-weight compounds of the cerebrospinal fluid, including metabolites of tryptophan, arachidonic acid, glucose; amino, polyunsaturated fatty and other organic acids; neuroactive steroids; drugs; and toxic metabolites. The literature data analysis revealed the absence of fully validated methods for cerebrospinal fluid analysis, and it presents opportunities for scientists to develop and validate analytical protocols using modern sample preparation techniques, such as microextraction by packed sorbent, dispersive liquid–liquid microextraction, and other potentially applicable techniques.     

Microextraction by packed sorbent (MEPS): a tutorial

This tutorial provides an overview on a new technique for sample preparation, microextraction by packed sorbent (MEPS). Not only the automation process by MEPS is the advantage but also the much smaller volumes of the samples, solvents and dead volumes in the system. Other significant advantages such as the speed and the simplicity of the sample preparation process are provided.In this tutorial the main concepts of MEPS will be elucidated. Different practical aspects in MEPS are addressed. The factors affecting MEPS performance will be discussed. The application of MEPS in clinical and pre-clinical studies for quantification of drugs and metabolites in blood, plasma and urine will be provided. A comparison between MEPS and other extraction techniques such as SPE, LLE, SPME and SBSE will be discussed.     

Liquid chromatography-electrospray ionization isotope dilution mass spectrometry analysis of paraquat and diquat using conventional and multilayer solid-phase extraction cartridges

The performance of alkyl-silica sorbent packed solid-phase extraction (SPE) cartridges and a mixed-mode, polymeric sorbent packed SPE cartridge (resin SPE cartridge) were evaluated for the sample preparation of paraquat and diquat in environmental water and vegetation matrices. Also the recoveries of the native and 2H-labeled paraquat and diquat were correlated to validate that the 2H-labeled species can be used for the isotopic dilution mass spectrometry (IDMS) analysis of paraquat and diquat. The results show that the extraction efficiency of alkyl-silica SPE is dependent on the carbon loading of the sorbent and deteriorates with an increasing sample pH. The resin SPE cartridge required no pH adjustment and showed excellent correlation between the native and 2H-labeled species, therefore, allowing us to develop the first liquid chromatography-electrospray ionization IDMS analytical method for the analysis of paraquat and diquat in environmental water and vegetation matrices. Method detection limits derived using standard EPA protocol were 0.2 and 0.1 microg/l for paraquat and diquat in water matrices, and 0.02 and 0.01 microg/g in vegetation matrices, respectively.     

Microextraction techniques in antibiotic monitoring in body fluids: Recent trends and future

The analysis of drugs in biomedical discipline targets a broad range of aims such as therapeutic drug monitoring, pharmacokinetic study to investigate the drug bioavailability, bioequivalence tests to evaluate the effect of formulation parameters, toxicology, and forensic science. Because of the low levels of typical antibiotics in plasma, blood, urine, exhalation samples, and other biological fluids as well as complex matrix of biological media, adequate sample preparation methods should be implemented for quantification of antibiotics. In this review, developments in well-established microextraction techniques for the clinical analysis of biological samples will be reviewed and discussed. This article presents an overview of microextraction methods for biological samples, focusing especially on antibiotics.     

Rapid identification and quantification of methamphetamine and amphetamine in hair by gas chromatography/mass spectrometry coupled with micropulverized extraction,aqueous acetylation and microextraction by packed sorbent

We developed a rapid identification and quantification method for the toxicological analysis of methamphetamine and amphetamine in human hair by gas chromatography/mass spectrometry coupled with a novel combination of micropulverized extraction, aqueous acetylation and microextraction by packed sorbent (MEPS) named MiAMi–GC/MS. A washed hair sample (1–5 mg) was micropulverized for 5 min in a 2 mL plastic tube with 250 μL of water. An anion-exchange sorbent was added to adsorb anionic interferences. After removing the residue with a membrane-filter unit, sodium carbonate and acetic anhydride was admixed in turn. Acetylation was completed in approximately 20 min at room temperature. The acetylated analytes in the reaction liquid were concentrated to an octadecylsilica sorbent packed in the needle of a syringe by a CombiPAL autosampler. Elution was carried out with 50 μL of methanol, and the entire eluate injected into a gas chromatograph using a programmable temperature vaporizing (PTV) technique. The time required for sample preparation and GC/MS analysis was approximately 1 h from a washed hair sample, and an evaporation process was not required. Ranges for quantification were 0.20–50 (ng/mg) each for methamphetamine and amphetamine using 1 mg of hair. Accuracy and relative standard deviation (RSD) were evaluated intraday and interday at three concentrations, and the results were within the limit of a guidance issued by U.S. Food and Drug Administration. For identification, full-scan mass spectra of methamphetamine and amphetamine were obtained using 5 mg of fortified hair samples at 0.2 ng/mg. The extraction device of MEPS was durable for at least 300 extractions, whereas the liner of the gas chromatograph should be replaced after 20–30 times use. The carry over was estimated to be about 1–2%. This sample-preparation method coupled with GC/MS is fast and labor-saving in comparison with conventional methods.