Recycling immunoaffinity chromatography for multiple analyte analysis in biological samples

The ability to isolate and measure multiple complex analytes in a single biological sample holds great potential in many biomedical fields, especially immunology and diagnostic clinical chemistry. We have developed a procedure involving recycling immunoaffinity chromatography for the simultaneous measurement of a number of analytes in a single sample. The procedure is based on the passage of a fluorochrome-labelled sample through a battery of small immunoaffinity columns, each column extracting a single analyte. Detection is achieved by acid elution of the bound analytes and laser-induced fluorescence. We have applied this system to a number of different biological fluids and found that it is capable of reliably isolating and measuring up to ten different cytokines in a 25-μl sample of human body fluid.     

Determination of a cardiac antiarrhythmic,tricyclic antipsychotics and antidepressants in human and animal urine by micellar electrokinetic capillary chromatography using a bile salt

A micellar electrokinetic capillary chromatographic method based on the use of sodium taurodeoxycholate has been developed to detect and quantitate a total of 26 tricyclic drugs. Detection limits in urine down to 4 ng/ml have been obtained. The method uses a simple liquid-liquid extraction and recovery of analytes followed by ultraviolet detection.     

Measuring environmental phenols and chlorinated organic chemicals in breast milk using automated on-line column-switching-high performance liquid chromatography-isotope dilution tandem mass spectrometry

Breast milk is one possible route of exposure to environmental chemicals, including phenols and chlorinated organic chemicals for breast-fed infants. We developed a highly sensitive method of analyzing breast milk for triclocarban (3,4,4'-trichlorocarbanilide) and eight phenolic compounds: bisphenol A (BPA), 4-tert-octylphenol (4-tOP), ortho-phenylphenol (OPP), 2,4-dichlorophenol, 2,5-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, and 2-hydroxy-4-metoxybenzophenone (BP-3). The method includes adding a solution containing a stable isotope of each chemical, enzymatic hydrolysis of the conjugated chemicals in the milk, and on-line solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry. It can also be used to measure the free (unconjugated) species by omitting the enzymatic deconjugation step. The method, validated using pooled breast milk samples, has inter-day coefficient of variations ranging from 4.8 to 18.9% for most analytes, and spiked recoveries generally about 100%. Detection limits for most analytes are below 1 ng/mL in 100 microL of breast milk. We tested the usefulness of the method by measuring concentrations of these nine compounds in 20 breast milk samples. BPA, OPP, and BP-3 were detected in more than 60% of the samples tested. The free species of these compounds appear to be most prevalent in milk.     

Ultrasensitive biosensing on the zepto-molar level

Detection of analytes on the zepto-molar (10(-21) M) level has been achieved using a field-effect bio-detector. By applying a gating voltage to enzymes immobilized on the working electrode of the detector, amplification of the biocatalytic current was observed. The amplification is attributed to the modification of the tunnel barrier between the enzyme and the electrode by the gating voltage-induced electric field which exists at the solution-electrode interface. The detection was demonstrated with the glucose oxidase (GOx)-glucose and alcohol dehydrogenase (ADH)-ethanol biocatalytic systems. Glucose at zepto-molar level was detected with zepto-molar detection resolution. Equivalently, 30 glucose molecules present in the sample were detected and the detection system responded distinctively to the incremental change in the number of glucose molecules in unit of 30 molecules. The enzyme's biospecificity was also preserved in the presence of the applied field. We present possible processes that could give rise to the electrical charges required to produce the observed current level.     

Detection of the isoflavone aglycones genistein and daidzein in urine using solid-phase microextraction–high-performance liquid chromatography–electrospray ionization mass spectrometry

An improved method of detection of the isoflavone aglycones, genistein and daidzein, is reported using solid-phase microextraction–high-performance liquid chromatography–electrospray ionization mass spectrometry (SPME–HPLC–ESI-MS). Extraction of the isoflavonoids from urine using SPME with a Carbowax–templated resin fiber coating allows rapid preconcentration of the analytes without the usual sample preparation required by other methods. Detection of the analytes is accomplished by HPLC–ESI-MS. Analysis of spiked samples of urine resulted in a linear range of 0.25 to 250 ng/ml for daidzein and 0.27 to 27.0 ng/ml for genistein. Limits of detection of daidzein and genistein were measured at 25.4 pg/ml for daidzein and 2.70 pg/ml for genistein. Daidzein and genistein were detected in urine following consumption of a soy drink.     

An improved method to discover adulteration of Styrian pumpkin seed oil

Pumpkin seed oil is rather expensive compared to other vegetable oils. Therefore, it is often adulterated by the addition of cheaper oils. In contrast to other edible oils, the content of Delta 5-sterols is very low, while Delta 7-sterols are dominating. The determination of Delta 5-sterols, especially beta-sitosterol has proven to be a good possibility to detect admixture of cheap vegetable oils to a level below which the economic profit is not significant any more. The presented method is a variant of an analysis concept, which has been published previously in European Food Research and Technology by Mandl et al. [Eur. Food Res. Technol. 209 (1999) 400. ]. It includes saponification of the triglycerides as a first step followed by separation of the potassium salts of the fatty acids from the unsaponifiable fraction by adsorption chromatography. In order to enhance gas chromatographic properties of the analytes, the hydroxyl function of the sterols is derivatized with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) to the trimethylsilylether. Finally, the analytes are separated on a capillary column of medium polarity (HP 35 MS) in a temperature programmed run within 18 min. Detection of the analytes was done by flame ionization. Special attention was set onto the precision and repeatability of the method.     

Rapid assay of cocaine, opiates and metabolites by gas chromatography—mass spectrometry

The simultaneous assay of cocaine, opiates and metabolites in small biological samples continues to be a difficult task. This report focuses upon tabulation of important techniques (extraction, derivatization, chromatographic conditions, detection mode, data acquisition) reported over the last decade that were used in the development of assays for these analytes. The most prevalent procedures for extraction of cocaine, opiates and metabolites were liquid—liquid and solid-phase extraction isolation methods. Following extraction analytes were derivatized and analyzed by gas chromatography—mass spectrometry. The technique most often used for chromatographic separation was fused-silica capillary column gas chromatography. Detection generally was performed by selected ion monitoring in the positive-ion electron-impact ionization mode, although full-scan acquisition and positive- and negative-ion chemical ionization methods have been used. It was apparent from the review that there is a continuing need for greater sensitivity and selectivity in the assay of highly potent opiates and for cocaine and metabolites.     

Simultaneous multi-analyte determination of estrone, isoproturon and atrazine in natural waters by the RIver ANAlyser (RIANA), an optical immunosensor

In most medical and environmental applications of biosensors, only single analytes are determined. However, the monitoring of several analytes is obviously preferable in order to gather more information about the sample under analysis. In line with this, different technologies are being developed to obtain multi-analyte sensors.In this paper, an analytical method for the simultaneous determination of three different contaminants-atrazine, isoproturon, and estrone-in natural waters by using an optical immunosensor prototype, the so-called "RIver ANAlyser" (RIANA), is described. RIANA is based on a rapid solid-phase fluoroimmunoassay that takes place at an optical transducer chip. The transducer surface is chemically modified with three analytes derivatives placed in different discrete locations. The sensor surface can be regenerated thus allowing the performance of several measurements with the same transducer. Each test cycle, including one regeneration step, is accomplished in 15 min. Detection limits achieved were 0.155, 0.046, and 0.084 microg/l, for atrazine, isoproturon, and estrone, respectively. Satisfactory repetition, with relative standard deviations between 1.06 and 6.98%, was obtained. Excluding a minor non-specifical binding of the isoproturon antibodies, no cross-reactivity effects were observed. Matrix effects were significant only in the case of wastewater samples. Biosensor measurements were validated using conventional liquid chromatography-mass spectrometry. The results obtained with both techniques were in good agreement.     

Gas chromatographic–mass spectrometric analysis of veterinary tranquillizers in urine: evaluation of method performance

A method for analysis of veterinary tranquillizers in urine using gas chromatography–mass spectrometry (GC–MS) is described. Detection limits are 5 μg/l for ketamine, azaperone and the phenothiazines (chlor-, aceto- and propionylpromazine), 10 μg/l for haloperidol, 20 μg/l for xylazine and 50 μg/l for azaperol, recoveries for all analytes were higher than 70%. Method performance in terms of within-batch, between-days and between-analysts reproducibility was studied and found to be acceptable. Compliance with European Union criteria for confirmation of GC–MS “positive” results is evaluated and discussed.     

Multi-analyte analysis of biological fluids with a recycling immunoaffinity column array.

A system for isolating and measuring up to 30 analytes in a single biological sample is described. The system is based on recycling a pre-labeled sample through an array of capillary immunoaffinity columns, each packed with glass beads, coated with a different antibody, thus enabling each column to isolate and extract a single analyte. Detection of the bound analytes is achieved by laser-induced fluorescence (LIF), using a laboratory-built scanning detector coupled to a fiber-optic spectrometer. The array can be regenerated up to 200 times, provided a suitable temperature is maintained. The individual immunoaffinity columns are able to bind between 2.9 and 3.6 ng of analyte, depending upon the individual column, with lower limits of detection (LOD) in the order of 1.6-2.8 pg/ml. The inter- and intra-assay coefficients of variation (CV) for all 30 columns in the array were less than 6.03+/-0.33 at analyte concentrations of 100 pg/ml. Comparison to standard enzyme-immunoassays demonstrated r(2) values in the range of 0.9151-0.9855 when analyzed by least-squares linear regression.     

Dual-signal analysis eliminates requirement for milk sample pretreatment

Detection of analytes in complex biological samples, such as milk and blood, normally requires sample pretreatment. These pretreatment regimes reduce assay throughput and increase testing costs. Technologies that make it possible to eliminate sample pretreatment are of great industrial interest. Here we report the development of a dual-signal flow injected analysis device which eliminates the need for sample pretreatment. The device employs thermal traducers to measure the signal from an enzyme and a reference column. This makes it possible to independently monitor and correct for non-specifically generated heat, thereby eliminating the need for sample pretreatment. The ability of the dual-signal device to determine urea and lactate in milk samples without any prior treatment was evaluated. The spiked milk samples, the urea assay had a linear range from 0.1 to 50mM (R=0.996), and the lactate assay had a linear range from 0.025 to 5.0mM (R=0.9998). The linear regression values for urea and lactate for 0.5%, 1.5% and 3.0% fat milk were at least 0.990. The dual-signal design improves assay reproducibility, accuracy and sensitivity. Addition benefits are shorter assay times and lowers costs, as well as reducing equipment and training requirements. The potential application of the technology for multi-analyte analysis in point of care and decentralized diagnostic testing in healthcare, agriculture and environmental areas is discussed.     

Sensitivity enhancement in liquid chromatography/atmospheric pressure ionization mass spectrometry using derivatization and mobile phase additives

High performance liquid chromatography with atmospheric pressure ionization (API) mass spectrometry has been essential to a large number of quantitative analytical applications for a variety of compounds. Poor detection sensitivity however is a problem observed for a number of analytes because detection sensitivity can be affected by many factors. The two most critical factors are the chemical and physical properties of the analyte and the composition of the mobile phase. In order to address these critical factors which may lead to poor sensitivity, either the structure of the analyte must be modified or the mobile phase composition optimized. The introduction of permanently charged moieties or readily ionized species may dramatically improve the ionization efficiency for electrospray ionization (ESI), and thus the sensitivity of detection. Detection sensitivity may also be enhanced via introduction of moieties with high proton affinity or electron affinity. Mobile phase component modification is an alternative way to enhance sensitivity by changing the form of the analytes in solution thereby improving ionization efficiency. pH adjustment and adduct formation have been commonly used to optimize detection conditions. The sensitivity of detection for analytes in bio-matrices could also be enhanced by decreasing ion-suppression from the matrix through derivatization or mobile phase addition. In this review, we will discuss detection-oriented derivatization as well as the application of mobile phase additives to enhance the sensitivity of detection in liquid chromatograph/atmospheric ionization/mass spectrometry (LC/API/MS), focusing in particular on the applications involving small molecules in bio-matrices.     

Determination of 2′-deoxy-5-iodouridine and its metabolite 5-iodouracil by high-performance liquid chromatography with ultraviolet absorbance detection in human serum

A new assay is described for 2′-deoxy-5-iodouridine, a drug employed as an antiviral agent by topical application. The parent drug, its systemic metabolite 5-iodouracil and an internal standard (5-iodouridine) were extracted from salted serum by an ethyl acetate partition at pH 6.7, back-extracted in alkalinized water and injected into a reversed-phase column. Potassium phosphate buffer—acetonitrile (95:5, v/v) eluted the analytes at a flow-rate of 1.5 ml/min. Detection was at 290 nm. The method proved to be linear in the 100–2000 ng/ml range.     

High-performance liquid chromatography of phosphatidic acid

This paper reviews existing high-performance liquid chromatographic (HPLC) methods for the analysis of phosphatidic acid (PA) in various sample matrices. In addition to the introductory background discussion on important aspects of PA in lipid biochemistry, the review provides comprehensive coverage in the areas of derivatization techniques, detection methods, and HPLC separation techniques. Conversions of PA to suitable derivatives enhance the detection sensitivity and improve the chromatographic behavior of the analytes. Detection methods include the use of state-of-the-art detectors and are discussed in terms of sensitivity, specificity, and compatibility with analytical systems. Pertinent normal-phase and reversed-phase HPLC data for PA are compiled from published methods.     

Glass-supported lipid/polydiacetylene films for colour sensing of membrane-active compounds

Glass-supported biomimetic lipid/polydiacetylene films were employed for colourimetric detection and analysis of amphiphilic and membrane-active molecules. The sensor films comprise lipid monolayers that constitute a biomimetic membrane platform, interspersed within polydiacetylene domains that function as the colour reporter. The optical detection scheme is based on visible blue–red transitions of polydiacetylene, induced by amphiphilic analytes interacting with the film. The colour transitions of the lipid/polydiacetylene films can be either detected by the naked eye, recorded spectroscopically, or registered through digital image analysis using conventional scanning devices. Digital image analysis, in particular, allows quantification of the colourimetric transformations. Detection threshold of micromolar concentration of a membrane-active cytolytic peptide is demonstrated.     

Detection of clinically relevant levels of protein analyte under physiologic buffer using planar field effect transistors

Electrochemical detection of protein binding at physiological salt concentration by planar field effect transistor platforms has yet to be documented convincingly. Here we report detection of streptavidin and clinically relevant levels of biotinylated monokine induced by interferon gamma (MIG) at physiological salt concentrations with AlGaN heterojunction field effect transistors (HFETs). The AlGaN HFETs are functionalized with a silane linker and analyte-specific affinity elements. Polarity of sensor responses is as expected from n-type HFETs to negatively and positively charged analytes. Sensitivity of the HFET sensors increases when salt concentration decreases, and the devices also exhibit dose-dependent responses to analyte. Detection of clinically relevant MIG concentrations at physiological salt levels demonstrates the potential for AlGaN devices to be used in development of in vivo biosensors.     

Sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous determination of paracetamol and guaifenesin in human plasma

A rapid and sensitive method for the simultaneous determination of paracetamol and guaifenesin in human plasma was developed and validated, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. After extracted from plasma samples by diethyl ether-dichloromethane (3:2, v/v), the analytes and internal standard osalmide were chromatographed on a C18 column. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI). The method was linear in the concentration range of 0.05-20.0 microg/ml for paracetamol and 5.0-2000.0 ng/ml for guaifenesin. The intra- and inter-day precision was within 14% for both paracetamol and guaifenesin. The assay accuracy was within +/-2.4% for the analytes. This is the first assay method described for the simultaneous determination of paracetamol and guaifenesin in plasma using one chromatographic run. The method was successfully employed in a pharmacokinetic study after an oral administration of a multicomponent formulation, containing 650 mg paracetamol, 200 mg guaifenesin, 60 mg pseudoephedrine and 20 mg dextrorphan.     

Multi-analyte assay for triazines using cross-reactive antibodies and neural networks

A biosensor system based on total internal reflectance fluorescence (TIRF) was used to discriminate a mixture of the triazines atrazine and simazine. Only cross-reactive antibodies were available for these two analytes. The biosensor is fully automated and can be regenerated allowing several hundreds of measurements without any user input. Even a remote control for online monitoring in the field is possible. The multivariate calibration of the sensor signal was performed using artificial neural networks, as the relationship between the sensor signals and the concentration of the analytes is highly non-linear. For the development of a multi-analyte immunoassay consisting of two polyclonal antibodies with cross-reactivity to atrazine and simazine and different derivatives immobilised on the transducer surface, the binding characteristics between these substances like binding capacity and cross-reactivity were characterised. The examination of three different measurement procedures showed that a two-step measurement using only one antibody per step allows a quantification of both analytes in a mixture with limits of detection of 0.2 microg/l for atrazine and 0.3 microg/l for simazine. The biosensor is suitable for online monitoring in the field and remote control is possible.     

Simultaneous analysis of cyclophosphamide, doxorubicin and doxorubicinol by liquid chromatography coupled to tandem mass spectrometry

A method for the simultaneous determination of cyclophosphamide (CP), doxorubicin (dox), and doxorubicinol (dol) was developed and validated to analyze 400 microL of plasma from patients receiving chemotherapeutic treatment with CP and dox. Final calibration ranges for the analytes were 0.440-60.0 microg/mL for cyclophosphamide, 7.20-984 ng/mL for dox and 3.04-104 ng/mL for dol. The samples were prepared using solid phase extraction and analyzed using a gradient separation over a Waters Symmetry C18, 2.1 by 30 mm (Milford, MA) column. Detection was achieved in positive mixed reaction monitoring mode on a triple quadrupole mass spectrometer.     

A GC-MS/MS method for the quantitative analysis of low levels of the tyrosine metabolites maleylacetone, succinylacetone, and the tyrosine metabolism inhibitor dichloroacetate in biological fluids and tissues

We developed a sensitive method to quantitate the tyrosine metabolites maleylacetone (MA) and succinylacetone (SA) and the tyrosine metabolism inhibitor dichloroacetate (DCA) in biological specimens. Accumulation of these metabolites may be responsible for the toxicity observed when exposed to DCA. Detection limits of previous methods are 200 ng/mL (1.2 pmol/microL) (MA) and 2.6 microg/mL (16.5 pmol/microL) (SA) but the metabolites are likely present in lower levels in biological specimens. To increase sensitivity, analytes were extracted from liver, urine, plasma and cultured nerve cells before and after dosing with DCA, derivatized to their pentafluorobenzyl esters, and analyzed via GC-MS/MS.