Recent advances in sample preparation techniques for effective bioanalytical methods

This paper reviews the recent developments in bioanalysis sample preparation techniques and gives an update on basic principles, theory, applications and possibilities for automation, and a comparative discussion on the advantages and limitation of each technique. Conventional liquid-liquid extraction (LLE), protein precipitation (PP) and solid-phase extraction (SPE) techniques are now been considered as methods of the past. The last decade has witnessed a rapid development of novel sample preparation techniques in bioanalysis. Developments in SPE techniques such as selective sorbents and in the overall approach to SPE, such as hybrid SPE and molecularly imprinted polymer SPE, have been addressed. Considerable literature has been published in the area of solid-phase micro-extraction and its different versions, e.g. stir bar sorptive extraction, and their application in the development of selective and sensitive bioanalytical methods. Techniques such as dispersive solid-phase extraction, disposable pipette extraction and micro-extraction by packed sorbent offer a variety of extraction phases and provide unique advantages to bioanalytical methods. On-line SPE utilizing column-switching techniques is rapidly gaining acceptance in bioanalytical applications. PP sample preparation techniques such as PP filter plates/tubes offer many advantages like removal of phospholipids and proteins in plasma/serum. Newer approaches to conventional LLE techniques (salting-out LLE) are also covered in this review article.     

A review of sample preparation methods for the pesticide residue analysis in foods

The pesticide residues in foods have received increasing attention as one of the most important food safety issues. Therefore, more strict regulations on the maximum residue limits (MRLs) for pesticides in foods have been established in many countries and health organizations, based on the sensitive and reliable analysis methods of pesticide residues. However, the analysis of pesticide residues is a continuing challenge mainly because of the small quantities of analytes as well as the large amounts of interfering substances which can be co-extracted with them, often leading to experimental errors and damage to the analytical instruments. Thus, extensive sample preparation is often required for the pesticide residue analysis for the effective extraction of the analytes and removal of the interferences. This paper focuses on reviewing the recent development in the sample preparation methods for the pesticide residue analysis in foods since 2006. The methods include: liquid-liquid extraction (LLE), supercritical-fluid extraction (SFE), pressurized-liquid extraction (PLE), microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), gel permeation chromatography (GPC), solid-phase extraction (SPE), molecularly imprinted polymers (MIPs), matrix solid-phase dispersion (MSPD), solid-phase micro-extraction (SPME), QuEChERS, cloud point extraction (CPE) and liquid phase micro-extraction (LPME), etc. Particularly their advantages, disadvantages and future perspectives will be discussed.     

超临界流体色谱在生物工程中应用的新进展

超临界流体色谱（SFC）是高效液相色谱（HPLC）和气相色谱（GC）的重要补充技术。用SFC可以分离多数不能用GC分离的低挥发性物质。与传统的HPLC相比，SFC的分离速度更快、效率更高。本文综述了SFC在生物分子分离分析方面应用的新进展。引用文献74篇。     

Simultaneous determination of prednisolone acetate, prednisolone, prednisone, cortisone and hydrocortisone in swine plasma using solid-phase and liquid-liquid extraction techniques

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of prednisolone acetate (PA), prednisolone (PO), prednisone (PN), cortisone and hydrocortisone in swine plasma is described. Extraction of the steroid mixture from swine plasma with dexamethasone as internal standard was accomplished by solid-phase extraction (SPE) or the more traditional liquid-liquid extraction (LLE) techniques. These compounds were analyzed by normal-phase HPLC with ultraviolet detection. Although a detectable sensitivity of 5 ng/ml is achieved by the SPE technique, the practical sensitivity is established as 10 ng/ml. Conversely, the practical sensitivity is 5 ng/ml for all compounds by the LLE technique. Calibration curves were found to be linear between 10 and 500 ng/ml by the SPE technique and between 5 and 100 ng/ml by the LLE technique. The average recovery of the steroids PA, PO and PN at 20 ng/ml is between 70 and 90%. PA is stable for up to 3 h in swine plasma at room temperature (22 degrees C) but is completely converted to PO within 24 h. PA is stable in swine plasma in an ice bath for over 24 h. The usefulness of this analytical technique is demonstrated by the intraperitoneal administration of 125 mg of PA to swine and the quantitative determination of PA, PO and PN in the plasma as a function of time.     

Development and validation of a high‐throughput assay for the quantification of multiple green tea‐derived catechins in human plasma

A rapid, accurate and robust method for the determination of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) concentrations in human plasma has been developed. The method utilizes protein precipitation following enzyme hydrolysis, with chromatographic separation and detection using reversed‐phase liquid chromatography–tandem mass spectrometry (LC–MS/MS). Traditional issues such as lengthy chromatographic runtimes, sample and extract stability, and lack of suitable internal standards have been addressed. The method has been evaluated using a comprehensive validation procedure, confirming linearity over appropriate concentration ranges, and inter/intra‐batch precision and accuracies within suitable thresholds (precisions within 13.8% and accuracies within 12.4%). Recoveries of analytes were found to be consistent between different matrix samples, compensated for using suitable internal markers and within the performance of the instrumentation used. Similarly, chromatographic interferences have been corrected using the internal markers selected. Stability of all analytes in matrix is demonstrated over 32 days and throughout extraction conditions. This method is suitable for high‐throughput sample analysis studies.     

Simultaneous determination of pantoprazole and its two metabolites in dog plasma by HPLC

A simple and sensitive high-performance liquid chromatography (HPLC) method is developed and validated for simultaneous determination of pantoprazole and its two metabolites (pantoprazole sulfone and pantoprazole thioether) in dog plasma and applied to a pharmacokinetic study in Beagle dogs. Following a protein precipitation procedure, the samples are separated using reversed-phase HPLC (C18) by a gradient of acetonitrile and ammonium acetate (pH 6.0) at a flow rate of 1.0 mL/min and quantitated using UV detection at 290 nm. Omeprazole is selected as the internal standard. The method has a lower limit of quantitation of 0.025 microg/mL for pantoprazole and its two metabolites, using 0.1-mL aliquots of plasma. The linear calibration curves are obtained in the concentration range of 0.025-10.0 microg/mL for three analytes. The intra- and interrun precision (relative standard deviation), calculated from quality control (QC) samples, is less than 13% for three analytes. The accuracy determined from QC samples is between -6.4% and 12%.     

Confirmatory analysis of acetylgestagens in plasma using liquid chromatography-tandem mass spectrometry

A confirmatory method has been developed and validated for the determination of chlormadinone acetate (CMA), megestrol acetate (MGA), melengestrol acetate (MLA) and medroxyprogesterone acetate (MPA) in bovine and porcine plasma. Analytes are extracted from plasma samples using matrix-assisted liquid-liquid extraction (LLE) on Extrelut NT columns followed by C18 solid-phase extraction (SPE). Analytes were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and quantification was performed using matrix-matched calibration standards in combination with deuterated internal standards. In accordance with Commission Decision 2002/657/EC, two ion transitions were monitored for each analyte. Decision limits (CCalpha) were estimated by analysing 20 blank plasma samples and ranged from 0.1 to 0.2 ng mL(-1). Detection capabilities (CCbeta) were estimated using 20 plasma samples fortified at 0.5 ng mL(-1) and were <0.5 ng mL(-1). In the range 0.5-2 ng mL(-1), the mean intra-laboratory reproducibility of the analytes ranged from 6 to 18% (%R.S.D.). Analytes were shown to be stable in fortified plasma samples for >8 months when stored at -20 degrees C.     

Micro-extraction of Xenobiotics and Biomolecules from Different Matrices on Nanostructures

Sample preparation is the backbone of any analytical procedure; it involves extraction and pre-concentration of the desired analytes; often at trace levels. The present article describes the applications of nanomaterials (carbon-based inorganic and polymeric materials) in miniaturized extraction such as solid phase micro-extraction, stir-bar sorptive extraction, liquid phase micro-extraction, and dispersive liquid phase micro-extraction in the analyses of aqueous samples. The nanoparticles used for micro-extractions are discussed on the basis of their chemical natures. The synthetic route and the preparation of nanomaterials are described along with the optimization strategies for micro-extraction. A comparison between the conventional materials and nanomaterials for micro-extraction is proposed. The key roles of the nanomaterials for the micro-extraction of different analytes such as drugs, pesticides, polycyclic aromatic hydrocarbons, proteins and peptides from aqueous samples are reported. The use of nanomaterials, combined with miniaturized micro-extraction techniques, proved to be highly promising for sample preparation of various matrices with analytes at trace levels.     

一种以含硅藻土的复合材料为介质的支撑液液萃取柱的制备及其应用

以含硅藻土的复合材料为支撑介质,开发了一种独特的支撑液液萃取柱;以一系列酸性、碱性和中性水溶液样品为模型化合物,结合高效液相色谱法(HPLC)对该萃取柱进行了系统评价,同时将其用于复杂基质样品的分析。结果表明: 经该支撑液液萃取柱预处理的苯甲酸、对硝基苯胺和对羟基苯甲酸甲酯水溶液的萃取回收率分别为90.6%、98.1%和97.7%,远超过对应样品经传统液液萃取法处理后的回收率（分别为71.9%、81.9%和83.9%）。对于复杂基质样品的分析,如雪碧中的防腐剂苯甲酸以及牛血清中的中性药物醋酸地塞米松、碱性药物马来酸氯苯那敏及酸性药物吲哚美辛等,样品的加标回收率均在80%和110%之间,相对标准偏差(RSD)均小于15%,符合生物样品的分析要求,且未出现传统液液萃取技术中常见的问题(如乳化现象)。所开发的支撑液液萃取柱具有快速、简单、耐受性好、易于实现自动化和高通量的特点,具有广泛的应用前景。     

Comparison of a GC Method and Two HPLC Methods for the Determination of p-Hydroxyphenytoin in Urine

Abstract

Three chromatographic methods for determining p-hydroxy-phenytoin (p-HPT) in urine were compared: (1) GC with derivatisation of the samples, (2) HPLC after extraction with ethyl acetate and (3) HPLC using a column switching system for direct injection of samples. In all three methods the p-HPT glucuronides were first hydrolysed using concentrated mineral acid at boiling point. For method (1) the acidic hydrolysate was adjusted to pH 7–8.5. Benzenetetrahydrofuran was used for extraction of p-HPT. The extract was evaporated to dryness, taken up in trimethyl-aniliniumhydroxide and injected. For method (2) the acidic hydrolysate was buffered with tri-sodium phosphate. An aliquot of the buffered solution was extracted with ethyl acetate. The extract was evaporated to dryness, taken up in methanol and injected. For method (3) the hydrolysate was diluted with water/acetonitrile (9:1), centrifuged and directly injected onto the pre-column for the sample washing step.     

The determination of total and unbound midazolam in human plasma. A comparison of high performance liquid chromatography, gas chromatography and gas chromatography/mass spectrometry

Midazolam concentrations in patients' plasma was determined after extraction with high performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). GC was selected for routine plasma assays in terms of selectivity, simplicity, precision, accuracy and sensitivity (0.02 microgram/mL); HPLC analysis was less sensitive (0.1 microgram/mL) than GC; GC/MS was used for analysis validation. Plasma protein binding of midazolam was determined by GC in patients' plasma after in vitro incubation with midazolam, ultrafiltration and extraction; 5% of the drug was unbound to plasma proteins. Midazolam distribution in lipoprotein fractions separated by ultracentrifugation of plasma obtained from patients on prolonged midazolam treatment was also assayed by GC.     

Application of negligible and full depletion micro-extraction in environmental sciences

Negligible depletion micro-extraction (nd-ME) and full depletion micro-extraction (fd-ME) have been developed for specific purposes in environmental studies. Nd-ME is mainly designed to measure the free concentration and therefore evaluate the bioavailability of analytes in environmental matrices, and achieved by using large ratio of sample to extraction phase or extraction phase with low distribution coefficients for analytes. On the contrary, fd-ME is adopted to avoid matrix effects by determining the total amount of the analyte in the samples, and performed by using very low ratio of sample to extraction phase or extraction phase with extremely large distribution coefficients for analytes. In this review article, we highlight the basic concepts and applications of nd-ME and fd-ME in environmental sciences.     

High-performance liquid chromatography with fluorescence detection and ultra-performance liquid chromatography with electrospray tandem mass spectrometry method for the determination of indoleamine neurotransmitters and their metabolites in sea lamprey plasma

We present a comparison of two sensitive methods, HPLC with fluorescence detector (HPLC/FLD) and UPLC with electrospray tandem mass spectrometry (UPLC/MS/MS), for the determination of indoleamine neurotransmitters (NTs) and their metabolites in sea lamprey plasma samples. Liquid–liquid extraction (LLE) and solid-phase extraction (SPE) were also tested for recovery and matrix effect. The recoveries of SPE determined by HPLC/FLD and UPLC/MS/MS ranged from 75 to 123% and 78 to 105%, respectively, while the recoveries of LLE ranged from 45 to 73% and 48 to 75%, respectively. SPE combined with HPLC/FLD and UPLC/MS/MS to determine the target analytes in plasma samples were validated of the sensitivity, reproducibility, accuracy and precision. Both methods exhibited excellent linearity in the range of 0.2–50 ng mL⁻¹ for all analytes. The limits of detection (LOD) varied from 0.04 ng mL⁻¹ to 0.13 ng mL⁻¹ for HPLC/FLD method and 0.003 ng mL⁻¹ to 0.02 ng mL⁻¹ for UPLC/MS/MS method. The inter-day accuracy ranged from 82.5 to 127.0% for HPLC/FLD and 93.0 to 113.0% for UPLC/MS/MS. The inter-day precision ranged from 9.9 to 32.3% for HPLC/FLD and 5.4 to 13.2% for UPLC/MS/MS. These results demonstrated that the values obtained by both methods were within the satisfactory range and the UPLC/MS/MS method provided more accurate and precise measurements than HPLC/FLD method. The comparison is of great importance to determine the available detectors, considering the complexity and expensiveness versus quality parameters. These two methods were applied to the analysis of four important indoleamine neurotransmitter analytes (5-hydroxytryptamine, 5-hydroxyindole-3-acetic acid, tryptamine and melatonin) in sea lamprey plasma samples.     

Liquid-phase microextraction with porous hollow fibers, a miniaturized and highly flexible format for liquid-liquid extraction

Since 1999, substantial research has been devoted to the development of liquid-phase microextraction (LPME) based on porous hollow fibers. With this technology, target analytes are extracted from aqueous samples, through a thin supported liquid membrane (SLM) sustained in the pores in the wall of a porous hollow fiber, and further into a microL volume of acceptor solution placed inside the lumen of the hollow fiber. After extraction, the acceptor solution is directly subjected to a final chemical analysis by liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (CE), or mass spectrometry (MS). In this review, LPME will be discussed with focus on extraction principles, historical development, fundamental theory, and performance. Also, major applications have been compiled, and recent forefront developments will be discussed.     

Automated determination of verapamil and norverapamil in human plasma with on-line coupling of dialysis to high-performance liquid chromatography and fluorometric detection

A fully automated method for the simultaneous determination of verapamil and its main metabolite norverapamil in human plasma is described. This method is based on on-line sample preparation using dialysis followed by clean-up and enrichment of the dialysate on a precolumn and subsequent HPLC analysis with fluorometric detection. All sample handling operations were performed automatically by a sample processor equipped with a robotic arm (ASTED system). The plasma samples were dialysed on a cellulose acetate membrane (cut-off: 15 kD) and the dialysate was purified and enriched on a short pre-column filled with cyanopropyl silica. Before starting dialysis, this trace enrichment column (TEC) was first conditioned with the HPLC mobile phase and then with pH 3.0 acetate buffer. 370 μl of plasma sample spiked with the internal standard (gallopamil) were dialysed in the static-pulsed mode. The solution at the donor side was pH 3.0 acetate buffer containing Triton X-100 while the acceptor solution was made of the same acetate buffer. When dialysis was discontinued, the analytes were desorbed from the TEC by the HPLC mobile phase and transferred to the C₁₈ analytical column by means of a switching valve. This mobile phase consisted of a mixture of acetonitrile, pH 3.0 acetate buffer and 2-aminoheptane. The influence of different parameters of the dialysis process on the recovery of verapamil and norverapamil has been studied. The effect of the volume, the aspirating and dispensing flow-rates of the dialysis solution has been investigated. The recoveries of verapamil and norverapamil in plasma were close to 75% and the limits of quantification were 5 ng/ml for both analytes. The method was found to be linear in the concentration range from 5 to 500 ng/ml (r²: 0.9996 for both analytes). The intra-day and inter-day reproducibilities at a concentration of 100 ng/ml were 2.3% and 5.6% for verapamil and 1.7% and 5.1% for norverapamil, respectively.     

Solid-phase extraction using C(18) bonded silica disks: interferences and analysis of chlorotriazines in seawater samples

The interferences in C(18) Empore extraction disks were obtained by processing 5 1. of HPLC water with average blanks of 1 ng/l. A C(19) alkane, plasticizers and the antioxidant Nonox A were identified in the blanks as possible interferences. The extraction of the components of the disks was carried out with methanol, acetonitrile and/or ethyl acetate with subsequent analysis by gas chromatography-mass spectrometry (GCMS). The identification of interferences was a requirement for the determination of the chlorotriazine herbicides atrazine and simazine, and of a transformation product, de-ethylatrazine, at concentration levels varying between 2 and 140 ng/l. Seawater samples of 3-28 parts-per-thousand were pre-filtered through a 47-mm diameter of 0.7 mum and subsequently with 0.45 mum glass-fibre filters to trap particulate matter, followed by Empore extraction disks of 500 mg C(18) bonded silica. Water volumes of 5 1. could be processed within 150 min. The disks were extracted with methanol, the extract was blown down under nitrogen, and the analytes were quantified by GC with nitrogen-phosphorus detection (NPD) with further confirmation using GCMS in the selected ion monitoring (SIM) mode. The proposed method has been applied to the determination of the environmental levels of atrazine and simazine in seawater samples of varying salinity. The recovery of de-ethylatrazine was 10%, so the method was not appropriate for this compound. The concentration of the herbicides has been plotted against the salinity values, showing a decrease in the levels as the salinity increases, with two inflexion points that indicate a non-conservative mixing with loss of the herbicides in the mixing zone of the estuary.     

High-performance liquid chromatographic method for routine determination of vitamins A and E and beta-carotene in plasma.

A simple and reliable reversed-phase high-performance liquid chromatographic (HPLC) method for the routine determination of vitamins A and E and beta-carotene in plasma (or serum) with wavelength-programmed ultraviolet-visible absorbance detection is described. A 200-microliters aliquot of serum or plasma sample, after deproteinization with ethanol, and containing tocopherol acetate as internal standard, was extracted with butanol-ethyl acetate. Sodium sulphate was added for dehydration. Analytes of extracted samples were found to be stable for at least four days. A 10-microliters aliquot of this organic extract was used for HPLC analysis. The mobile phase was methanol-butanol-water (89.5:5:5.5, v/v) and the flow-rate was set at 1.5 ml/min. The analytes of interest were well separated from other plasma constituents within 22 min at 45 degrees C. The lowest detection limits of vitamins A and E and beta-carotene were 0.02, 0.5 and 0.1 microgram/ml, respectively. The recovery and reproducibility of the present method were around 90%. The method is sensitive, specific and can be used for epidemiological studies and for routine determination of vitamin deficiency. Several important factors that may affect the analysis are also discussed in this paper.     

Basic drug analysis by strong cation-exchange liquid chromatography-tandem mass spectrometry: simultaneous analysis of amisulpride, and of metamfetamine and amfetamine in serum/plasma

In the HPLC of basic drugs and metabolites, good efficiency and peak shape can often be attained using strong cation‐exchange packings with isocratic 100% methanol eluents containing an ionic modifier at an appropriate pH* and ionic strength. Solvent extracts can be analysed directly, and use of ammonium acetate as modifier facilitates the use of atmospheric pressure chemical ionization (APCI)–tandem mass spectrometry, selected reaction monitoring mode. For the analysis of amisulpride and of metamfetamine/amfetamine in plasma (200 µL) after single oral doses in man, a column packed with Waters Spherisorb S5SCX (5 µm average particle size, 100 × 2.1 mm i.d.) was used with methanolic ammonium acetate (40 mmol/L, pH* 6.0, flow rate 0.5 mL/min) as eluent (35°C). Deuterated internal standards were used for each analyte. Detection was by positive‐mode APCI. Responses for all analytes were linear over the calibration ranges. Intra‐assay precision (RSD) was 2–18%, and inter‐assay precision was 2–12%. The limit of detection was 0.5 µg/L for all analytes. No significant matrix effects or isobaric interferences were noted. The total analysis time was 7 min. Similar methodology can be applied to a wide range of basic analytes using MS/MS detection. Copyright © 2010 John Wiley & Sons, Ltd.     

气相色谱-质谱法检测饮用水新生含氮消毒副产物氯代乙酰胺

建立了气相色谱-质谱(GC-MS)法检测饮用水中新生致突变含氮消毒副产物(N-DBPs)二乙酰胺(DCAcAm)和三乙酰胺(TCAcAm)的分析方法.考察了GC/MS和GC/ECD两种检测仪器及不同样品前处理技术对氯代乙酰胺(CAcAms)的检测效果.对比发现,酸催化水解+GC/ECD的分析方法容易受其它消毒副产物(DBPs)的影响,直接液液萃取(LLE)+GC/MS更适合CAcAms的分析;乙酸乙酯(ETAC)的萃取效果优于DBPs分析常用萃取剂甲基叔丁基醚(MTBE).在选定条件下,DAcAm和TAcAm在10～1000 μg/L的范围内线性关系良好,r＞0.9995; 方法回收率在82.0%～111.9%之间;RSD小于10%;检出限(MDL)和测定限(RQL)均低于1 μg/L.